**Weed and Disease Control and Peanut Response Following Post—Emergence Herbicide and Fungicide Combinations**

W. James Grichar, Peter A. Dotray and Jason E. Woodward

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55949

### **1. Introduction**

Peanut, or groundnut (*Arachis hypogaea* L.), is a species in the legume or "bean" family (Faba‐ ceae). Hypogaea means "under the earth" [1]. Peanuts are known by many other local names such as earthnuts, goober peas, monkey nuts, pygmy nuts and pig nuts [2,3]. Peanut was probably first domesticated and cultivated in the valleys of Paraguay [3].

The domesticated peanut is an amphidiploid or allotetraploid, meaning that it has two sets of chromosomes from two different species, thought to be *A*. *duranensis* and *A. ipaensis*. These likely combined in the wild to form the tetraploid species, *A*. *monticola*, which gave rise to the domesticated peanut [4,5]. This domestication might have taken place in Paraguay or Bolivia, where the wildest strains are found today. Archeologists have dated the oldest specimens to about 7,600 years, found in Peru [3,4]. Cultivation spread as far as Mesoamerica where the Spanish conquistadors found the tlalcacahuatl (Nahuatl = "peanut", whence Mexican Spanish, cacahuate and French, cacahuète) being offered for sale in the marketplace of Tenochtitlan (Mexico City). The plant was later spread worldwide by European traders [3].

Peanuts grow best in light, sandy loam soil. They require 120 to 150 days of warm weather, and an annual rainfall of 380 to 650 mm or the equivalent in irrigation water [6]. It is an annual herbaceous plant growing 30 to 50 cm tall. The leaves are opposite, pinnate with four leaflets (two opposite pairs; no terminal leaflet), each leaflet 1 to 7 cm long and 1 to 3 cm wide. The orange-veined, yellow-petaled, pea-like flower (2 to 4 cm across) of *A*. *hypogaea* is borne in axillary clusters above ground. Following self-pollination, the flowers fade and wither. The stalk at the base of the ovary, called the pedicel, elongates rapidly, and turns downward.

© 2013 Grichar et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Grichar et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Santín-Montanyá et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

Continued stalk growth pushes the ovary underground where the mature fruit develops into a legume pod (the peanut). The fruits or pods have wrinkled shells that are constricted between pairs of the one to four (usually two) seeds per pod [4,5].

system becomes intertwined with the peanut plant, causing peanut pods to be stripped from the vine during digging. Peanuts that become detached from the plant remain unharvested

Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations

 China 15.64 India 5.85 United States 1.89 Nigeria 1.55 Senegal 1.29 Indonesia 1.25 Burma 1.14 Argentina 1.05 Sudan 0.85 Chad 0.47 Ghana 0.44 Vietnam 0.44 Congo Kinshasa 0.37 Burkino Faso 0.35 Mali 0.28 Malawi 0.27 Guinea 0.26 Cameroon 0.24 Brazil 0.23 Egypt 0.19

Total 34.05

The dinitroaniline herbicides are registered for use in over forty crops [19]. These herbi‐ cides provide excellent control of annual grasses [10,18,20] and are the only soil-applied herbicides registered for use in peanut that will provide full-season control of Texas millet [10,21,22]. Peanut tolerance to the dinitroaniline herbicides has been questioned previously [23,24,25]. Greenhouse studies showed that ethalfluralin inhibited seedling growth more than pendimethalin at equivalent rates applied preplant incorporated; however, injury by these herbicides following preemergence applications were similar [26]. In runner peanuts, which are more prone to peg injury compared to Spanish peanut [27], proper herbicide incorpora‐

Source: USDA Foreign Agricultural Service; Table 13 Peanut Area, Yield, and Production (Created 8/10/2012)

**Production (Million metric tons)**

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103

in or on the soil [18].

**Rank Country**

**Table 1.** Worldwide peanut production.

Harvesting occurs in two stages [6]. In modern, mechanized systems, a machine called a digger is used to cut off the main or tap root of the peanut plant by cutting through the soil just below the level of the peanut pods. The machine lifts the plant from the ground, shakes and inverts the plant, leaving it upside down on the ground to keep the peanut pods out of the soil. This allows the peanuts to dry slowly to a bit less than a third of their original moisture level over a period of three to four days depending on weather conditions [6]. Prior to mechanization, peanuts were pulled and inverted by hand [6]. The second stage consists of the use of a combine to remove peanuts from the vine.

World peanut production totals approximately 34 million metric tons per year (Table 1). China leads in production of peanuts, having a share of about 46% of overall world production, followed by India (17%), and the United States (6 %) [7]. The United States is one of the world's leading exporters, with average annual exports of between 200,000 and 250,000 metric tons. Argentina and China are other significant exporters [7].

Peanut production requires the use of a wide range of agrichemical products to control weed and diseases and optimize crop growth and development [8-10]. Peanut has several unique features that contribute to challenging weed management [10]. Peanut cultivars grown in the United States require a fairly long growing season (140 to 160 days), depending on cultivar and geographical region [10,11]. Consequently, soil-applied herbicides may not provide season-long control and mid-to-late season weed emergence can occur. Peanut also has a prostrate growth habit, a relatively shallow canopy, and is slow to shade inter-rows allowing weeds to be more competitive [10,12]. Additionally, peanut fruit develop underground on pegs originating from branches that grow along the soil surface. This prostrate growth habit and pattern of fruit development restricts cultivation to an early season control option [10,13]. With conventional row spacing (91 to 102 cm), complete ground cover may not be attained until 8 to 10 weeks after planting. In some areas of the United States peanut growing region, complete canopy closure may never occur.

Pigweed (*Amaranthus* spp.) is listed as one of the ten most common weeds in most peanutgrowing states in the United States, with Palmer amaranth (*Amaranthus palmeri* S. Wats) ranked as the fourth most common weed in South Carolina [14]. Palmer amaranth is not generally ranked as a troublesome weed in most crops in the United States; however, it is a common weed in many crops produced around the world. Palmer amaranth is currently found in the southern half of the United States [15] while in Texas, Palmer amaranth can be found in all areas of the state [16] and is a severe problem in many peanut fields when not properly controlled [17]. Texas millet (*Urochloa texana* (Buckley) R. D. Webster) is a large seeded, vigorous, fast growing annual grass commonly found in peanut fields in parts of Florida, South Carolina, Oklahoma, and Texas [14]. It is listed as one of the most trouble‐ some weeds in all peanut growing states except Alabama and Georgia [14]. During the digging operation, the peanut plant is lifted out of the ground and inverted. A heavy stand of Palmer amaranth or Texas millet can reduce the effectiveness of the process. The tight fibrous root system becomes intertwined with the peanut plant, causing peanut pods to be stripped from the vine during digging. Peanuts that become detached from the plant remain unharvested in or on the soil [18].


Source: USDA Foreign Agricultural Service; Table 13 Peanut Area, Yield, and Production (Created 8/10/2012)

**Table 1.** Worldwide peanut production.

Continued stalk growth pushes the ovary underground where the mature fruit develops into a legume pod (the peanut). The fruits or pods have wrinkled shells that are constricted between

Harvesting occurs in two stages [6]. In modern, mechanized systems, a machine called a digger is used to cut off the main or tap root of the peanut plant by cutting through the soil just below the level of the peanut pods. The machine lifts the plant from the ground, shakes and inverts the plant, leaving it upside down on the ground to keep the peanut pods out of the soil. This allows the peanuts to dry slowly to a bit less than a third of their original moisture level over a period of three to four days depending on weather conditions [6]. Prior to mechanization, peanuts were pulled and inverted by hand [6]. The second stage consists of the use of a combine

World peanut production totals approximately 34 million metric tons per year (Table 1). China leads in production of peanuts, having a share of about 46% of overall world production, followed by India (17%), and the United States (6 %) [7]. The United States is one of the world's leading exporters, with average annual exports of between 200,000 and 250,000 metric tons.

Peanut production requires the use of a wide range of agrichemical products to control weed and diseases and optimize crop growth and development [8-10]. Peanut has several unique features that contribute to challenging weed management [10]. Peanut cultivars grown in the United States require a fairly long growing season (140 to 160 days), depending on cultivar and geographical region [10,11]. Consequently, soil-applied herbicides may not provide season-long control and mid-to-late season weed emergence can occur. Peanut also has a prostrate growth habit, a relatively shallow canopy, and is slow to shade inter-rows allowing weeds to be more competitive [10,12]. Additionally, peanut fruit develop underground on pegs originating from branches that grow along the soil surface. This prostrate growth habit and pattern of fruit development restricts cultivation to an early season control option [10,13]. With conventional row spacing (91 to 102 cm), complete ground cover may not be attained until 8 to 10 weeks after planting. In some areas of the United States peanut growing region,

Pigweed (*Amaranthus* spp.) is listed as one of the ten most common weeds in most peanutgrowing states in the United States, with Palmer amaranth (*Amaranthus palmeri* S. Wats) ranked as the fourth most common weed in South Carolina [14]. Palmer amaranth is not generally ranked as a troublesome weed in most crops in the United States; however, it is a common weed in many crops produced around the world. Palmer amaranth is currently found in the southern half of the United States [15] while in Texas, Palmer amaranth can be found in all areas of the state [16] and is a severe problem in many peanut fields when not properly controlled [17]. Texas millet (*Urochloa texana* (Buckley) R. D. Webster) is a large seeded, vigorous, fast growing annual grass commonly found in peanut fields in parts of Florida, South Carolina, Oklahoma, and Texas [14]. It is listed as one of the most trouble‐ some weeds in all peanut growing states except Alabama and Georgia [14]. During the digging operation, the peanut plant is lifted out of the ground and inverted. A heavy stand of Palmer amaranth or Texas millet can reduce the effectiveness of the process. The tight fibrous root

pairs of the one to four (usually two) seeds per pod [4,5].

Argentina and China are other significant exporters [7].

complete canopy closure may never occur.

to remove peanuts from the vine.

102 Herbicides - Current Research and Case Studies in Use

The dinitroaniline herbicides are registered for use in over forty crops [19]. These herbi‐ cides provide excellent control of annual grasses [10,18,20] and are the only soil-applied herbicides registered for use in peanut that will provide full-season control of Texas millet [10,21,22]. Peanut tolerance to the dinitroaniline herbicides has been questioned previously [23,24,25]. Greenhouse studies showed that ethalfluralin inhibited seedling growth more than pendimethalin at equivalent rates applied preplant incorporated; however, injury by these herbicides following preemergence applications were similar [26]. In runner peanuts, which are more prone to peg injury compared to Spanish peanut [27], proper herbicide incorpora‐ tion was needed to prevent injury [28]. Merkle [27] stated that sporadic injury to runner peanut from trifluralin was due to the failure to properly incorporate the herbicide. No differences were observed in a study examining peanut growth, yield, and grade effects with ethalfluralin, pendimethalin, or trifluralin in two different studies [24,29]. In Florida, ethalfluralin did not cause peanut injury at any rate or application timing [23]. Dinitroani‐ line injury on peanut includes swollen hypocotyl, abnormal lateral root growth, and stunted plants [18,28].

large crabgrass [*Digitaria sanguinalis* (L.) Scop.], southern crabgrass [*Digitaria ciliaris* (Retz.)

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Peanut is susceptible to numerous fungal diseases caused by foliar and soilborne pathogens. Chlorothalonil has been the most widely used fungicide in the United States peanut production areas for control of early leaf spot caused by *Cercospora arachidicola* S. Hori, late leaf spot caused by *Cercosporidium personatum* Berk. & M.A. Curtis, and rust caused by *Puccinia arachidis* Speg. for over 30 years [49,50]. Despite its widespread use across the peanut belt, chlorothalonil continues to provide effective control of foliar diseases [50,51]; however, it has no activity against any of the soilborne diseases such as southern stem rot caused by *Sclerotium rolfsii* Sacc. or Rhizoctonia pod or stem rot caused by *Rhizoctonia solani* Kühn [49,52,53]. Within the past 15 years, several fungicides, including the sterol biosynthesis inhibitor fungicide, tebucona‐ zole, along with the strobilurin fungicides azoxystrobin and pyraclostrobin have been registered for use in peanut for control of both leaf spot and soilborne diseases [49,52-55].

Depending on the fungicide, the calendar spray regime in the southeastern United States may result in seven applications [50,52] while in the southwest United States peanut growing region a maximum of five fungicide applications may be applied during the growing season [53,56]. Chlorothalonil is used to fill the remaining treatment slots in an azoxystrobin, pyraclostrobin, tebuconazole program to minimize the risk of fungal pathogens developing resistance to

Prothioconazole is a sterol biosynthesis inhibitor fungicide in the new triazolinthione class of fungicides [58] that has shown activity against the leaf spot pathogens, *C*. *arachidicola* and *C*. *personatum*, as well as the soilborne pathogens *S. rolfsii* and *R. solani* [59]. Prothioconazole has shown promise for control of cereal diseases in Europe when applied alone or in combination with strobilurin fungicides [58]. In addition, the activity of this fungicide on foliar diseases is of special interest because populations of both *C. arachidicola* and *C. personatum* have displayed reduced sensitivity to tebuconazole and noticeable reductions in efficacy of that fungicide [59]. Prothioconazole plus tebuconazole received registration for use in peanut during the 2008

Management strategies to protect peanut from various weeds, insects, and fungi require multiple applications of herbicides, insecticides, or fungicides. Timing of application of herbicides and fungicides may coincide during the growing season, and co-application of these pesticides is desirable if herbicide or fungicide performance and peanut tolerance are not compromised [61]. Potential interactions related to physiological effects on plants and other organisms, application variables such as adjuvant, water quality, commercial formulation, and

Considerable research has been conducted over the past several years to define interactions among pesticides including interactions of herbicides in mixture with other herbicides and

environmental stress can affect pesticide compatibility [61].

Koel.], and broadleaf signalgrass [*Brachiaria platyphylla* (Griseb.) Nash] [46].

triazole or strobilurin fungicides [57].

growing season [60].

**2. Research needs**

Metolachlor is commonly used in peanut for control of small-seeded broadleaf weeds, some annual grasses, and yellow nutsedge [30]. *S*-metolachlor is labeled for either preplant incor‐ porated (PPI), POST-plant incorporated, preemergence (PRE), postemergence (POST), or layby in peanut [31]. The registered rate for the southwest United States is 1.1 to 1.4 kg/ha [31]. However, many growers have reported peanut stunting when soil applications of metolachlor have been followed by rain [30]. Grichar et al. [30] reported that POST applications of meto‐ lachlor followed by (fb) irrigation within 24 hour could be effective for yellow nutsedge control and reduce the chance of peanut injury from soil applications of metolachlor. Combinations of factors, such as herbicide rate, moisture conditions at planting, soil organic matter, and pH may affect peanut injury by chloroacetamide herbicides such as *S*-metolachlor [32-35]. Cardina and Swann [32] reported that metolachlor often delayed peanut emergence and reduced peanut growth when irrigation followed planting. However, yield loss was observed only when metolachlor was applied at a 3X rate.

Several postemergence herbicides are used to control weed escapes. Imazethapyr and imazapic are imidazolinone herbicides registered for use in peanut. Imazethapyr may be applied PPI, PRE, ground cracking (GC), or POST for effective weed control [10]. Imazetha‐ pyr applied PPI or PRE controls many troublesome weeds such as coffee senna (*Cassia occidentalis* L.), common lambsquarters (*Chenopodium album* L.), morningglory species (*Ipomoea* spp.), pigweed species (*Amaranthus* spp.) including Palmer amaranth, prickly sida (*Sida spinosa* L.), purple and yellow nutsedge (*Cyperus rotundus* L. and *C. esculentus* L., respective‐ ly), spurred anoda [*Anoda cristata* (L.) Schlecht.], and wild poinsettia (*Euphorbia heterophylla* L.) [29,36-39].

Imazethapyr applied POST provides broad spectrum and most consistent control when applied within 10 days of weed emergence [37,40,41]. Imazethapyr and imazapic are the only POST herbicides to effectively control both yellow and purple nutsedge [29,42]. Control is most effective when imazethapyr is applied to the soil or to yellow nutsedge that is no more than 12 cm tall [10,42,43].

Imazapic is similar to imazethapyr and controls all the weeds controlled by imazethapyr [10,44-46]. In addition, imazapic provides control and suppression of Florida beggarweed [*Desmodium tortuosum* (S.W.) D.C.] and sicklepod [*Senna obtusifolia* (L.) Irwin & Barneby), which are not adequately controlled by imazethapyr [47]. Imazethapyr provides consistent control of many broadleaf and sedge species if applied within 10 days after emergence, but imazapic has a longer effectiveness period when applied POST [10,42,46,48]. Imazapic also is effective for control of rhizome and seedling johnsongrass [*Sorghum halepense* (L.) Pers.], Texas millet, large crabgrass [*Digitaria sanguinalis* (L.) Scop.], southern crabgrass [*Digitaria ciliaris* (Retz.) Koel.], and broadleaf signalgrass [*Brachiaria platyphylla* (Griseb.) Nash] [46].

Peanut is susceptible to numerous fungal diseases caused by foliar and soilborne pathogens. Chlorothalonil has been the most widely used fungicide in the United States peanut production areas for control of early leaf spot caused by *Cercospora arachidicola* S. Hori, late leaf spot caused by *Cercosporidium personatum* Berk. & M.A. Curtis, and rust caused by *Puccinia arachidis* Speg. for over 30 years [49,50]. Despite its widespread use across the peanut belt, chlorothalonil continues to provide effective control of foliar diseases [50,51]; however, it has no activity against any of the soilborne diseases such as southern stem rot caused by *Sclerotium rolfsii* Sacc. or Rhizoctonia pod or stem rot caused by *Rhizoctonia solani* Kühn [49,52,53]. Within the past 15 years, several fungicides, including the sterol biosynthesis inhibitor fungicide, tebucona‐ zole, along with the strobilurin fungicides azoxystrobin and pyraclostrobin have been registered for use in peanut for control of both leaf spot and soilborne diseases [49,52-55].

Depending on the fungicide, the calendar spray regime in the southeastern United States may result in seven applications [50,52] while in the southwest United States peanut growing region a maximum of five fungicide applications may be applied during the growing season [53,56]. Chlorothalonil is used to fill the remaining treatment slots in an azoxystrobin, pyraclostrobin, tebuconazole program to minimize the risk of fungal pathogens developing resistance to triazole or strobilurin fungicides [57].

Prothioconazole is a sterol biosynthesis inhibitor fungicide in the new triazolinthione class of fungicides [58] that has shown activity against the leaf spot pathogens, *C*. *arachidicola* and *C*. *personatum*, as well as the soilborne pathogens *S. rolfsii* and *R. solani* [59]. Prothioconazole has shown promise for control of cereal diseases in Europe when applied alone or in combination with strobilurin fungicides [58]. In addition, the activity of this fungicide on foliar diseases is of special interest because populations of both *C. arachidicola* and *C. personatum* have displayed reduced sensitivity to tebuconazole and noticeable reductions in efficacy of that fungicide [59]. Prothioconazole plus tebuconazole received registration for use in peanut during the 2008 growing season [60].

Management strategies to protect peanut from various weeds, insects, and fungi require multiple applications of herbicides, insecticides, or fungicides. Timing of application of herbicides and fungicides may coincide during the growing season, and co-application of these pesticides is desirable if herbicide or fungicide performance and peanut tolerance are not compromised [61]. Potential interactions related to physiological effects on plants and other organisms, application variables such as adjuvant, water quality, commercial formulation, and environmental stress can affect pesticide compatibility [61].

### **2. Research needs**

tion was needed to prevent injury [28]. Merkle [27] stated that sporadic injury to runner peanut from trifluralin was due to the failure to properly incorporate the herbicide. No differences were observed in a study examining peanut growth, yield, and grade effects with ethalfluralin, pendimethalin, or trifluralin in two different studies [24,29]. In Florida, ethalfluralin did not cause peanut injury at any rate or application timing [23]. Dinitroani‐ line injury on peanut includes swollen hypocotyl, abnormal lateral root growth, and stunted

Metolachlor is commonly used in peanut for control of small-seeded broadleaf weeds, some annual grasses, and yellow nutsedge [30]. *S*-metolachlor is labeled for either preplant incor‐ porated (PPI), POST-plant incorporated, preemergence (PRE), postemergence (POST), or layby in peanut [31]. The registered rate for the southwest United States is 1.1 to 1.4 kg/ha [31]. However, many growers have reported peanut stunting when soil applications of metolachlor have been followed by rain [30]. Grichar et al. [30] reported that POST applications of meto‐ lachlor followed by (fb) irrigation within 24 hour could be effective for yellow nutsedge control and reduce the chance of peanut injury from soil applications of metolachlor. Combinations of factors, such as herbicide rate, moisture conditions at planting, soil organic matter, and pH may affect peanut injury by chloroacetamide herbicides such as *S*-metolachlor [32-35]. Cardina and Swann [32] reported that metolachlor often delayed peanut emergence and reduced peanut growth when irrigation followed planting. However, yield loss was observed only

Several postemergence herbicides are used to control weed escapes. Imazethapyr and imazapic are imidazolinone herbicides registered for use in peanut. Imazethapyr may be applied PPI, PRE, ground cracking (GC), or POST for effective weed control [10]. Imazetha‐ pyr applied PPI or PRE controls many troublesome weeds such as coffee senna (*Cassia occidentalis* L.), common lambsquarters (*Chenopodium album* L.), morningglory species (*Ipomoea* spp.), pigweed species (*Amaranthus* spp.) including Palmer amaranth, prickly sida (*Sida spinosa* L.), purple and yellow nutsedge (*Cyperus rotundus* L. and *C. esculentus* L., respective‐ ly), spurred anoda [*Anoda cristata* (L.) Schlecht.], and wild poinsettia (*Euphorbia heterophylla*

Imazethapyr applied POST provides broad spectrum and most consistent control when applied within 10 days of weed emergence [37,40,41]. Imazethapyr and imazapic are the only POST herbicides to effectively control both yellow and purple nutsedge [29,42]. Control is most effective when imazethapyr is applied to the soil or to yellow nutsedge that is no more than

Imazapic is similar to imazethapyr and controls all the weeds controlled by imazethapyr [10,44-46]. In addition, imazapic provides control and suppression of Florida beggarweed [*Desmodium tortuosum* (S.W.) D.C.] and sicklepod [*Senna obtusifolia* (L.) Irwin & Barneby), which are not adequately controlled by imazethapyr [47]. Imazethapyr provides consistent control of many broadleaf and sedge species if applied within 10 days after emergence, but imazapic has a longer effectiveness period when applied POST [10,42,46,48]. Imazapic also is effective for control of rhizome and seedling johnsongrass [*Sorghum halepense* (L.) Pers.], Texas millet,

plants [18,28].

104 Herbicides - Current Research and Case Studies in Use

L.) [29,36-39].

12 cm tall [10,42,43].

when metolachlor was applied at a 3X rate.

Considerable research has been conducted over the past several years to define interactions among pesticides including interactions of herbicides in mixture with other herbicides and fungicides [62-65]. Peanut fungicides are applied beginning approximately 30 to 60 days after planting and can be applied until a few weeks prior to digging. Efficacy of clethodim and sethoxydim can be reduced by co-application with copper-containing fungicides or azoxy‐ strobin, chlorothalonil, and pyraclostrobin [8,66,67]. Fluazinam and tebuconazole did not reduce grass control compared with graminicides applied alone [8,9,66]. Efficacy of herbicides that control dicotyledonous weeds and sedges are not generally affected by fungicides [66]. Weed species and size, and plant stress can affect the magnitude of interactions between herbicides and fungicides [66].

sethoxydim at 0.21 kg ai/ha while the broadleaf weed study included aciflurofen at 0.42 kg ai/ ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides evaluated included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168

Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations

Herbicides and fungicides were applied alone and in combination to determine efficacy against various weeds. A crop oil concentrate (Agri-Dex, a blend of 83% paraffin-based petroleum oil and 17% surfactant) at 2.3 L/ha was added to each treatment except in 2007 at Yoakum where a non-ionic surfactant (X-77, 90% nonionic surfactant) at 0.25% v/v was added. Herbicide and fungicides at Yoakum were applied with a CO2-pressurized backpack sprayer equipped with TeeJet 11002 DG flat fan spray tips (Spraying Systems Company, P.O. Box 7900, North Avenue, Wheaton, IL 60188) that delivered a spray volume of 190 L/ha at 180 kPa while on the Texas High Plains locations, fungicides and herbicides were applied with a CO2 pressurized backpack sprayer using TeeJet 110015 TT flat fan nozzles calibrated to deliver a spray volume of 94 L/ha at 207 kPa. At Yoakum, the peanut variety Tamrun OL02 [68] was planted in each year at a seeding rate of 112 kg/ha. At the Texas High Plains locations, Flavor Runner 458 [69]

Texas millet and southern crabgrass were present at Yoakum in 2007 and 2009 while broadleaf signalgrass was present in 2008. Texas millet was present at Lamesa in 2007. Palmer amaranth was present at Yoakum in 2007, 2008, and 2009, Lamesa in 2007, and Halfway in 2008 and 2009. Smellmelon (*Cucumis melo* L. var. Dudaim Naud.) was present at Yoakum in 2007, 2008, and 2009 while horse purslane (*Trianthema portulacastrum* L.) was present at Yoakum only in 2009. When present, all field plots were naturally infested with dense populations of Texas millet

. Typically, treatments were applied when annual grasses were 10 to 26 cm tall, Palmer amaranth was 15 to 30 cm tall, horse purslane was 10 to 20 cm tall, and smellmelon was 15 to 30 cm in length. No attempt was made to harvest peanut in the efficacy studies due to the

Studies also were conducted under weed-free conditions at the Lamesa and Halfway in 2008 and 2009. Plots were maintained weed-free with ethalfluralin (Sonalan HFP®, Dow Agro‐ Sciences, 9330 Zionsville Road, Indianapolis, IN 46268) at 0.84 kg/ha applied preplant incor‐ porated. At Lamesa, Flavor Runner 458 was planted in 2008 while Tamrun OL02 was planted in 2009; at Halfway, the Spanish market type, OLin [70] was planted both years of the study. Seeding rate for the runner market cultivars (Flavor Runner 458, Tamrun OL02) was 90 kg/ha while OLin was planted at 100 kg/ha. Peanut phytotoxicity ratings were recorded throughout the growing season and peanut yield was obtained by digging each plot separately, air-drying in the field for 4 to 7 days, and harvesting pods from each plot with a combine. Weights were recorded after soil and trash were removed from plot samples were adjusted to 10% moisture.

, smellmelon at 6 to 8 plants/m2

, southern crabgrass at 6 to 8 plants/m2

, or Palmer amaranth at 4 to 6 plants/

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107

, horse

kg ai/ha.

was planted at the rate of 100 kg/ha.

purslane at 6 to 8 plants/m2

*3.1.2. Weed-free studies*

m2

and broadleaf signalgrass at 4 to 6 plants/m2

difficulty in digging weedy plots [10,13,17].

Additional research was conducted to define potential interactions of various postemergence herbicides and fungicides when used in combination on peanut for control of various broadleaf weeds and annual grasses. Therefore, the purpose of this research was to determine interac‐ tions of postemergence grass (clethodim and sethoxydim) and broadleaf herbicides (lactofen, imazethapyr, imazapic, aciflurofen, and 2,4-DB) with commonly used peanut fungicides (boscalid, fluazinam, pyraclostrobin, tebuconazole, or prothioconazole plus tebuconazole) for annual grass and broadleaf weed control in peanut as well as the response to foliar and soilborne disease development.

## **3. Research methods with tank-mix combinations for weed and disease control**

#### **3.1. Weed control with tank-mix combinations**

Field studies were conducted in two different peanut growing regions of Texas from 2007 through 2010 to determine weed efficacy and peanut response to applications of herbicides and fungicides applied alone and in combination. Field studies at south Texas were conducted at the Texas A&M AgriLife Research site near Yoakum and on the Texas Southern High Plains at Lamesa or Halfway. Soils at the Yoakum site were a Tremona loamy fine sand (thermic Aquic Arenic Paleustalfs) with less than 1% organic matter and pH 7.0 to 7.2. The location near Lamesa was at the Agricultural Complex for Research and Extension Center (AG-CARES) on a Amarillo fine sandy loam (fine-loamy, mixed, superactive, thermic Aridic Paleustalf) with 0.4% organic matter and pH 7.8. The Halfway location was located west of Plainview at the Texas A&M AgriLife Research and Extension Center on a Acuff clay loam (fine-loamy, mixed, thermic Aridic Paleustolls) with less than 1.0% organic matter and pH 7.9.

The experimental design was a randomized complete block with a factorial arrangement of two grass or five broadleaf herbicides by three fungicides with three replications. All studies included a non-treated control. Each plot consisted of two rows spaced 97 or 101 cm apart and 7.6 m long.

#### *3.1.1. Weed efficacy studies*

Weed efficacy studies were divided into two groups: 1) a grass herbicide study and 2) a broadleaf weed study. The grass herbicide study included clethodim at 0.14 kg ai/ha or sethoxydim at 0.21 kg ai/ha while the broadleaf weed study included aciflurofen at 0.42 kg ai/ ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides evaluated included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168 kg ai/ha.

Herbicides and fungicides were applied alone and in combination to determine efficacy against various weeds. A crop oil concentrate (Agri-Dex, a blend of 83% paraffin-based petroleum oil and 17% surfactant) at 2.3 L/ha was added to each treatment except in 2007 at Yoakum where a non-ionic surfactant (X-77, 90% nonionic surfactant) at 0.25% v/v was added. Herbicide and fungicides at Yoakum were applied with a CO2-pressurized backpack sprayer equipped with TeeJet 11002 DG flat fan spray tips (Spraying Systems Company, P.O. Box 7900, North Avenue, Wheaton, IL 60188) that delivered a spray volume of 190 L/ha at 180 kPa while on the Texas High Plains locations, fungicides and herbicides were applied with a CO2 pressurized backpack sprayer using TeeJet 110015 TT flat fan nozzles calibrated to deliver a spray volume of 94 L/ha at 207 kPa. At Yoakum, the peanut variety Tamrun OL02 [68] was planted in each year at a seeding rate of 112 kg/ha. At the Texas High Plains locations, Flavor Runner 458 [69] was planted at the rate of 100 kg/ha.

Texas millet and southern crabgrass were present at Yoakum in 2007 and 2009 while broadleaf signalgrass was present in 2008. Texas millet was present at Lamesa in 2007. Palmer amaranth was present at Yoakum in 2007, 2008, and 2009, Lamesa in 2007, and Halfway in 2008 and 2009. Smellmelon (*Cucumis melo* L. var. Dudaim Naud.) was present at Yoakum in 2007, 2008, and 2009 while horse purslane (*Trianthema portulacastrum* L.) was present at Yoakum only in 2009. When present, all field plots were naturally infested with dense populations of Texas millet and broadleaf signalgrass at 4 to 6 plants/m2 , southern crabgrass at 6 to 8 plants/m2 , horse purslane at 6 to 8 plants/m2 , smellmelon at 6 to 8 plants/m2 , or Palmer amaranth at 4 to 6 plants/ m2 . Typically, treatments were applied when annual grasses were 10 to 26 cm tall, Palmer amaranth was 15 to 30 cm tall, horse purslane was 10 to 20 cm tall, and smellmelon was 15 to 30 cm in length. No attempt was made to harvest peanut in the efficacy studies due to the difficulty in digging weedy plots [10,13,17].

#### *3.1.2. Weed-free studies*

fungicides [62-65]. Peanut fungicides are applied beginning approximately 30 to 60 days after planting and can be applied until a few weeks prior to digging. Efficacy of clethodim and sethoxydim can be reduced by co-application with copper-containing fungicides or azoxy‐ strobin, chlorothalonil, and pyraclostrobin [8,66,67]. Fluazinam and tebuconazole did not reduce grass control compared with graminicides applied alone [8,9,66]. Efficacy of herbicides that control dicotyledonous weeds and sedges are not generally affected by fungicides [66]. Weed species and size, and plant stress can affect the magnitude of interactions between

Additional research was conducted to define potential interactions of various postemergence herbicides and fungicides when used in combination on peanut for control of various broadleaf weeds and annual grasses. Therefore, the purpose of this research was to determine interac‐ tions of postemergence grass (clethodim and sethoxydim) and broadleaf herbicides (lactofen, imazethapyr, imazapic, aciflurofen, and 2,4-DB) with commonly used peanut fungicides (boscalid, fluazinam, pyraclostrobin, tebuconazole, or prothioconazole plus tebuconazole) for annual grass and broadleaf weed control in peanut as well as the response to foliar and

**3. Research methods with tank-mix combinations for weed and disease**

Field studies were conducted in two different peanut growing regions of Texas from 2007 through 2010 to determine weed efficacy and peanut response to applications of herbicides and fungicides applied alone and in combination. Field studies at south Texas were conducted at the Texas A&M AgriLife Research site near Yoakum and on the Texas Southern High Plains at Lamesa or Halfway. Soils at the Yoakum site were a Tremona loamy fine sand (thermic Aquic Arenic Paleustalfs) with less than 1% organic matter and pH 7.0 to 7.2. The location near Lamesa was at the Agricultural Complex for Research and Extension Center (AG-CARES) on a Amarillo fine sandy loam (fine-loamy, mixed, superactive, thermic Aridic Paleustalf) with 0.4% organic matter and pH 7.8. The Halfway location was located west of Plainview at the Texas A&M AgriLife Research and Extension Center on a Acuff clay loam (fine-loamy, mixed,

The experimental design was a randomized complete block with a factorial arrangement of two grass or five broadleaf herbicides by three fungicides with three replications. All studies included a non-treated control. Each plot consisted of two rows spaced 97 or 101 cm apart and

Weed efficacy studies were divided into two groups: 1) a grass herbicide study and 2) a broadleaf weed study. The grass herbicide study included clethodim at 0.14 kg ai/ha or

thermic Aridic Paleustolls) with less than 1.0% organic matter and pH 7.9.

herbicides and fungicides [66].

106 Herbicides - Current Research and Case Studies in Use

soilborne disease development.

**3.1. Weed control with tank-mix combinations**

**control**

7.6 m long.

*3.1.1. Weed efficacy studies*

Studies also were conducted under weed-free conditions at the Lamesa and Halfway in 2008 and 2009. Plots were maintained weed-free with ethalfluralin (Sonalan HFP®, Dow Agro‐ Sciences, 9330 Zionsville Road, Indianapolis, IN 46268) at 0.84 kg/ha applied preplant incor‐ porated. At Lamesa, Flavor Runner 458 was planted in 2008 while Tamrun OL02 was planted in 2009; at Halfway, the Spanish market type, OLin [70] was planted both years of the study. Seeding rate for the runner market cultivars (Flavor Runner 458, Tamrun OL02) was 90 kg/ha while OLin was planted at 100 kg/ha. Peanut phytotoxicity ratings were recorded throughout the growing season and peanut yield was obtained by digging each plot separately, air-drying in the field for 4 to 7 days, and harvesting pods from each plot with a combine. Weights were recorded after soil and trash were removed from plot samples were adjusted to 10% moisture. Weed control and peanut phytotoxicity, expressed as chlorosis and necrosis of leaf tissue, was visually estimated on a scale of 0 to 100 (0 indicating no weed kill or leaf chlorosis or necrosis and 100 indicating complete weed or peanut kill), relative to the non-treated control. Weed control was recorded approximately four weeks after POST herbicide applications while peanut phytotoxicity was recorded 5 to 14 days after herbicide application.

variety Tamrun OL02 [68] was planted in 2008 and 2009 while Florida 07 [71] was planted in 2010 at the rate of 112 kg/ha. Planting dates were June 16, 2008, July 1, 2009, and May 24, 2010.

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109

Studies conducted in central Texas focused on early leaf spot and Sclerotinia blight caused by *Sclerotina minor* Jagger. These studies included the herbicides clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha and the fungicides boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Agridex at 1.0% v/v was included in all treatments. Each plot consisted of two rows spaced 91 cm apart and 7.9 m long. Fungicides and herbicides were applied alone and in combination with a CO2-pressurized backpack sprayer equipped with two 8002VS flat fan spray nozzles per row in 140 L of water/ha at a pressure of 134 kPa. The runner-type variety

Typical peanut injury resulted in rapid damage to plant tissue after application and manifested as small necrotic lesions. The visible injury on leaflets with 2,4-DB was common and consisted of typical 2,4-DB damage which consisted of elongated leaflets with a slightly faded appear‐ ance [10]. This symptomology was not visible on new growth and remained visible on lower leaves throughout the growing season. Peanut phytotoxicity ratings were recorded 7 days after treatment at Yoakum. Peanut injury was estimated visually on a scale of 0 to 100 (0 indicating no leaf chlorosis or necrosis and 100 indicating complete peanut kill), relative to the non-treated control. Severity of leaf spot was rated in the center two rows using the Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot [49,59]. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots in lower versus upper canopy of the plots; whereas values 4 through 10 reflect increasing levels of defoliation [51]. The leaf spot rating was recorded immediately

Loci of southern stem rot or Sclerotinia blight (where applicable) were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii* or *S. minor* [72]. Plots were harvested in south Texas in 2008 and 2010, but not in 2009 due to extremely wet conditions which persisted during late October and November and prevented digging of individual plots (Table 1). Plots were harvested in

All test areas were maintained weed-free with a preemergence tank-mix application of pendimethalin at 1.06 kg ai/ha plus *S*-metolachlor at 1.42 kg ai/ha. Overhead sprinkler irrigation was applied on a 1- to 2-week schedule throughout the growing season as needed.

Peanut yields were obtained by digging each plot separately, air-drying in the field for 4 to 7 days, and harvesting pods from each plot with a combine. Weights were recorded after soil and trash were removed from plot samples were adjusted to 10% moisture. Leaf spot ratings and incidence of soilborne disease development were used for comparison of tank-mix combinations. Data were analyzed using PROC GLM with SAS (SAS Institute, Inc., Cary, NC) and a model statement appropriate for a factorial design. Treatments means were separated

by Fisher's protected least significant difference test at P≤0.05.

Flavor Runner 458 [69] was planted each year of the study at 95 kg/ha.

prior to peanut digging.

2008 and 2009 in central Texas.

*3.2.2. Data analysis*

#### *3.1.3. Data analysis*

Weed control and peanut injury data were transformed to the arcsine square root prior to analysis of variance, but are expressed in their original form for clarity because the transfor‐ mation did not alter interpretation. Visual estimates of weed control and peanut injury, and yield were subjected to analysis of variance to test effects of POST herbicide and fungicide. Means were compared with the appropriate Fisher's Protected LSD test at the 5% probability level. The non-treated was not included in weed efficacy or peanut injury analysis but was included in peanut yield analysis.

#### **3.2. Disease control with tank-mix combinations**

Studies were conducted in two different peanut growing regions of Texas to determine disease control and peanut response to applications of herbicides and fungicides applied alone and in combination. Field studies at south Texas were conducted at the Texas A&M AgriLife Research site near Yoakum while the central Texas studies were conducted at the Texas A&M AgriLife Research and Extension Center near Stephenville. Soils at the Yoakum site were described previously. This site has been in continuous peanut for over forty years so there was a high concentration of soil-borne and foliar disease inoculum. The soil at the Stephenville site was a Windthorst loamy sand (fine mixed thermic Udic Paleustalfs) with less than 1% organic matter and pH 7.6.

#### *3.2.1. Disease efficacy studies*

Studies in south Texas were conducted from 2008 to 2010 on early leaf spot and southern blight. These studies included the fungicides pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168 kg ai/ha and the herbicides aciflurofen at 0.42 kg ai/ha, clethodim at 0.14 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, sethoxydim at 0.21 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and herbicides were applied alone and in combination to determine efficacy against foliar and soilborne diseases. No adjuvant was included in these studies in 2008 or 2009; however, in 2010 a crop oil concentrate (Agri-Dex, a blend of 83% paraffin-based petroleum oil and 17% surfactant) at 2.3 L/ha was added to each treatment.

Fungicides and herbicides alone and in combination were applied with a CO2-pressurized backpack sprayer equipped with three D2-23 hollow-cone spray nozzles per row in 140 L of water/ha at a pressure of 504 kPa. The experimental design was a randomized complete block with a factorial arrangement of seven herbicides by three fungicides. All studies included a non-treated control. Each plot consisted of four rows spaced 97 cm apart and 6.3 m long. The variety Tamrun OL02 [68] was planted in 2008 and 2009 while Florida 07 [71] was planted in 2010 at the rate of 112 kg/ha. Planting dates were June 16, 2008, July 1, 2009, and May 24, 2010.

Studies conducted in central Texas focused on early leaf spot and Sclerotinia blight caused by *Sclerotina minor* Jagger. These studies included the herbicides clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha and the fungicides boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Agridex at 1.0% v/v was included in all treatments. Each plot consisted of two rows spaced 91 cm apart and 7.9 m long. Fungicides and herbicides were applied alone and in combination with a CO2-pressurized backpack sprayer equipped with two 8002VS flat fan spray nozzles per row in 140 L of water/ha at a pressure of 134 kPa. The runner-type variety Flavor Runner 458 [69] was planted each year of the study at 95 kg/ha.

Typical peanut injury resulted in rapid damage to plant tissue after application and manifested as small necrotic lesions. The visible injury on leaflets with 2,4-DB was common and consisted of typical 2,4-DB damage which consisted of elongated leaflets with a slightly faded appear‐ ance [10]. This symptomology was not visible on new growth and remained visible on lower leaves throughout the growing season. Peanut phytotoxicity ratings were recorded 7 days after treatment at Yoakum. Peanut injury was estimated visually on a scale of 0 to 100 (0 indicating no leaf chlorosis or necrosis and 100 indicating complete peanut kill), relative to the non-treated control. Severity of leaf spot was rated in the center two rows using the Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot [49,59]. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots in lower versus upper canopy of the plots; whereas values 4 through 10 reflect increasing levels of defoliation [51]. The leaf spot rating was recorded immediately prior to peanut digging.

Loci of southern stem rot or Sclerotinia blight (where applicable) were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii* or *S. minor* [72]. Plots were harvested in south Texas in 2008 and 2010, but not in 2009 due to extremely wet conditions which persisted during late October and November and prevented digging of individual plots (Table 1). Plots were harvested in 2008 and 2009 in central Texas.

All test areas were maintained weed-free with a preemergence tank-mix application of pendimethalin at 1.06 kg ai/ha plus *S*-metolachlor at 1.42 kg ai/ha. Overhead sprinkler irrigation was applied on a 1- to 2-week schedule throughout the growing season as needed.

#### *3.2.2. Data analysis*

Weed control and peanut phytotoxicity, expressed as chlorosis and necrosis of leaf tissue, was visually estimated on a scale of 0 to 100 (0 indicating no weed kill or leaf chlorosis or necrosis and 100 indicating complete weed or peanut kill), relative to the non-treated control. Weed control was recorded approximately four weeks after POST herbicide applications while

Weed control and peanut injury data were transformed to the arcsine square root prior to analysis of variance, but are expressed in their original form for clarity because the transfor‐ mation did not alter interpretation. Visual estimates of weed control and peanut injury, and yield were subjected to analysis of variance to test effects of POST herbicide and fungicide. Means were compared with the appropriate Fisher's Protected LSD test at the 5% probability level. The non-treated was not included in weed efficacy or peanut injury analysis but was

Studies were conducted in two different peanut growing regions of Texas to determine disease control and peanut response to applications of herbicides and fungicides applied alone and in combination. Field studies at south Texas were conducted at the Texas A&M AgriLife Research site near Yoakum while the central Texas studies were conducted at the Texas A&M AgriLife Research and Extension Center near Stephenville. Soils at the Yoakum site were described previously. This site has been in continuous peanut for over forty years so there was a high concentration of soil-borne and foliar disease inoculum. The soil at the Stephenville site was a Windthorst loamy sand (fine mixed thermic Udic Paleustalfs) with less than 1% organic matter

Studies in south Texas were conducted from 2008 to 2010 on early leaf spot and southern blight. These studies included the fungicides pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168 kg ai/ha and the herbicides aciflurofen at 0.42 kg ai/ha, clethodim at 0.14 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, sethoxydim at 0.21 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and herbicides were applied alone and in combination to determine efficacy against foliar and soilborne diseases. No adjuvant was included in these studies in 2008 or 2009; however, in 2010 a crop oil concentrate (Agri-Dex, a blend of 83% paraffin-based petroleum oil and 17% surfactant) at 2.3 L/ha was added to each treatment.

Fungicides and herbicides alone and in combination were applied with a CO2-pressurized backpack sprayer equipped with three D2-23 hollow-cone spray nozzles per row in 140 L of water/ha at a pressure of 504 kPa. The experimental design was a randomized complete block with a factorial arrangement of seven herbicides by three fungicides. All studies included a non-treated control. Each plot consisted of four rows spaced 97 cm apart and 6.3 m long. The

peanut phytotoxicity was recorded 5 to 14 days after herbicide application.

*3.1.3. Data analysis*

and pH 7.6.

*3.2.1. Disease efficacy studies*

included in peanut yield analysis.

108 Herbicides - Current Research and Case Studies in Use

**3.2. Disease control with tank-mix combinations**

Peanut yields were obtained by digging each plot separately, air-drying in the field for 4 to 7 days, and harvesting pods from each plot with a combine. Weights were recorded after soil and trash were removed from plot samples were adjusted to 10% moisture. Leaf spot ratings and incidence of soilborne disease development were used for comparison of tank-mix combinations. Data were analyzed using PROC GLM with SAS (SAS Institute, Inc., Cary, NC) and a model statement appropriate for a factorial design. Treatments means were separated by Fisher's protected least significant difference test at P≤0.05.

## **4. Effects of tank mix combinations on weed control, peanut phytotoxicity, and peanut yield**

that large crabgrass control was reduced when sethoxydim was applied with azoxystrobin or pyraclostrobin, but not fluazinam, propiconazole plus trifloxystrobin, or tebuconazole.

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111

At Yoakum in 2007 and 2008 and Halfway in 2008 there was an herbicide by fungicide interaction; therefore, those data are presented as a 2-way interaction of broadleaf herbicide by fungicide (Table 3). However, only herbicide effects were significant at Lamesa in 2007 and

In 2007 at Yoakum, lactofen, aciflurofen, and imazapic alone controlled Palmer amaranth at least 91% while 2,4-DB and imazethapyr alone provided 83% and 68% control, respectively (Table 3). Lactofen plus tebuconazole and aciflurofen plus either the premix of prothioconazole plus tebuconazole or tebuconazole reduced Palmer amaranth control over each respective herbicide applied alone. In 2008 at Yoakum, all herbicides alone controlled Palmer amaranth at least 92%. Reduced control from each respective herbicide alone was noted with acifluorfen plus either pyraclostrobin or tebuconazole and imazethapyr or 2,4-DB plus pyraclostrobin. At the Halfway location, lactofen and aciflurofen alone provided poor control (≤ 25%) of Palmer amaranth while imazethapyr, imazapic, and 2,4-DB controlled Palmer amaranth at least 77% (Table 3). Only the combination of 2,4-DB plus the premix of prothioconazole plus tebucona‐

At Lamesa, all herbicides controlled Palmer amaranth less than 60% while at Yoakum there was no difference in Palmer amaranth control following all herbicide treatments (Table 4). At the Halfway location, lactofen, imazapic, and imazethapyr controlled Palmer amaranth at least

Grichar [74] reported that imazapic at 0.04 to 0.07 kg/ha controlled Palmer amaranth at least 95% when applied to weeds that were less than 15 cm tall while imazethapyr provided at least 90% control in 2 of 3 years. In other research, Jordan et al. [66] reported that smooth pigweed

There was an herbicide by fungicide interaction for horse purslane in 2009. Lactofen and 2,4- DB alone and in combination with fungicides provided almost complete control of horse purslane (Table 3). Aciflurofen alone controlled 97% horse purslane while antagonism was noted with acifluorfen plus the premix of prothioconazole plus tebuconazole combinations. All imazethapyr plus fungicide combinations reduced horse purslane control compared to imazethapyr alone. Imazapic alone or in combination failed to control horse purslane.

Horse purslane can be a stronger competitor with peanut early in the growing season than common purslane due to a more upright growth than that of common purslane [75]. Grichar [75] reported that aciflurofen and lactofen alone or combinations of these herbicides with 2,4- DB controlled horse purslane at least 70% when evaluated 21 days after treatment (DAT), but no greater than 75% control was observed when rated up to 115 DAT. In later work, Grichar

98% while 2,4-DB and aciflurofen controlled this weed 75% and 54%, respectively.

(*A. hybridus* L.) control by imazethapyr was reduced by tank mixing with fungicides.

*4.1.2. Palmer amaranth control*

*4.1.3. Horse purslane control*

Halfway and Yoakum in 2009 (Table 4).

zole reduced control when compared to 2,4-DB alone.

#### **4.1. Weed efficacy with combinations of herbicides plus fungicides.**

There was no herbicide by fungicide by year interaction for Texas millet, southern crabgrass, or broadleaf signalgrass control; therefore, that data are combined over clethodim and sethoxydim herbicides.

#### *4.1.1. Annual grass control*

No differences in broadleaf signalgrass, Texas millet, or southern crabgrass control were noted between clethodim or sethoxydim when applied alone or in combination with any of the fungicides (Table 2). Grichar [73] reported that clethodim and sethoxydim controlled 3 to 10 cm tall Texas millet and southern crabgrass at least 85%. Clethodim applied to 15 to 25 cm tall Texas millet or southern crabgrass provided no better than 89% Texas millet control while southern crabgrass control varied from 51 to 95% [73]. Sethoxydim applied to the same height Texas millet or southern crabgrass controlled Texas millet 79 to 87% and southern crabgrass control was no better than 76% [73].


a Herbicides and rates included clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha. Data were combined over fungicides due to a lack of interaction.

b Texas millet present in south Texas in 2007 and 2009 and at Lamesa in 2007. Southern crabgrass present in south Texas in 2007 and 2009. Broadleaf signalgrass present in south Texas in 2008.

c Texas millet, *Urochloa texana* (Buckley) R. D. Webster; Southern crabgrass, *Digitaria ciliaris* (Retz.) Koeler; broadleaf signalgrass, *Brachiaria platyphylla* (Griseb.) Nash.

d NS, not significant at the 5% level of probability.

**Table 2.** Annual grass control with clethodim and sethoxydim.a,b

Lancaster et al. [8,9] reported large crabgrass control was reduced with clethodim when applied with pyraclostrobin, chlorothalonil, and azoxystrobin; however, fluazinam, propico‐ nazole plus trifloxystrobin, and tebuconazole did not reduce large crabgrass control by clethodim. Similarly, Jordan et al. [66] reported that azoxystrobin and chlorothalonil, but not tebuconazole, reduced annual grass control by clethodim. Also Lancaster et al. [8,9] reported that large crabgrass control was reduced when sethoxydim was applied with azoxystrobin or pyraclostrobin, but not fluazinam, propiconazole plus trifloxystrobin, or tebuconazole.

### *4.1.2. Palmer amaranth control*

**4. Effects of tank mix combinations on weed control, peanut phytotoxicity,**

There was no herbicide by fungicide by year interaction for Texas millet, southern crabgrass, or broadleaf signalgrass control; therefore, that data are combined over clethodim and

No differences in broadleaf signalgrass, Texas millet, or southern crabgrass control were noted between clethodim or sethoxydim when applied alone or in combination with any of the fungicides (Table 2). Grichar [73] reported that clethodim and sethoxydim controlled 3 to 10 cm tall Texas millet and southern crabgrass at least 85%. Clethodim applied to 15 to 25 cm tall Texas millet or southern crabgrass provided no better than 89% Texas millet control while southern crabgrass control varied from 51 to 95% [73]. Sethoxydim applied to the same height Texas millet or southern crabgrass controlled Texas millet 79 to 87% and southern crabgrass

> **Texasc millet**

**kg ai/ha %** Clethodim 0.14 96 96 98 Sethoxydim 0.21 95 96 98 LSD (0.05) NSd NS NS

 Herbicides and rates included clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha +

b Texas millet present in south Texas in 2007 and 2009 and at Lamesa in 2007. Southern crabgrass present in south

Texas millet, *Urochloa texana* (Buckley) R. D. Webster; Southern crabgrass, *Digitaria ciliaris* (Retz.) Koeler; broadleaf

Lancaster et al. [8,9] reported large crabgrass control was reduced with clethodim when applied with pyraclostrobin, chlorothalonil, and azoxystrobin; however, fluazinam, propico‐ nazole plus trifloxystrobin, and tebuconazole did not reduce large crabgrass control by clethodim. Similarly, Jordan et al. [66] reported that azoxystrobin and chlorothalonil, but not tebuconazole, reduced annual grass control by clethodim. Also Lancaster et al. [8,9] reported

tebuconazole at 0.168 kg ai/ha. Data were combined over fungicides due to a lack of interaction.

Texas in 2007 and 2009. Broadleaf signalgrass present in south Texas in 2008.

**Table 2.** Annual grass control with clethodim and sethoxydim.a,b

**Southern crabgrass**

**Broadleaf signalgrass**

**4.1. Weed efficacy with combinations of herbicides plus fungicides.**

**and peanut yield**

110 Herbicides - Current Research and Case Studies in Use

sethoxydim herbicides.

*4.1.1. Annual grass control*

control was no better than 76% [73].

**Herbicide Rate**

signalgrass, *Brachiaria platyphylla* (Griseb.) Nash. d NS, not significant at the 5% level of probability.

a

c

At Yoakum in 2007 and 2008 and Halfway in 2008 there was an herbicide by fungicide interaction; therefore, those data are presented as a 2-way interaction of broadleaf herbicide by fungicide (Table 3). However, only herbicide effects were significant at Lamesa in 2007 and Halfway and Yoakum in 2009 (Table 4).

In 2007 at Yoakum, lactofen, aciflurofen, and imazapic alone controlled Palmer amaranth at least 91% while 2,4-DB and imazethapyr alone provided 83% and 68% control, respectively (Table 3). Lactofen plus tebuconazole and aciflurofen plus either the premix of prothioconazole plus tebuconazole or tebuconazole reduced Palmer amaranth control over each respective herbicide applied alone. In 2008 at Yoakum, all herbicides alone controlled Palmer amaranth at least 92%. Reduced control from each respective herbicide alone was noted with acifluorfen plus either pyraclostrobin or tebuconazole and imazethapyr or 2,4-DB plus pyraclostrobin. At the Halfway location, lactofen and aciflurofen alone provided poor control (≤ 25%) of Palmer amaranth while imazethapyr, imazapic, and 2,4-DB controlled Palmer amaranth at least 77% (Table 3). Only the combination of 2,4-DB plus the premix of prothioconazole plus tebucona‐ zole reduced control when compared to 2,4-DB alone.

At Lamesa, all herbicides controlled Palmer amaranth less than 60% while at Yoakum there was no difference in Palmer amaranth control following all herbicide treatments (Table 4). At the Halfway location, lactofen, imazapic, and imazethapyr controlled Palmer amaranth at least 98% while 2,4-DB and aciflurofen controlled this weed 75% and 54%, respectively.

Grichar [74] reported that imazapic at 0.04 to 0.07 kg/ha controlled Palmer amaranth at least 95% when applied to weeds that were less than 15 cm tall while imazethapyr provided at least 90% control in 2 of 3 years. In other research, Jordan et al. [66] reported that smooth pigweed (*A. hybridus* L.) control by imazethapyr was reduced by tank mixing with fungicides.

#### *4.1.3. Horse purslane control*

There was an herbicide by fungicide interaction for horse purslane in 2009. Lactofen and 2,4- DB alone and in combination with fungicides provided almost complete control of horse purslane (Table 3). Aciflurofen alone controlled 97% horse purslane while antagonism was noted with acifluorfen plus the premix of prothioconazole plus tebuconazole combinations. All imazethapyr plus fungicide combinations reduced horse purslane control compared to imazethapyr alone. Imazapic alone or in combination failed to control horse purslane.

Horse purslane can be a stronger competitor with peanut early in the growing season than common purslane due to a more upright growth than that of common purslane [75]. Grichar [75] reported that aciflurofen and lactofen alone or combinations of these herbicides with 2,4- DB controlled horse purslane at least 70% when evaluated 21 days after treatment (DAT), but no greater than 75% control was observed when rated up to 115 DAT. In later work, Grichar


*4.1.4. Smellmelon control*

interaction.

c

germination of seed and smellmelon growth [76].

*4.1.5. Peanut phytotoxicity with tank mix combinations*

0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha.

d NS, not significant at the 5% level of probability.

**Table 4.** Weed control with various postemergence herbicides.a,b

b Palmer amaranth present at Lamesa in 2007, Yoakum in 2009, and Halfway in 2009.

Palmer amaranth, *Amaranthus palmeri* S. Wats.; smellmelon, *Cucumis melo* L. var. Dudaim Naud.

*4.1.5.1. Clethodim/sethoxydim plus fungicide combinations*

Only herbicides were significant for smellmelon control (Table 4). No difference in smellmelon control was noted with any herbicides in 2007, while in 2008 imazethapyr produced the worst control. In 2009, lactofen controlled less smellmelon than imazapic. Grichar [76] reported that imazapic provided the most consistent control of smellmelon while acifluorfen, imazethapyr, imazapic, and lactofen controlled at least 80% smellmelon in some years but in other years control was less than 70%. Imazapic at 0.04 to 0.07 kg/ha controlled smellmelon greater than 90% in corn (*Zea mays* L.) regardless whether applied PRE, early POST, or late POST [77]. Grichar [78] reported that imazapic provided consistent control (> 85%) of citronmelon (*Citrullus lanatus* var. *citroides*) in peanut. Typically, season-long smellmelon control with 2,4- DB is poor. This can be attributed to lack of any residual activity of 2,4-DB and continued

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No peanut phytotoxicity was noted with any graminicide by fungicide combinations at Yoakum or Halfway (data not shown); however, at Lamesa there was a treatment by year

Lactofen 49 94 98 93 99 89 Aciflurofen 38 90 54 88 99 96 Imazethapyr 28 88 99 88 91 95 Imazapic 25 90 98 99 98 98 2,4-DB 59 96 75 93 99 96 LSD (0.05) 6 NSd 12 NS 4 9

<sup>a</sup> Data are pooled over herbicides due to a lack of interaction. Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at

In 2007 (with Texas millet pressure) and in 2009 (weed-free), peanut phytotoxicity (up to 12%) was evident with clethodim and sethoxydim combinations with either pyraclostrobin, tebuconazole, and the premix of prothioconazole + tebuconazole up to two weeks after

**Palmer amaranthc Smellmelon Lamesa Yoakum Halfway 2007 2008 2009 %**

a Agri-Dex at 2.3 L/ha was added to each treatment except in 2007 at Yoakum where X-77 at 0.25% v/v was added. b Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha. c Palmer amaranth, *Amaranthus palmeri* S. Wats.; horse purslane, *Trianthema portulacastrum* L.

d Present only in 2009.

**Table 3.** Palmer amaranth and horse purslane control with herbicide-fungicide combinations.a,b

[76] reported that in one year, lactofen applied to horse purslane less than 15 cm tall controlled this weed 93% while in another year, lactofen applied to horse purslane less than 15 cm tall or 20 to 30 cm tall provided at least 93% control while acifluorfen applied to horse purslane less than 15 cm tall controlled this weed 77%.

#### *4.1.4. Smellmelon control*

Only herbicides were significant for smellmelon control (Table 4). No difference in smellmelon control was noted with any herbicides in 2007, while in 2008 imazethapyr produced the worst control. In 2009, lactofen controlled less smellmelon than imazapic. Grichar [76] reported that imazapic provided the most consistent control of smellmelon while acifluorfen, imazethapyr, imazapic, and lactofen controlled at least 80% smellmelon in some years but in other years control was less than 70%. Imazapic at 0.04 to 0.07 kg/ha controlled smellmelon greater than 90% in corn (*Zea mays* L.) regardless whether applied PRE, early POST, or late POST [77]. Grichar [78] reported that imazapic provided consistent control (> 85%) of citronmelon (*Citrullus lanatus* var. *citroides*) in peanut. Typically, season-long smellmelon control with 2,4- DB is poor. This can be attributed to lack of any residual activity of 2,4-DB and continued germination of seed and smellmelon growth [76].

#### *4.1.5. Peanut phytotoxicity with tank mix combinations*

#### *4.1.5.1. Clethodim/sethoxydim plus fungicide combinations*

No peanut phytotoxicity was noted with any graminicide by fungicide combinations at Yoakum or Halfway (data not shown); however, at Lamesa there was a treatment by year interaction.


<sup>a</sup> Data are pooled over herbicides due to a lack of interaction. Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha.

b Palmer amaranth present at Lamesa in 2007, Yoakum in 2009, and Halfway in 2009.

c Palmer amaranth, *Amaranthus palmeri* S. Wats.; smellmelon, *Cucumis melo* L. var. Dudaim Naud.

d NS, not significant at the 5% level of probability.

[76] reported that in one year, lactofen applied to horse purslane less than 15 cm tall controlled

**Palmer amaranth**<sup>c</sup>

**Yoakum Yoakum Halfway Yoakum**

78 93 17 100

57 97 30 58

82 98 82 25

86 99 94 7

98 100 28 100

**2007 2008**

Herbicide Fungicide %

Prothioconazole + tebuconazole

112 Herbicides - Current Research and Case Studies in Use

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

Lactofen

Acifluorfen

Imazethapyr

Imazapic

2,4-DB

a

c

d Present only in 2009.

Lactofen - 93 92 22 100 Lactofen Pyraclostrobin 100 100 18 100

Lactofen Tebuconazole 70 93 17 99 Acifluorfen - 91 97 25 97 Acifluorfen Pyraclostrobin 73 80 18 80

Acifluorfen Tebuconazole 60 85 18 99 Imazethapyr - 68 100 77 80 Imazethapyr Pyraclostrobin 88 86 75 0 Prothioconazole 98

Imazethapyr Tebuconazole 87 93 80 10 Imazapic - 94 97 96 13 Imazapic Pyraclostrobin 94 99 94 0

Imazapic Tebuconazole 97 93 94 0 2,4-DB - 83 96 88 100 2,4-DB Pyraclostrobin 67 87 87 100

2,4-DB Tebuconazole 100 97 83 99 LSD (0.05) 20 9 18 32

Palmer amaranth, *Amaranthus palmeri* S. Wats.; horse purslane, *Trianthema portulacastrum* L.

**Table 3.** Palmer amaranth and horse purslane control with herbicide-fungicide combinations.a,b

 Agri-Dex at 2.3 L/ha was added to each treatment except in 2007 at Yoakum where X-77 at 0.25% v/v was added. b Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha.

**Horse purslaned**

this weed 93% while in another year, lactofen applied to horse purslane less than 15 cm tall or

20 to 30 cm tall provided at least 93% control while acifluorfen applied to horse purslane less

than 15 cm tall controlled this weed 77%.

**Table 4.** Weed control with various postemergence herbicides.a,b

In 2007 (with Texas millet pressure) and in 2009 (weed-free), peanut phytotoxicity (up to 12%) was evident with clethodim and sethoxydim combinations with either pyraclostrobin, tebuconazole, and the premix of prothioconazole + tebuconazole up to two weeks after application (Table 5). In 2007, clethodim, sethoxydim, or tebuconazole alone or clethodim or sethoxydim in combination with tebuconazole caused no phytotoxicity. All other combina‐ tions resulted in at least 3% phytotoxicity. Either graminicide in combination with prothioco‐ nazole plus tebuconazole or prothioconazole plus tebuconazole alone caused the greatest phytotoxicity. In 2009, similar results were noted; however, pyraclostrobin alone or in combination with either graminicide caused the greatest injury (Table 5). Subsequent new growth did not exhibit adverse effects of any tank-mix combination and was 2% or less, four weeks after application (data not shown).

In 2008 at Yoakum, lactofen alone and in combination with prothioconazole plus tebucona‐ zole or tebuconazole alone caused at least 10% peanut phytotoxicity while aciflurofen alone or in combination with any of the fungicides caused 4 to 7% phytotoxicity. At Lamesa, combinations with aciflurofen, lactofen, and 2,4-DB caused the greatest injury (Table 6). Imazethapyr or imazapic alone or in combination with pyraclostrobin resulted in no injury. Imazethapyr plus tebuconazole caused no injury while imazapic plus tebuconazole result‐

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In 2009 at Lamesa, imazapic, imazethapyr, and 2,4-DB alone resulted in no injury; however, imazapic plus either pyraclostrobin or prothioconazole plus tebuconazole, imazapic plus pyraclostrobin, and 2,4-DB plus any fungicide resulted in 5 to 15% phytotoxicity (Table 6). Slight peanut phytotoxicity was also noted with the fungicides pyraclostrobin and tebucona‐ zole. At Halfway, peanut injury with aciflurofen or lactofen was greater than at Lamesa with the exception of lactofen plus pyraclostrobin which caused 9 to 10% injury at both locations

Under weed-free conditions, when using either the grass or broadleaf herbicides with fungicides, no negative response with respect to peanut yield was noted when compared with the non-treated control for either runner or Spanish market types (data not shown). Most studies conducted on herbicide-fungicide interactions on peanut have focused on either weed efficacy or disease control and few have reported on effect on peanut yield. No studies could be found that reported any peanut yield reductions with clethodim or sethoxydim under weedfree conditions. Although lactofen at 0.22 kg/ha caused peanut leaf bronzing and spotting [74], lactofen produced a similar yield when compared to the untreated, weed-free control [79]. Richburg et al. [80] reported no yield differences with runner, Spanish, or Virginia peanut cultivars with imazethapyr at 0.07 kg/ha in Georgia or Texas. No reduction in peanut grade or yield following imazapic treatments have been observed in several studies [76,81,82]. Grichar et al. [83] reported that single and multiple applications of 2,4-DB at 0.45 kg/ha did

> **Phytotoxictyc 2008 2009 Yoakum Lamesa Lamesa Halfway**

12 10 8 22

ed in 10% injury.

(Table 6).

not affect runner-type yield.

Lactofen

*4.1.6. Peanut yield as influenced by tank mix combinations*

**Herbicide Fungicide %**

Prothioconazole + tebuconazole



a Herbicides and rates included clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha.

**Table 5.** Peanut phytotoxicity with graminicide plus fungicide combinations at Lamesa in 2007 and 2009.a

#### *4.1.5.2. Aciflurofen, imazapic, imazethapyr, lactofen, or 2,4-DB plus fungicide combinations*

Phytotoxicity observations were not recorded in the weed efficacy studies with the exception of Yoakum in 2008; however, phytotoxicity ratings were recorded in the weed-free studies conducted at Lemasa in 2008 and 2009 and Halfway in 2009. In these studies, there was a significant herbicide by fungicide interaction; therefore, data are presented separately by location. Phytotoxicity varied across locations and treatments but in most instances was greater with the use of aciflurofen or lactofen.

In 2008 at Yoakum, lactofen alone and in combination with prothioconazole plus tebucona‐ zole or tebuconazole alone caused at least 10% peanut phytotoxicity while aciflurofen alone or in combination with any of the fungicides caused 4 to 7% phytotoxicity. At Lamesa, combinations with aciflurofen, lactofen, and 2,4-DB caused the greatest injury (Table 6). Imazethapyr or imazapic alone or in combination with pyraclostrobin resulted in no injury. Imazethapyr plus tebuconazole caused no injury while imazapic plus tebuconazole result‐ ed in 10% injury.

In 2009 at Lamesa, imazapic, imazethapyr, and 2,4-DB alone resulted in no injury; however, imazapic plus either pyraclostrobin or prothioconazole plus tebuconazole, imazapic plus pyraclostrobin, and 2,4-DB plus any fungicide resulted in 5 to 15% phytotoxicity (Table 6). Slight peanut phytotoxicity was also noted with the fungicides pyraclostrobin and tebucona‐ zole. At Halfway, peanut injury with aciflurofen or lactofen was greater than at Lamesa with the exception of lactofen plus pyraclostrobin which caused 9 to 10% injury at both locations (Table 6).

#### *4.1.6. Peanut yield as influenced by tank mix combinations*

application (Table 5). In 2007, clethodim, sethoxydim, or tebuconazole alone or clethodim or sethoxydim in combination with tebuconazole caused no phytotoxicity. All other combina‐ tions resulted in at least 3% phytotoxicity. Either graminicide in combination with prothioco‐ nazole plus tebuconazole or prothioconazole plus tebuconazole alone caused the greatest phytotoxicity. In 2009, similar results were noted; however, pyraclostrobin alone or in combination with either graminicide caused the greatest injury (Table 5). Subsequent new growth did not exhibit adverse effects of any tank-mix combination and was 2% or less, four

**Herbicide Fungicide 2007 2009**

Clethodim - 2 0 Clethodim Pyraclostrobin 3 13 Clethodim Tebuconazole 0 0

Sethoxydim - 2 0 Sethoxydim Pyraclostrobin 5 12 Sethoxydim Tebuconazole 0 0


Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

+ tebuconazole

**Table 5.** Peanut phytotoxicity with graminicide plus fungicide combinations at Lamesa in 2007 and 2009.a

*4.1.5.2. Aciflurofen, imazapic, imazethapyr, lactofen, or 2,4-DB plus fungicide combinations*

LSD (0.05) 3 3

 Herbicides and rates included clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha +

Phytotoxicity observations were not recorded in the weed efficacy studies with the exception of Yoakum in 2008; however, phytotoxicity ratings were recorded in the weed-free studies conducted at Lemasa in 2008 and 2009 and Halfway in 2009. In these studies, there was a significant herbicide by fungicide interaction; therefore, data are presented separately by location. Phytotoxicity varied across locations and treatments but in most instances was greater


**%**

10 8

12 3

8 0

weeks after application (data not shown).

114 Herbicides - Current Research and Case Studies in Use

Clethodim

Sethoxydim

tebuconazole at 0.168 kg ai/ha.

with the use of aciflurofen or lactofen.

a

Under weed-free conditions, when using either the grass or broadleaf herbicides with fungicides, no negative response with respect to peanut yield was noted when compared with the non-treated control for either runner or Spanish market types (data not shown). Most studies conducted on herbicide-fungicide interactions on peanut have focused on either weed efficacy or disease control and few have reported on effect on peanut yield. No studies could be found that reported any peanut yield reductions with clethodim or sethoxydim under weedfree conditions. Although lactofen at 0.22 kg/ha caused peanut leaf bronzing and spotting [74], lactofen produced a similar yield when compared to the untreated, weed-free control [79]. Richburg et al. [80] reported no yield differences with runner, Spanish, or Virginia peanut cultivars with imazethapyr at 0.07 kg/ha in Georgia or Texas. No reduction in peanut grade or yield following imazapic treatments have been observed in several studies [76,81,82]. Grichar et al. [83] reported that single and multiple applications of 2,4-DB at 0.45 kg/ha did not affect runner-type yield.



**5. Effects of tank mix combinations on foliar and soilborne disease control,**

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Rainfall in south Texas was below average in 2008 and the early to mid-part of the 2009 peanut growing season (May through August); however, rainfall amounts were above average for the latter portion of the 2009 season (September through November). Rainfall amounts for 2010 were above average for May, July, August, and September (Table 7). In central Texas, rainfall amounts in 2008 were below average for all months (May through November) with the exception of July which was slightly above average while in 2009 rainfall was below average

**Month 2008 2009 2010 60-yr avg 2008 2009 30-yr avg**

May 1.3 16.3 118.4 112.2 76.5 65.5 117.6 June 65.3 3.8 95.0 109.2 30.5 8.6 100.0 July 54.9 5.3 200.7 65.8 47.2 79.0 34.7 August 57.9 42.7 89.4 78.7 50.3 2.0 58.3 September 2.5 114.0 223.3 102.6 55.9 10.6 70.5 October 14.2 352.6 0 94.5 32.5 127.3 72.3 November 25.9 111.3 71.1 75.4 40.4 25.9 54.5 Total 222.0 646.0 797.9 638.4 333.3 318.9 507.9

There was an herbicide by fungicide interaction for early leaf spot control in 2008 and 2009. In 2010, the main plots of herbicide and fungicide were significant for early leaf spot control; therefore, that data were averaged over herbicides and fungicides only. Foliar disease development was moderate in 2008 due to extreme drought and hot conditions that persisted throughout the 2008 and the early portion of the 2009 growing seasons. Typically, early leaf spot epidemics are favored by temperatures of approximately 16 to 250 C and long periods of high relative humidity are required for infections to occur [84]. All herbicides alone, with the exception of sethoxydim and lactofen, were not different from the non-treated control with respect to early leaf spot development in 2008 (Table 8). All fungicides alone or in combination with any of the herbicides produced leaf spot levels that were less than the non-treated control. When individual fungicides were compared with the respective fungicide plus herbicide

**South Texas Central Texas**

**mm**

**peanut phytotoxicity, and peanut yield**

**5.1. Disease control with tank mix combinations**

for all months with the exception of July and October (Table 7).

**Table 7.** Rainfall amounts in south Texas and central Texas from 2008 through 2010

*5.1.1. Early leaf spot control in South Texas*

a Agri-Dex at 2.3 L/ha was added to each treatment.

b Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha. c Rating index: 0=no leaf chlorosis or necrosis, 100=plants completely dead.

**Table 6.** Peanut phytotoxicity with herbicide-fungicide combinations when rated 12 to 15 days after treatment.a,b

## **5. Effects of tank mix combinations on foliar and soilborne disease control, peanut phytotoxicity, and peanut yield**

#### **5.1. Disease control with tank mix combinations**

**Phytotoxictyc 2008 2009 Yoakum Lamesa Lamesa Halfway**

7 9 5 23

0 4 5 12

0 10 0 3

1 12 10 20

0 1 0 13

Lactofen Tebuconazole 12 12 7 25 Acifluorfen - 5 5 5 20 Acifluorfen Pyraclostrobin 4 5 5 17

Acifluorfen Tebuconazole 6 9 5 22 Imazethapyr - 0 0 0 0 Imazethapyr Pyraclostrobin 2 0 7 0

Imazethapyr Tebuconazole 0 0 0 0 Imazapic - 0 0 0 0 Imazapic Pyraclostrobin 0 0 8 0

Imazapic Tebuconazole 0 10 0 3 2,4-DB - 0 5 0 10 2,4-DB Pyraclostrobin 3 12 15 18

2,4-DB Tebuconazole 0 6 5 5 - Pyraclostrobin 0 0 10 3


LSD (0.05) 3 1 2 5

b Herbicides and rates included aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Fungicides and rates included pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha.

**Table 6.** Peanut phytotoxicity with herbicide-fungicide combinations when rated 12 to 15 days after treatment.a,b

Prothioconazole + tebuconazole

116 Herbicides - Current Research and Case Studies in Use

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

+ tebuconazole

Rating index: 0=no leaf chlorosis or necrosis, 100=plants completely dead.


Agri-Dex at 2.3 L/ha was added to each treatment.

Acifluorfen

Imazethapyr

Imazapic

2,4-DB

a

c

Rainfall in south Texas was below average in 2008 and the early to mid-part of the 2009 peanut growing season (May through August); however, rainfall amounts were above average for the latter portion of the 2009 season (September through November). Rainfall amounts for 2010 were above average for May, July, August, and September (Table 7). In central Texas, rainfall amounts in 2008 were below average for all months (May through November) with the exception of July which was slightly above average while in 2009 rainfall was below average for all months with the exception of July and October (Table 7).


**Table 7.** Rainfall amounts in south Texas and central Texas from 2008 through 2010

#### *5.1.1. Early leaf spot control in South Texas*

There was an herbicide by fungicide interaction for early leaf spot control in 2008 and 2009. In 2010, the main plots of herbicide and fungicide were significant for early leaf spot control; therefore, that data were averaged over herbicides and fungicides only. Foliar disease development was moderate in 2008 due to extreme drought and hot conditions that persisted throughout the 2008 and the early portion of the 2009 growing seasons. Typically, early leaf spot epidemics are favored by temperatures of approximately 16 to 250 C and long periods of high relative humidity are required for infections to occur [84]. All herbicides alone, with the exception of sethoxydim and lactofen, were not different from the non-treated control with respect to early leaf spot development in 2008 (Table 8). All fungicides alone or in combination with any of the herbicides produced leaf spot levels that were less than the non-treated control. When individual fungicides were compared with the respective fungicide plus herbicide treatments some differences were noted. Pyraclostrobin alone resulted in less early leaf spot than pyraclostrobin plus either imazapic, lactofen, or sethoxydim. No differences were noted between tebuconazole alone or in combination with any herbicide. Prothioconazole plus tebuconazole alone resulted in less early leaf spot than prothioconazole plus tebuconazole in combination with acifluorfen (Table 8).

**Leaf spot b Southern**

Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations

**Fungicide Herbicide 2008 2009 2010 2009 2010 2008**

+ tebuconazole Sethoxydim 3.8 6.7 <sup>21</sup> <sup>0</sup> <sup>6</sup> <sup>2380</sup>

+ tebuconazole Imazethapyr 3.0 6.8 <sup>39</sup> <sup>0</sup> <sup>0</sup> <sup>1500</sup>

+ tebuconazole Imazapic 3.5 6.9 <sup>17</sup> <sup>0</sup> <sup>1</sup> <sup>2080</sup>

+ tebuconazole 2,4-DB 3.5 7.0 <sup>16</sup> <sup>1</sup> <sup>9</sup> <sup>1510</sup> LSD (0.05) 1.0 0.6 31 1 2 780

 Fungicides and rates: pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioco‐ nazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha,

<sup>b</sup> Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots in lower versus upper canopy of the plots. Values 4 through 10 reflect increasing levels of defoliation. <sup>c</sup> Loci of southern stem rot were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii*. Percent incidence based on number of loci/12.7

d Peanut phytotoxicity ratings (leaf chlorosis and necrosis) ratings were taken 7 days after treatment. Peanut injury was visually estimated on a scale of 0 to 100 (0 indicating no leaf chlorosis or necrosis and 100 indicating complete

Although early-season rainfall was below normal in 2009, September rainfall was above normal leading to conditions for late-season development of high levels of foliar diseases. No differences were noted between the non-treated control and any herbicide with respect to early leaf spot control (Table 8). All fungicides alone or in combination with herbicides resulted in less early leaf spot than the non-treated control. When fungicides were compared alone or in combination, pyraclostrobin alone resulted in less early leaf spot than the combination of

**Table 8.** Disease control and peanut response to fungicide-herbicide combinations in south Texas.a

+ tebuconazole Prothioconazole + tebuconazole

Prothioconazole

Prothioconazole + tebuconazole

Prothioconazole + tebuconazole

Prothioconazole

Prothioconazole

Prothioconazole

lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha.

peanut kill), relative to the non-treated control.

a

m rows.

**blight c Phytotoxicity d Yield**

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119

**Florida scale % Incidence % kg/ha**

Clethodim 3.7 6.7 55 0 1 1890

Lactofen 3.3 6.5 31 10 7 2470

Acifluorfen 4.2 7.3 21 5 8 2020


treatments some differences were noted. Pyraclostrobin alone resulted in less early leaf spot than pyraclostrobin plus either imazapic, lactofen, or sethoxydim. No differences were noted between tebuconazole alone or in combination with any herbicide. Prothioconazole plus tebuconazole alone resulted in less early leaf spot than prothioconazole plus tebuconazole in

**Leaf spot b Southern**

**Fungicide Herbicide 2008 2009 2010 2009 2010 2008**


**blight c Phytotoxicity d Yield**

**Florida scale % Incidence % kg/ha**

combination with acifluorfen (Table 8).

118 Herbicides - Current Research and Case Studies in Use


a Fungicides and rates: pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioco‐ nazole at 0.084 kg ai/ha + tebuconazole at 0.168 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha.

<sup>b</sup> Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots in lower versus upper canopy of the plots. Values 4 through 10 reflect increasing levels of defoliation.

<sup>c</sup> Loci of southern stem rot were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii*. Percent incidence based on number of loci/12.7 m rows.

d Peanut phytotoxicity ratings (leaf chlorosis and necrosis) ratings were taken 7 days after treatment. Peanut injury was visually estimated on a scale of 0 to 100 (0 indicating no leaf chlorosis or necrosis and 100 indicating complete peanut kill), relative to the non-treated control.

**Table 8.** Disease control and peanut response to fungicide-herbicide combinations in south Texas.a

Although early-season rainfall was below normal in 2009, September rainfall was above normal leading to conditions for late-season development of high levels of foliar diseases. No differences were noted between the non-treated control and any herbicide with respect to early leaf spot control (Table 8). All fungicides alone or in combination with herbicides resulted in less early leaf spot than the non-treated control. When fungicides were compared alone or in combination, pyraclostrobin alone resulted in less early leaf spot than the combination of


Weather conditions in 2010 were conducive for development of early leaf spot (Table 7). When herbicides were compared, aciflurofen, imazethapyr, and lactofen resulted in greater early leaf spot than where no herbicide was used (Table 9). All fungicides resulted in less early leaf spot

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Early leaf spot data was collected only in 2008 and neither fungicide nor herbicide effects were significant. Due to dry conditions, early leaf spot pressure was moderate and there were no differences with any factors (Table 10). Management of early and late leaf spot of peanut is essential for peanut production in most areas of the world [59]. In the southeastern United States, control of these diseases is heavily reliant upon multiple fungicide applications [59,84] while far fewer applications are necessary in the southwestern United States [53,56,85].

Control of southern blight was not significant for any factor in 2008; however, in 2010 there was a fungicide by herbicide interaction. Since peanut were not dug in 2009, no southern blight ratings were taken. In 2008, no differences were noted with respect to development of southern blight (Table 8). In 2010, under low to moderate pressure, sethoxydim alone produced the highest levels of southern blight with over 85% disease incidence (Table 9). No differences were noted between fungicides alone or the combinations of a fungicide with a herbicide.

Sclerotinia blight control was significant for both fungicides in both years; whereas herbicides did not impact disease control. Sclerotinia blight pressure was moderate to heavy in each year (Table 10). In 2008, fluazinam provided the best control of Sclerotinia blight compared with the non-treated control while both boscalid and fluazinam reduced Sclerotinia blight com‐ pared to the non-treated control in 2009. Fluazinam has provided good to excellent disease control depending on the rate applied [86-88]. Smith et al. [89] reported in field studies that the application of boscalid or fluazinam that preceded the largest incremental increase in disease incidence provided the best control of disease or increased yield. They advised that disease advisories or intensive scouting should be used to determine when epidemics initiate

In south Texas, peanut phytotoxicity ratings were recorded in 2009 and 2010 and an herbicide by fungicide interaction was observed in each year. In 2009, lactofen alone or in combination with any fungicide resulted in the greatest amount of foliar chlorosis or necrosis (Table 8). The addition of a fungicide to lactofen reduced phytotoxicity 10 to 64% compared with lactofen alone. Lactofen is classified as a diphenyl ether (cell membrane disruptor), which interferes with protoporphyrinogen IX oxidase and causes accumulation of protoporphyrin IX [90]. Protoporphyrinogen IX is a potent photosensitizer that generates high levels of singlet oxygen

than where no fungicide was used.

*5.1.3. Southern blight control*

*5.1.4.Sclerotinia blight control*

so that a fungicide can be applied prior to infection.

**5.2. Peanut phytotoxicity with tank mix combinations**

*5.1.2. Early leaf spot control in central Texas*

a Fungicides and rates: pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha.

<sup>b</sup> Loci of southern stem rot were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii*. Percent incidence based on number of loci/12.7 m rows.

c Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots in lower versus upper canopy of the plots. Values 4 through 10 reflect increasing levels of defoliation.

d Abbreviation: NS, not significant at the 5% level of significance.

**Table 9.** Disease control and peanut response to herbicides and fungicides in south Texas.a

pyraclostrobin plus imazapic while tebuconazole alone resulted in less leaf spot than tebuco‐ nazole plus either imazapic or aciflurofen. No differences were noted between prothioconazole plus tebuconazole alone or in combination with any herbicides.

Weather conditions in 2010 were conducive for development of early leaf spot (Table 7). When herbicides were compared, aciflurofen, imazethapyr, and lactofen resulted in greater early leaf spot than where no herbicide was used (Table 9). All fungicides resulted in less early leaf spot than where no fungicide was used.

#### *5.1.2. Early leaf spot control in central Texas*

Early leaf spot data was collected only in 2008 and neither fungicide nor herbicide effects were significant. Due to dry conditions, early leaf spot pressure was moderate and there were no differences with any factors (Table 10). Management of early and late leaf spot of peanut is essential for peanut production in most areas of the world [59]. In the southeastern United States, control of these diseases is heavily reliant upon multiple fungicide applications [59,84] while far fewer applications are necessary in the southwestern United States [53,56,85].

### *5.1.3. Southern blight control*

Control of southern blight was not significant for any factor in 2008; however, in 2010 there was a fungicide by herbicide interaction. Since peanut were not dug in 2009, no southern blight ratings were taken. In 2008, no differences were noted with respect to development of southern blight (Table 8). In 2010, under low to moderate pressure, sethoxydim alone produced the highest levels of southern blight with over 85% disease incidence (Table 9). No differences were noted between fungicides alone or the combinations of a fungicide with a herbicide.

#### *5.1.4.Sclerotinia blight control*

pyraclostrobin plus imazapic while tebuconazole alone resulted in less leaf spot than tebuco‐ nazole plus either imazapic or aciflurofen. No differences were noted between prothioconazole

**Southern**

**% Incidence**

Herbicide

120 Herbicides - Current Research and Case Studies in Use

Fungicide

Prothioconazole + tebuconazole

at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha.

a

rows. c

**Herbicide 2008 2010 2010**

No herbicide 24 6.5 3635 Aciflurofen 13 7.6 3047 Clethodim 16 6.6 3302 Imazapic 13 6.8 3581 Imazethapyr 15 7.4 3387 Lactofen 17 7.3 3048 Sethoxydim 15 6.8 3240 2,4-DB 28 6.8 3461 LSD (0.05) NS c 0.5 NS

No fungicide 22 8.8 2834 Pyraclostrobin 16 6.1 3490 Tebuconazole 17 6.5 3401

LSD (0.05) NSd 0.5 419

in lower versus upper canopy of the plots. Values 4 through 10 reflect increasing levels of defoliation.

**Table 9.** Disease control and peanut response to herbicides and fungicides in south Texas.a

 Fungicides and rates: pyraclostrobin at 0.27 kg ai/ha, tebuconazole at 0.23 kg ai/ha, and the premix of prothioconazole at 0.084 kg ai/ha plus tebuconazole at 0.168 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen

<sup>b</sup> Loci of southern stem rot were counted immediately after peanut plants were inverted. A locus represented 31 cm or less of linear row with one or more plants infected with *S. rolfsii*. Percent incidence based on number of loci/12.7 m

 Florida leaf spot scoring system where 1 = no leaf spot, and 10 = plants completely defoliated and dead because of leaf spot. Values of 1 through 4 on the scale reflect increasing incidence of leaflets with spots, and occurrence of spots

**blight b Leaf spot c Yield**

**Florida**

16 6.5 3627

**scale Kg/ha**

plus tebuconazole alone or in combination with any herbicides.

d Abbreviation: NS, not significant at the 5% level of significance.

Sclerotinia blight control was significant for both fungicides in both years; whereas herbicides did not impact disease control. Sclerotinia blight pressure was moderate to heavy in each year (Table 10). In 2008, fluazinam provided the best control of Sclerotinia blight compared with the non-treated control while both boscalid and fluazinam reduced Sclerotinia blight com‐ pared to the non-treated control in 2009. Fluazinam has provided good to excellent disease control depending on the rate applied [86-88]. Smith et al. [89] reported in field studies that the application of boscalid or fluazinam that preceded the largest incremental increase in disease incidence provided the best control of disease or increased yield. They advised that disease advisories or intensive scouting should be used to determine when epidemics initiate so that a fungicide can be applied prior to infection.

#### **5.2. Peanut phytotoxicity with tank mix combinations**

In south Texas, peanut phytotoxicity ratings were recorded in 2009 and 2010 and an herbicide by fungicide interaction was observed in each year. In 2009, lactofen alone or in combination with any fungicide resulted in the greatest amount of foliar chlorosis or necrosis (Table 8). The addition of a fungicide to lactofen reduced phytotoxicity 10 to 64% compared with lactofen alone. Lactofen is classified as a diphenyl ether (cell membrane disruptor), which interferes with protoporphyrinogen IX oxidase and causes accumulation of protoporphyrin IX [90]. Protoporphyrinogen IX is a potent photosensitizer that generates high levels of singlet oxygen


a Fungicides and rates: boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Data combined over fungicides due to a lack of interaction. b Leaf spot assessed using the Florida 1-10 scale where 1=no disease and 10=completely dead. Leaf spot present only in 2008.

c Loci of Sclerotinia blight were counted just prior to peanut plants being inverted. A locus represents 31 cm or less of linear row with one or more plants exhibiting disease symptoms or signs of *S. minor*.

d NS, not significant at the 5% level of probability.

**Table 10.** Foliar disease and Sclerotinia blight control with fungicides in central Texas.a

in the presence of molecular oxygen and light, leading to light-induced oxidative breakdown of cell constituents [90]. Aciflurofen, also a diphenyl ether herbicide, caused injury similar to lactofen; however, this injury was not as great as that observed with lactofen (Table 8). Peanut and soybean (*Glycine max* L.) tolerance to aciflurofen and lactofen is based on metabolism, which often results in some leaf bronzing and spotting of leaves and plant growth can be temporarily reduced [79,91].

the hot, dry conditions during the growing season and relatively low disease pressure. In 2010,

Fungicides and rates: boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Herbicides and rates included clethodim


Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations


Clethodim - 2713 2099 Clethodim Fluazinam 3408 3337 Clethodim Boscalid 2973 3060 Sethoxydim - 2930 2351 Sethoxydim Fluazinam 2778 2930 Sethoxydim Boscalid 2865 4240

LSD (0.05) NS b 855

**Table 11.** Peanut yield as influenced by fungicide and herbicide alone and in combinations in central Texas.a

**Yield 2008 2009 Kg/ha**

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123

In central Texas, there was no difference with any factor in 2008; however, a significant fungicide by herbicide interaction was observed in 2009. In 2008, there were no differences with any factor for yield while in 2009 there was a fungicide by herbicide interaction; however, yields were extremely variable (Table 11). Damicone and Jackson [92] reported that yield reductions of over 50% can occur following severe outbreaks of Sclerotinia blight. All boscalid or fluazinam treatments improved peanut yield over the non-treated control . Boscalid alone or in combination with sethoxydim produced greater yield than fluazinam alone or fluazinam in combination with sethoxydim. This agrees with the results of Smith et al. [89] who reported that in both field and greenhouse studies, boscalid performed marginally better than fluazi‐

**6. Conclusions of using tank mix combinations on weed efficacy and**

Adding fungicides to either clethodim or sethoxydim did not have an effect on annual grass efficacy. No phytotoxicity was noted on peanut and yield was not affected with any gramini‐

all fungicides improved peanut yield over the non-treated control (Table 9).

**Herbicide Fungicide**

at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha.

b NS, Not significant at the 5% level.

nam.

a

**peanut response**

In 2010, aciflurofen and lactofen exhibited similar phytotoxicity symptoms as exhibited in 2009; however, more phytotoxicity overall was noted with other fungicide-herbicide combinations than was seen in 2009. This increase in phytotoxicity was probably due to the addition of Agridex to all treatments in 2010, which was not added in 2008 or 2009. Phytotoxicity was noted with pyraclostrobin, which is never seen (authors personal observations). Pyraclostrobin and prothioconazole plus tebuconazole combinations with herbicides were more phytotoxic than tebuconazole combinations with herbicides. With tebuconazole, other than aciflurofen or lactofen, only the combination of tebuconazole plus 2,4-DB resulted in observed phytotoxcity. However, with pyraclostrobin or prothioconazole plus tebuconazole, phytotoxicity resulted from combinations with either clethodim, sethoxydim, imazethapyr, or imazapic in addition to aciflurofen or lactofen (Table 8).

#### **5.3. Peanut yield with tank mix combinations**

In south Texas, there was a fungicide by herbicide interaction for peanut yield in 2008; therefore, data are presented as an interaction while in 2010 only fungicide treatment was significant. In 2008, no treatments affected peanut yield when compared with the non-treated control (Table 8). Only pyraclostrobin alone or tebuconazole plus 2,4-DB resulted in an increase in yield over the non-treated control. The lack of response to fungicides is probably related to Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations http://dx.doi.org/10.5772/55949 123


a Fungicides and rates: boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha and sethoxydim at 0.21 kg ai/ha.

b NS, Not significant at the 5% level.

in the presence of molecular oxygen and light, leading to light-induced oxidative breakdown of cell constituents [90]. Aciflurofen, also a diphenyl ether herbicide, caused injury similar to lactofen; however, this injury was not as great as that observed with lactofen (Table 8). Peanut and soybean (*Glycine max* L.) tolerance to aciflurofen and lactofen is based on metabolism, which often results in some leaf bronzing and spotting of leaves and plant growth can be

 Fungicides and rates: boscalid at 0.49 kg ai/ha and fluazinam at 0.88 kg ai/ha. Herbicides and rates included clethodim at 0.14 kg ai/ha, sethoxydim at 0.21 kg ai/ha, aciflurofen at 0.42 kg ai/ha, imazapic at 0.07 kg ai/ha, imazethapyr at 0.07 kg ai/ha, lactofen at 0.22 kg ai/ha, or 2,4-DB at 0.42 kg ai/ha. Data combined over fungicides due to a lack of interaction. b Leaf spot assessed using the Florida 1-10 scale where 1=no disease and 10=completely dead. Leaf spot present only

Loci of Sclerotinia blight were counted just prior to peanut plants being inverted. A locus represents 31 cm or less of

None 5.4 31.9 39.3 Boscalid 5.1 24.3 18.5 Fluazinam 5.6 16.6 12.7 LSD (0.05) NSd 13.9 8.0

**Florida scale %**

**Sclerotinia blight c 2008 2009**

In 2010, aciflurofen and lactofen exhibited similar phytotoxicity symptoms as exhibited in 2009; however, more phytotoxicity overall was noted with other fungicide-herbicide combinations than was seen in 2009. This increase in phytotoxicity was probably due to the addition of Agridex to all treatments in 2010, which was not added in 2008 or 2009. Phytotoxicity was noted with pyraclostrobin, which is never seen (authors personal observations). Pyraclostrobin and prothioconazole plus tebuconazole combinations with herbicides were more phytotoxic than tebuconazole combinations with herbicides. With tebuconazole, other than aciflurofen or lactofen, only the combination of tebuconazole plus 2,4-DB resulted in observed phytotoxcity. However, with pyraclostrobin or prothioconazole plus tebuconazole, phytotoxicity resulted from combinations with either clethodim, sethoxydim, imazethapyr, or imazapic in addition

In south Texas, there was a fungicide by herbicide interaction for peanut yield in 2008; therefore, data are presented as an interaction while in 2010 only fungicide treatment was significant. In 2008, no treatments affected peanut yield when compared with the non-treated control (Table 8). Only pyraclostrobin alone or tebuconazole plus 2,4-DB resulted in an increase in yield over the non-treated control. The lack of response to fungicides is probably related to

temporarily reduced [79,91].

d NS, not significant at the 5% level of probability.

122 Herbicides - Current Research and Case Studies in Use

a

in 2008. c

to aciflurofen or lactofen (Table 8).

**5.3. Peanut yield with tank mix combinations**

**Fungicide Leaf spot b**

linear row with one or more plants exhibiting disease symptoms or signs of *S. minor*.

**Table 10.** Foliar disease and Sclerotinia blight control with fungicides in central Texas.a

**Table 11.** Peanut yield as influenced by fungicide and herbicide alone and in combinations in central Texas.a

the hot, dry conditions during the growing season and relatively low disease pressure. In 2010, all fungicides improved peanut yield over the non-treated control (Table 9).

In central Texas, there was no difference with any factor in 2008; however, a significant fungicide by herbicide interaction was observed in 2009. In 2008, there were no differences with any factor for yield while in 2009 there was a fungicide by herbicide interaction; however, yields were extremely variable (Table 11). Damicone and Jackson [92] reported that yield reductions of over 50% can occur following severe outbreaks of Sclerotinia blight. All boscalid or fluazinam treatments improved peanut yield over the non-treated control . Boscalid alone or in combination with sethoxydim produced greater yield than fluazinam alone or fluazinam in combination with sethoxydim. This agrees with the results of Smith et al. [89] who reported that in both field and greenhouse studies, boscalid performed marginally better than fluazi‐ nam.

### **6. Conclusions of using tank mix combinations on weed efficacy and peanut response**

Adding fungicides to either clethodim or sethoxydim did not have an effect on annual grass efficacy. No phytotoxicity was noted on peanut and yield was not affected with any gramini‐ cide -fungicide combinations. Lancaster et al. [8] reported that pyraclostrobin and tebucona‐ zole did not reduce the amount of 14C-labled clethodim or sethoxydim absorbed in large crabgrass. Although tebuconazole did not reduce efficacy of either graminicide in the field, pyraclostrobin reduced efficacy of clethodim and sethoxydim in some instances. They concluded that reduced absorption was not the mechanism for reduced large crabgrass control but may be the result of a biological response or a chemical interaction. Pyraclostrobin is a strobilurin fungicide which inhibits fungal respiration and acts systemically within the plant [93]. Therefore, the formulated product is not likely to remain on leaf surfaces and interfere with herbicide absorption [8,9]. With Palmer amaranth, antagonism was noted 33% of the time with aciflurofen plus either pyraclostrobin or tebuconazole and 2,4-DB plus pyraclostrobin. Horse purslane also exhibited reduced control with herbicide-fungicides while smellmelon showed no effects of these combinations. Peanut leaf phytotoxicity was most evident with combinations that included aciflurofen or lactofen but this is to be expected since these two herbicides can cause bronzing and leaf spotting when applied alone.

**Acknowledgements**

technical assistance.

**Author details**

**References**

W. James Grichar1\*, Peter A. Dotray2

any. 2007;94(12) 1963–1971.

1973. p17-45.

p135-173.

p299-326.

\*Address all correspondence to: w-grichar@tamu.edu

1 Texas A&M AgriLife Research, Corpus Christi, TX, USA

2 Texas Tech University, Texas A&M AgriLife Research, Lubbock,TX, USA

3 Texas Tech University, Texas A&M AgriLife Extension Service, Lubbock, TX, USA

The National Peanut Board through the Texas Peanut Producers Board provided funds for this research. Kevin Brewer, Dwayne Drozd, Lyndell Gilbert, Bill Klesel, and Ira Yates provided

Weed and Disease Control and Peanut Response Following Postemergence Herbicide and Fungicide Combinations

http://dx.doi.org/10.5772/55949

125

and Jason E. Woodward3

[1] Anonymous. World Geography of the Peanut, University of Georgia. 2004-01-02. http://www.lanra.uga.edu/peanut/knowledgebase/ (accessed 22 Aug 2012.

[2] Seijo G, Lavia GI, Fernandez A, Krapovickas A, Ducasse DA, Bertioli DJ, Moscone EA. Genomic relationships between the cultivated peanut (Arachis hypogaea, Legu‐ minosae) and its close relatives revealed by double GISH. American Journal of Bot‐

[3] Hammons R. Early History and Origin of the Peanut. In: Tripp L. (ed.) Peanuts-Cul‐ ture and Uses. Roanoke: American Peanut Research and Education Association;

[4] Gregory WC, Gregory MP, Krapovickas A, Smith BW, Yarbrough JA. Structures and Genetic Resources of Peanuts. In: Tripp L. (ed.) Peanuts-Culture and Uses. Roanoke:

[5] Hammons R. Genetics of Arachis hypogaea. In: Tripp L. (ed.) Peanuts-Culture and Uses. Roanoke: American Peanut Research and Education Association; 1973.

[6] Sturke DG, Buchanan GA. Cultural Practices. In: Tripp L. (ed.) Peanuts-Culture and Uses. Roanoke: American Peanut Research and Education Association; 1973.

American Peanut Research and Education Association; 1973. p47-134.

### **7. Conclusion of tank mix combinations on disease control and peanut response**

Control of early leaf spot was reduced with pyraclostrobin plus imazapic combinations compared with pyraclostrobin alone in two of three years while pyraclostrobin plus either sethoxydim or lactofen, tebuconazole plus either clethodim or aciflurofen or the premix of prothioconazole plus tebuconazole in combination with aciflurofen reduced leaf spot control over the respective fungicide in one of three years. Fungicide-herbicide combinations did not affect southern blight or Sclerotinia blight disease development over the respective fungicide alone. Peanut phytotoxicity was greatest with aciflurofen or lactofen combinations. Under early leaf spot and southern blight or Sclerotinia blight disease pressure, no negative response was noted for peanut yield with any fungicide-herbicide combinations over the respective fungicide alone.

Many variables can affect interactions of herbicides with fungicides. Adjuvant selection, herbicide and fungicide rate, commercial formulation, active ingredient, spray volume, water quality, and environmental conditions can affect interactions [61]. Applying a higher rate of the herbicide that may be adversely affected can compensate for interactions [94-96]. Applying ammonium sulfate with bentazon reduced the negative effect of adding bentazon to clethodim or sethoxydim [97,98,99]. Differential response to clethodim has been noted when applied with different formulations of chlorothalonil [66]. Applying graminicides in higher spray volumes can hasten the negative influence of herbicides and fungicides on weed control by graminicides [66,100,101]. Environmental conditions that affect plant response to herbicides or fungicides can influence the magnitude of interactions. Negative effects of interactions associated with the efficacy of systemic herbicides, especially graminicides, are increased when grasses are stressed and the physiological processes that reduce absorption and translocation occur [63,102-105].

### **Acknowledgements**

cide -fungicide combinations. Lancaster et al. [8] reported that pyraclostrobin and tebucona‐ zole did not reduce the amount of 14C-labled clethodim or sethoxydim absorbed in large crabgrass. Although tebuconazole did not reduce efficacy of either graminicide in the field, pyraclostrobin reduced efficacy of clethodim and sethoxydim in some instances. They concluded that reduced absorption was not the mechanism for reduced large crabgrass control but may be the result of a biological response or a chemical interaction. Pyraclostrobin is a strobilurin fungicide which inhibits fungal respiration and acts systemically within the plant [93]. Therefore, the formulated product is not likely to remain on leaf surfaces and interfere with herbicide absorption [8,9]. With Palmer amaranth, antagonism was noted 33% of the time with aciflurofen plus either pyraclostrobin or tebuconazole and 2,4-DB plus pyraclostrobin. Horse purslane also exhibited reduced control with herbicide-fungicides while smellmelon showed no effects of these combinations. Peanut leaf phytotoxicity was most evident with combinations that included aciflurofen or lactofen but this is to be expected since these two

**7. Conclusion of tank mix combinations on disease control and peanut**

Control of early leaf spot was reduced with pyraclostrobin plus imazapic combinations compared with pyraclostrobin alone in two of three years while pyraclostrobin plus either sethoxydim or lactofen, tebuconazole plus either clethodim or aciflurofen or the premix of prothioconazole plus tebuconazole in combination with aciflurofen reduced leaf spot control over the respective fungicide in one of three years. Fungicide-herbicide combinations did not affect southern blight or Sclerotinia blight disease development over the respective fungicide alone. Peanut phytotoxicity was greatest with aciflurofen or lactofen combinations. Under early leaf spot and southern blight or Sclerotinia blight disease pressure, no negative response was noted for peanut yield with any fungicide-herbicide combinations over the respective

Many variables can affect interactions of herbicides with fungicides. Adjuvant selection, herbicide and fungicide rate, commercial formulation, active ingredient, spray volume, water quality, and environmental conditions can affect interactions [61]. Applying a higher rate of the herbicide that may be adversely affected can compensate for interactions [94-96]. Applying ammonium sulfate with bentazon reduced the negative effect of adding bentazon to clethodim or sethoxydim [97,98,99]. Differential response to clethodim has been noted when applied with different formulations of chlorothalonil [66]. Applying graminicides in higher spray volumes can hasten the negative influence of herbicides and fungicides on weed control by graminicides [66,100,101]. Environmental conditions that affect plant response to herbicides or fungicides can influence the magnitude of interactions. Negative effects of interactions associated with the efficacy of systemic herbicides, especially graminicides, are increased when grasses are stressed and the physiological processes that reduce absorption and translocation occur

herbicides can cause bronzing and leaf spotting when applied alone.

**response**

124 Herbicides - Current Research and Case Studies in Use

fungicide alone.

[63,102-105].

The National Peanut Board through the Texas Peanut Producers Board provided funds for this research. Kevin Brewer, Dwayne Drozd, Lyndell Gilbert, Bill Klesel, and Ira Yates provided technical assistance.

### **Author details**

W. James Grichar1\*, Peter A. Dotray2 and Jason E. Woodward3

\*Address all correspondence to: w-grichar@tamu.edu

1 Texas A&M AgriLife Research, Corpus Christi, TX, USA

2 Texas Tech University, Texas A&M AgriLife Research, Lubbock,TX, USA

3 Texas Tech University, Texas A&M AgriLife Extension Service, Lubbock, TX, USA

### **References**


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**Chapter 6**

**Weed Management in Cereals in**

Additional information is available at the end of the chapter

**1.1. The weed problem on cereal arable fields**

and ability to occupy sites disturbed by humans.

José Luis Tenorio-Pasamón

http://dx.doi.org/10.5772/55970

**1. Introduction**

production.

**Semi-Arid Environments: A Review**

Inés Santín-Montanyá, Encarnación Zambrana-Quesada and

With growing concern about the environment, and the increased public interest in environ‐ mental conservation, traditional agriculture has led to profound changes in in recent years. Cereals are the most important crop in dry-land areas of southern Europe. In Spain, nearly 5.5 million ha of winter cereals are sown each year [1]. Research in agriculture has undergone a paradigm shift, favoring systems aimed at improving the performance of cropping systems without deleterious effects to the environment. To achieve this, weed managers continually develop comprehensive programs for crop protection, in which an essential component is the use of crops more competitive with weeds [2], in order to maintain the stability of agricultural

The selection of a crop is not an easy task and it involves the consideration of numerous environmental and socioeconomic factors. Additionally, in any cropping system, we always can observe the presence of weeds that invade, persist and survive. They are unwanted and we refer to them as plants "out of place". There are numerous definitions of a weed: a plant that is out of place and not intentionally sown; a plant that grows where it is not wanted or welcomed; a plant whose virtues have not yet been discovered; a plant that is competitive, persistent, pernicious, and interferes negatively with human activity. Weeds possess one or more of the following characteristics that allow them to survive and increase in nature: abundant seed production; rapid population establishment; seed dormancy; long-term survival of buried seed; adaptation for spread; presence of vegetative reproductive structures

> © 2013 Santín-Montanyá et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Chapter 6**

## **Weed Management in Cereals in Semi-Arid Environments: A Review**

Inés Santín-Montanyá, Encarnación Zambrana-Quesada and José Luis Tenorio-Pasamón

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55970

### **1. Introduction**

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37-45.

#### **1.1. The weed problem on cereal arable fields**

With growing concern about the environment, and the increased public interest in environ‐ mental conservation, traditional agriculture has led to profound changes in in recent years. Cereals are the most important crop in dry-land areas of southern Europe. In Spain, nearly 5.5 million ha of winter cereals are sown each year [1]. Research in agriculture has undergone a paradigm shift, favoring systems aimed at improving the performance of cropping systems without deleterious effects to the environment. To achieve this, weed managers continually develop comprehensive programs for crop protection, in which an essential component is the use of crops more competitive with weeds [2], in order to maintain the stability of agricultural production.

The selection of a crop is not an easy task and it involves the consideration of numerous environmental and socioeconomic factors. Additionally, in any cropping system, we always can observe the presence of weeds that invade, persist and survive. They are unwanted and we refer to them as plants "out of place". There are numerous definitions of a weed: a plant that is out of place and not intentionally sown; a plant that grows where it is not wanted or welcomed; a plant whose virtues have not yet been discovered; a plant that is competitive, persistent, pernicious, and interferes negatively with human activity. Weeds possess one or more of the following characteristics that allow them to survive and increase in nature: abundant seed production; rapid population establishment; seed dormancy; long-term survival of buried seed; adaptation for spread; presence of vegetative reproductive structures and ability to occupy sites disturbed by humans.

© 2013 Santín-Montanyá et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Therefore, to control effectively we should ask: *why do weeds emerge;* and *what factors limit their development?*. There is abundant evidence that the presence of weeds reduce crop yields; weeds compete for environmental resources, especially water, light and soil nutrients, resulting in decreased crop yield or reducing the crops quality by contaminating the commodity, inter‐ fering with harvest, serving as hosts for crop diseases or providing shelter for insects to overwinter, limiting the choice of crop rotation sequences and cultural practices. The most important parameters that characterize the infestation of weeds in a crop and that determine the competitive relationships between them are the density and time of weed competition. Their competitive ability is associated with the establishment of a dense infestation, and is caused by the different habits of growth of weeds and crops. Weeds have developed a number of features that allow them to survive and even dominate in adverse environmental conditions. Also, to learn more about competition exerted by weeds is necessary to know their life cycle, and we can observe three major life cycle groups in cereal arable fields:

be used most effectively. Understanding some of these factors enables you to use herbicides

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**1.** When any plant is established and persists in a given area, it is likely to have established a presence of seeds, tubers, rhizomes or other means propagative in the place; that environmental conditions are favorable for reproductive success; and competes successfully with established plant populations. Furthermore, morphologi‐ cal and physiological differences between plants being constantly selected will likely be the most suited to climate, soil and agricultural management, for their establish‐ ment and persistence and will likely dominate [6]. Yenish [7] pointed out that it is not economical nor practical to try to eradicate the most problematic species already established, when the presence of them is high in the soil seed bank; in most cases, they can be kept under control with the application of herbicides. In a period of about five years we may reduce the seed bank to less than 5%, but we should also consid‐ er that in a single year without control, their seed production may be sufficient to

**2.** The weed composition in different communities is not always the same, and it changes over time; this has been called succession. According to this theory, when the habitat remains relatively constant, we do not record considerable changes in the community. When the conditions are modified, the species adapted to the "original conditions" are replaced by those that the new environment is more conducive for their development. At the same time, the presence of new species modifies the new environmental conditions and favors the establishment of other species [8]. In agricultural land the succession process is different than in natural areas since agricultural practices constantly disrupt natural succession process, and the dynamic successional cycle begins. With the suspen‐ sion of agricultural operations, successional processes in vegetative populations are

**3.** The practices used by the farmers to produce their crops each year favor the development of certain species of weeds so that populations that occur in different plots reflect

**4.** The competitive damage to the crop depends on the species, the density of each range, the proximity in which it is growing when they emerge to the crop plant and the duration of the competition. There are many species that do become problematic during a crop cycle in a particular field, depending on crop. However, it has been found that the early

**5.** Herbicides are available in the market, which when selected appropriately for each particular problem, can efficiently control weeds. To succeed, it is not enough to acquire and apply herbicides recommended for cultivation, it is necessary to take into account the

agricultural management provided to crops that year and previous years.

stages of crop development are more sensitive to competition by weeds.

factors that affect the efficiency of action of these herbicides, such as:

to their maximum advantage. Urzúa [5] recorded the following precepts:

exceed 50% of the original population [7].

restored [9].

#### **Annuals**

*Summer annuals* germinate in the spring, mature, produce seed, and die in one growing season.

*Winter annuals* germinate in late summer or fall, mature, produce seed, and then die the following spring or summer.

#### **Biennials**

Weeds grow from seed anytime during the growing season. They normally produce a rosette of leaves close to the soil surface the first year, then flower, mature, and die during the second year. A true biennial never produces flowers or seeds the first year. There are relatively few biennial weeds.

#### **Perennials**

*Simple perennials* form a deep taproot and spread primarily by seed dispersal.

*Creeping perennials* may be either herbaceous or woody and can spread by both vegetative structures as well as by seed.

When we study the competition process between species, we must consider what resources are limiting in the environment, which will account for more competition. Since weeds are so prevalent in many areas of the landscape, management techniques are necessary to maintain order. Weed management is most successful when it involves an integrated approach using a variety of methods. The common methods used to manage weeds include prevention and cultural, mechanical, biological, and chemical means.

Herbicides remain the predominant weed management tool with the greatest influence on weed selection over the last 60 years [3]. Reliance on chemicals for weed control has increased significantly in the last decades [4]. However, herbicide use also carries risks that include environmental, ecological, and human health effects. It is important to understand both the benefits and disadvantages associated with chemical weed control before selecting the appropriate control. Many factors determine when, where, and how a particular herbicide can be used most effectively. Understanding some of these factors enables you to use herbicides to their maximum advantage. Urzúa [5] recorded the following precepts:

Therefore, to control effectively we should ask: *why do weeds emerge;* and *what factors limit their development?*. There is abundant evidence that the presence of weeds reduce crop yields; weeds compete for environmental resources, especially water, light and soil nutrients, resulting in decreased crop yield or reducing the crops quality by contaminating the commodity, inter‐ fering with harvest, serving as hosts for crop diseases or providing shelter for insects to overwinter, limiting the choice of crop rotation sequences and cultural practices. The most important parameters that characterize the infestation of weeds in a crop and that determine the competitive relationships between them are the density and time of weed competition. Their competitive ability is associated with the establishment of a dense infestation, and is caused by the different habits of growth of weeds and crops. Weeds have developed a number of features that allow them to survive and even dominate in adverse environmental conditions. Also, to learn more about competition exerted by weeds is necessary to know their life cycle,

*Summer annuals* germinate in the spring, mature, produce seed, and die in one growing season.

*Winter annuals* germinate in late summer or fall, mature, produce seed, and then die the

Weeds grow from seed anytime during the growing season. They normally produce a rosette of leaves close to the soil surface the first year, then flower, mature, and die during the second year. A true biennial never produces flowers or seeds the first year. There are relatively few

*Creeping perennials* may be either herbaceous or woody and can spread by both vegetative

When we study the competition process between species, we must consider what resources are limiting in the environment, which will account for more competition. Since weeds are so prevalent in many areas of the landscape, management techniques are necessary to maintain order. Weed management is most successful when it involves an integrated approach using a variety of methods. The common methods used to manage weeds include prevention and

Herbicides remain the predominant weed management tool with the greatest influence on weed selection over the last 60 years [3]. Reliance on chemicals for weed control has increased significantly in the last decades [4]. However, herbicide use also carries risks that include environmental, ecological, and human health effects. It is important to understand both the benefits and disadvantages associated with chemical weed control before selecting the appropriate control. Many factors determine when, where, and how a particular herbicide can

*Simple perennials* form a deep taproot and spread primarily by seed dispersal.

and we can observe three major life cycle groups in cereal arable fields:

**Annuals**

**Biennials**

biennial weeds.

**Perennials**

following spring or summer.

134 Herbicides - Current Research and Case Studies in Use

structures as well as by seed.

cultural, mechanical, biological, and chemical means.


In addition, the selected herbicide must fulfill other requisites about their mode of action, which are:


Herbicides provide a convenient, economical, and effective way to help manage weeds. They allow fields to be planted with less tillage, allow earlier planting dates, and provide additional time to perform the other tasks that farm or personal life require. However, if herbicides are not applied in a timely and appropriate manner in terms of dosage and coverage, or resistant weeds are present, they can have ineffective control.

> herbicides and the development of resistant weeds has led to seek integrated weed management systems more. Integrated weed management requires more knowledge on how weed community compositions respond to changing agronomic practices after one crop rotation cycle with different practices. Gerard [16] observed that the prediction of the distribution and abundance of weed infestations likely in each field could help to plan and carry out timely control measures in an efficient and economical manner, in accordance with ecology and the interests of the society. The above statement is framed within what is known as "integrated management of weeds", where the main objective is to cause displacement of species difficult to control, by others less problematic and / or reduce the density of populations of noxious weeds at levels that do not cause damage. Therefore, such rationalization goes through the realization of a good diagnosis of the situation, by using a series of agronomic practices that hinder the development of weed populations most problematic and the use of clear decision criteria based on scientific knowledge.

**CHANGES IN WEED COMMUNITY** 

CLIMATE FACTORS

HUMAN FACTORS

**Figure 1.** Factors involved in changes of weed community present in a field.

SOIL FACTORS

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TECHNOLOGICAL FACTORS

Cereals are the most important crop in dry-land areas of southern Europe. In Spain, nearly 5.5 million ha of winter cereals are sown each year [1]. In Mediterranean areas, weed species are adapted to crops and to management techniques like soil disturbance by tillage. However, the agricultural intensification in the last decades is a process occurring at different scales, which reduces biodiversity, simplifies communities, leads to a loss of ecosystem services [17- 19] and reduces species richness [20]. At the landscape scale, farming intensification has caused the replacement of most natural habitats with arable fields [21], which leads to large, uniformly cropped areas with low spatial heterogeneity [22, 23]. At the field scale, intensification is related to the farming practices performed: i.e., high amount of external inputs (mainly chemical

*1.1.1. Weed ecology in dry land cereal agriculture*

In this context, long-term experiments, carried out for decades, are considered very important in agricultural research when evaluating the sustainability of crop systems in which are being developed programs of integrated crop protection, in order to maintain stability of agricultural production. The weed vegetation in an agricultural area can change quickly and vary greatly among fields and regions. The factors that influence the weed community are numerous and are difficult to evaluate each factor independently, in a culture system (Figure 1): climatic factors relevant to the persistence of plants, soil factors, which involved the physical and chemical properties of soil, human factors, which are involved in various legislative measures and the use and farm management and technological factors, where one is constantly inno‐ vating and researching systems tillage, crop rotation, herbicides, fertilization, and irrigation.

Intensification of land use has also been identified as a major cause of the current biodiversity decline in agro ecosystems [10, 11]. For instance, arable weeds have suffered a severe decline over all Europe, which has developed concerns over the sustainability and environmental consequences of the intensification of land use in agricultural systems [12]. Plant diversity in dry land Mediterranean cereal fields is affected by agricultural intensification at any of these abovementioned scales, as reflected by a decrease of plant species richness and changes in species composition [13]. But the ecological implications of these changes still remain uncer‐ tain, because in such agro systems there is a high variability in the local occurrence of plant species [14, 15].

Historically in central semiarid Spain, arable fields have been dominated by cereal production. In this region, tillage intensity has markedly decreased in order to decrease soil loss. There has been an increasing trend towards utilizing conservation tillage systems and the use of herbicides in winter cereals holds a prominent place in the overall use of pesticides in Spain. However, in recent years, climate change, grain prices, cost of

**Figure 1.** Factors involved in changes of weed community present in a field.

**•** In post-emergence applications, the species present, their size, age, growth rate and

**•** In pre-emergence applications, soil type (texture, pH and organic matter content), soil

In addition, the selected herbicide must fulfill other requisites about their mode of action,

Herbicides provide a convenient, economical, and effective way to help manage weeds. They allow fields to be planted with less tillage, allow earlier planting dates, and provide additional time to perform the other tasks that farm or personal life require. However, if herbicides are not applied in a timely and appropriate manner in terms of dosage and coverage, or resistant

In this context, long-term experiments, carried out for decades, are considered very important in agricultural research when evaluating the sustainability of crop systems in which are being developed programs of integrated crop protection, in order to maintain stability of agricultural production. The weed vegetation in an agricultural area can change quickly and vary greatly among fields and regions. The factors that influence the weed community are numerous and are difficult to evaluate each factor independently, in a culture system (Figure 1): climatic factors relevant to the persistence of plants, soil factors, which involved the physical and chemical properties of soil, human factors, which are involved in various legislative measures and the use and farm management and technological factors, where one is constantly inno‐ vating and researching systems tillage, crop rotation, herbicides, fertilization, and irrigation. Intensification of land use has also been identified as a major cause of the current biodiversity decline in agro ecosystems [10, 11]. For instance, arable weeds have suffered a severe decline over all Europe, which has developed concerns over the sustainability and environmental consequences of the intensification of land use in agricultural systems [12]. Plant diversity in dry land Mediterranean cereal fields is affected by agricultural intensification at any of these abovementioned scales, as reflected by a decrease of plant species richness and changes in species composition [13]. But the ecological implications of these changes still remain uncer‐ tain, because in such agro systems there is a high variability in the local occurrence of plant

Historically in central semiarid Spain, arable fields have been dominated by cereal production. In this region, tillage intensity has markedly decreased in order to decrease soil loss. There has been an increasing trend towards utilizing conservation tillage systems and the use of herbicides in winter cereals holds a prominent place in the overall use of pesticides in Spain. However, in recent years, climate change, grain prices, cost of

moisture at the time of application and weed species to be controlled.

environmental conditions.

136 Herbicides - Current Research and Case Studies in Use

**•** Penetrate into the weed.

**•** Affect any vital function.

species [14, 15].

**•** Control weeds with a sufficient dose.

**•** Move to where conduct its physiological action.

weeds are present, they can have ineffective control.

which are:

herbicides and the development of resistant weeds has led to seek integrated weed management systems more. Integrated weed management requires more knowledge on how weed community compositions respond to changing agronomic practices after one crop rotation cycle with different practices. Gerard [16] observed that the prediction of the distribution and abundance of weed infestations likely in each field could help to plan and carry out timely control measures in an efficient and economical manner, in accordance with ecology and the interests of the society. The above statement is framed within what is known as "integrated management of weeds", where the main objective is to cause displacement of species difficult to control, by others less problematic and / or reduce the density of populations of noxious weeds at levels that do not cause damage. Therefore, such rationalization goes through the realization of a good diagnosis of the situation, by using a series of agronomic practices that hinder the development of weed populations most problematic and the use of clear decision criteria based on scientific knowledge.

#### *1.1.1. Weed ecology in dry land cereal agriculture*

Cereals are the most important crop in dry-land areas of southern Europe. In Spain, nearly 5.5 million ha of winter cereals are sown each year [1]. In Mediterranean areas, weed species are adapted to crops and to management techniques like soil disturbance by tillage. However, the agricultural intensification in the last decades is a process occurring at different scales, which reduces biodiversity, simplifies communities, leads to a loss of ecosystem services [17- 19] and reduces species richness [20]. At the landscape scale, farming intensification has caused the replacement of most natural habitats with arable fields [21], which leads to large, uniformly cropped areas with low spatial heterogeneity [22, 23]. At the field scale, intensification is related to the farming practices performed: i.e., high amount of external inputs (mainly chemical fertilizers and herbicides), low complexity of crop-rotational schemes and improvements in seed-cleaning techniques [24].

due to their abundance (associated with a huge seed production and a high persistence of these seed on the soil surface) than competitiveness with the crop (relatively low). Similarly, the Cruciferae family (*Sinapis arvensis* L., *Diplotaxis erucoides* L. and Raphanus spp.) produces high numbers of seeds although the competition with the crop can be quite high. These species had adapted to conventional tillage, but the increasing use of herbicides has reduced their popu‐ lations while favoring the presence of other species: "Cleavers" (*Galium aparine* L.), which are fast growing and can outcompete almost completely the cereal plants; "Speedwell" (*Veronica hederifolia* L. and *Veronica persica* L.), "Chamomile" (*Matricaria chamomilla* L.), *Polygonum aviculare* L., etc. Other species of the genus Tussilago, Epilobium, Conyza, Artemisia, Lactuca, etc., have problems in soils subjected to periodic disturbances, and they have also adapted to no-tillage fields. Also, the species Chenopodium spp., Amaranthus spp., Salsola spp., etc. can invade the cereal fallows, which can require investment in specific herbicides for control. Finally, perennial weeds base their success on their bodies' underground reserves that enable rapid development at the beginning of spring. They are represented by the bindweed (Con‐

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In this paper, we will not create a weed inventory or abundance, but focus on identifying the most significant risks to which crop will face during its development. Before herbicide treatment, it is imperative to carry out a diagnosis as accurate as possible of the weed situation. This idea is according to the National Academy of Sciences (1980): "to induce population changes in response to agricultural management, it is necessary to know the biology of the species involved and environmental modifications that causes each agronomic practice". This requires knowledge of the dynamics of weed populations that cause *a favorable succession* and it is necessary to know the majority of weed species present in the plots treated. In this sense,

First, it is necessary to select the most appropriate treatments taking into account the efficacy and selectivity of the products available on the market. In view of the problems identified in each field, we will need to find which products adequately control all high-risk species. In Spain there are over 30 different active ingredients for use in cereal crops and over two times that many commercial products (with various formulations and/or combinations of active substances). The selection of products to be used will be dictated by the timing of treatments. Table 1 lists some of the most widely used herbicides in cereals and their application times. We should note that the application of these products is not always carried out in isolation, so it is important to know if there is a problem of incompatibility between products (relatively frequent event). There would also be possible to find problems of sensitivity of crops because not all products are equally safe for barley and wheat, and even within the same crop, there

In the case of herbicides used in pre-emergence, this decision will have to be made based on the problems identified in previous years. In that sense, it is highly desirable to have some information about the history of the field, i.e. crops that were planted, cultural practices, herbicides used, and what kind of weed problems developed. This information will help us to identify the type and severity of the problem to be faced in the coming season. Since weed

decisions regarding herbicide treatments should be based on four main points:

volvulus spp.) and several thistles (Cirsium spp.).

are differences in sensitivity in some varieties.

In this sense, the patterns of weed species composition in cereal fields are often attributable to a complex number of interacting factors and multivariate analysis has been used in many studies to discuss them. The selection of weeds is constantly evolving in response to crop management practices; therefore, these practices have an important role in the flora composi‐ tion and its fluctuations in the short and long term at the field level. Management practices, geographical gradients and climatic factors have been found to be the driving factors to explain weed species composition and richness in Northern Europe [25] and in Central Europe [26-29]. Thus, changes in flora may be the result, among other factors, of complex interactions between agronomic practices (choice of species, tillage systems, and strategies for weed control) and environmental factors (soil quality, temperature, and rainfall). It is well known that sometimes, the use of some methods of control, or changes in them by others, causes a change in the composition of the flora, and we can say that weed communities are not static, producing the phenomenon known as *Flora Inversion*.

Although major weeds can be quite different from one region to another, from one farm to another and even between different locations of the same farm, we can select a few species that are widespread throughout the Spanish geography which represent a serious threat by the competitiveness, by the difficulty of control and by the rapid expansion of their populations. Among them we can mention four annual grasses:

*Avena sterilis* L. and *A. fatua* L. ("Wild oats"), these weeds are found throughout the peninsula, has an almost identical cycle of cereals, germinating simultaneously with them and for a fairly long period of time and matures at the same time as grain crops. These attributes, combined with its ability to emerge from depths relatively high (up to 25 cm) and the prolonged persistence of seeds in the soil (over 3 years) facilitates the development and presence in tilled fields. However, the main reason for its spread is its ability to cause high losses in cereal yields.

*Lolium rigidum* Gaudin and *Lolium multiflorum* L. ("Ryegrass") are widespread geographically, being especially prevalent in cereals. These species germinate with the first rains of autumn, usually beginning their nascence before sowing of cereal. If the first plants were not completely destroyed by seedbed preparation tillage or pre-plant herbicides they can become great competitors with the crop. Most seeds germinate the following year of their production, making containment or eradication of their population easier than in the case of the other grass.

Bromus spp. and Phalaris spp. were a very common species in the margins of roads and cultivated fields until the arrival of conservation agriculture. With tillage reduction or elimination, they have been introduced in the fields quickly causing major problems. These species are well adapted to emerge from the soil surface zone. Its emergence period is very short, beginning with the first rains of autumn, and almost all seeds germinate the following year.

Besides the grasses mentioned above, there are some dicotyledonous annuals that are harmful, either because of their abundance, their competitiveness or difficulties involved in their control. In the case of the "poppy" (Papaver spp. and Hypecoum spp.) the problem is more due to their abundance (associated with a huge seed production and a high persistence of these seed on the soil surface) than competitiveness with the crop (relatively low). Similarly, the Cruciferae family (*Sinapis arvensis* L., *Diplotaxis erucoides* L. and Raphanus spp.) produces high numbers of seeds although the competition with the crop can be quite high. These species had adapted to conventional tillage, but the increasing use of herbicides has reduced their popu‐ lations while favoring the presence of other species: "Cleavers" (*Galium aparine* L.), which are fast growing and can outcompete almost completely the cereal plants; "Speedwell" (*Veronica hederifolia* L. and *Veronica persica* L.), "Chamomile" (*Matricaria chamomilla* L.), *Polygonum aviculare* L., etc. Other species of the genus Tussilago, Epilobium, Conyza, Artemisia, Lactuca, etc., have problems in soils subjected to periodic disturbances, and they have also adapted to no-tillage fields. Also, the species Chenopodium spp., Amaranthus spp., Salsola spp., etc. can invade the cereal fallows, which can require investment in specific herbicides for control. Finally, perennial weeds base their success on their bodies' underground reserves that enable rapid development at the beginning of spring. They are represented by the bindweed (Con‐ volvulus spp.) and several thistles (Cirsium spp.).

fertilizers and herbicides), low complexity of crop-rotational schemes and improvements in

In this sense, the patterns of weed species composition in cereal fields are often attributable to a complex number of interacting factors and multivariate analysis has been used in many studies to discuss them. The selection of weeds is constantly evolving in response to crop management practices; therefore, these practices have an important role in the flora composi‐ tion and its fluctuations in the short and long term at the field level. Management practices, geographical gradients and climatic factors have been found to be the driving factors to explain weed species composition and richness in Northern Europe [25] and in Central Europe [26-29]. Thus, changes in flora may be the result, among other factors, of complex interactions between agronomic practices (choice of species, tillage systems, and strategies for weed control) and environmental factors (soil quality, temperature, and rainfall). It is well known that sometimes, the use of some methods of control, or changes in them by others, causes a change in the composition of the flora, and we can say that weed communities are not static, producing the

Although major weeds can be quite different from one region to another, from one farm to another and even between different locations of the same farm, we can select a few species that are widespread throughout the Spanish geography which represent a serious threat by the competitiveness, by the difficulty of control and by the rapid expansion of their populations.

*Avena sterilis* L. and *A. fatua* L. ("Wild oats"), these weeds are found throughout the peninsula, has an almost identical cycle of cereals, germinating simultaneously with them and for a fairly long period of time and matures at the same time as grain crops. These attributes, combined with its ability to emerge from depths relatively high (up to 25 cm) and the prolonged persistence of seeds in the soil (over 3 years) facilitates the development and presence in tilled fields. However, the main reason for its spread is its ability to cause high losses in cereal yields. *Lolium rigidum* Gaudin and *Lolium multiflorum* L. ("Ryegrass") are widespread geographically, being especially prevalent in cereals. These species germinate with the first rains of autumn, usually beginning their nascence before sowing of cereal. If the first plants were not completely destroyed by seedbed preparation tillage or pre-plant herbicides they can become great competitors with the crop. Most seeds germinate the following year of their production, making containment or eradication of their population easier than in the case of the other grass. Bromus spp. and Phalaris spp. were a very common species in the margins of roads and cultivated fields until the arrival of conservation agriculture. With tillage reduction or elimination, they have been introduced in the fields quickly causing major problems. These species are well adapted to emerge from the soil surface zone. Its emergence period is very short, beginning with the first rains of autumn, and almost all seeds germinate the following

Besides the grasses mentioned above, there are some dicotyledonous annuals that are harmful, either because of their abundance, their competitiveness or difficulties involved in their control. In the case of the "poppy" (Papaver spp. and Hypecoum spp.) the problem is more

seed-cleaning techniques [24].

138 Herbicides - Current Research and Case Studies in Use

phenomenon known as *Flora Inversion*.

year.

Among them we can mention four annual grasses:

In this paper, we will not create a weed inventory or abundance, but focus on identifying the most significant risks to which crop will face during its development. Before herbicide treatment, it is imperative to carry out a diagnosis as accurate as possible of the weed situation. This idea is according to the National Academy of Sciences (1980): "to induce population changes in response to agricultural management, it is necessary to know the biology of the species involved and environmental modifications that causes each agronomic practice". This requires knowledge of the dynamics of weed populations that cause *a favorable succession* and it is necessary to know the majority of weed species present in the plots treated. In this sense, decisions regarding herbicide treatments should be based on four main points:

First, it is necessary to select the most appropriate treatments taking into account the efficacy and selectivity of the products available on the market. In view of the problems identified in each field, we will need to find which products adequately control all high-risk species. In Spain there are over 30 different active ingredients for use in cereal crops and over two times that many commercial products (with various formulations and/or combinations of active substances). The selection of products to be used will be dictated by the timing of treatments. Table 1 lists some of the most widely used herbicides in cereals and their application times. We should note that the application of these products is not always carried out in isolation, so it is important to know if there is a problem of incompatibility between products (relatively frequent event). There would also be possible to find problems of sensitivity of crops because not all products are equally safe for barley and wheat, and even within the same crop, there are differences in sensitivity in some varieties.

In the case of herbicides used in pre-emergence, this decision will have to be made based on the problems identified in previous years. In that sense, it is highly desirable to have some information about the history of the field, i.e. crops that were planted, cultural practices, herbicides used, and what kind of weed problems developed. This information will help us to identify the type and severity of the problem to be faced in the coming season. Since weed


herbicides (2.4-D, MCPA, etc.) for overall control of dicots is almost negligible, the use of specific herbicides against Galium spp. or Avena spp. may be a considerable investment.

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Third, it is necessary to estimate the economic benefits of a treatment application. This involves estimating the expected yields in the crop (and its sale value) and the losses that would be avoided by such treatment. In this sense, while the application of herbicides in areas of high productivity (yields higher than 4 t/ha) is usually economically profitable in more marginal areas (with an income below 2 t/ha) these benefits are rather dubious. Similarly, in meteoro‐ logically favorable years higher investments in inputs may provide higher profits. In relation to avoidable losses, we should consider the competitiveness of the dominant species (it´s not the same having a plot infested by Avena spp. or it infested by Papaver spp.), and the level of

Finally, we must consider the potential side effects arising from the application of such treatment. This section is not only to consider the effects on the environment (pollution of waterways, loss of biodiversity) but also the risk of resistance. The emergence of resistance as a result of poor practices is increasingly common. Continued application of the same product (or products belonging to the same chemical family or families with the same mode of action) over a certain period of time leads, sooner or later, to the emergence of resistant weed biotypes. The best strategy to prevent the emergence of weed resistance is the integrated use of preven‐

**•** Use of crop rotations, using spring crops needed to eliminate resistant biotypes before

**•** Avoid seeds with resistance movement from one field to another, carefully cleaning tillage

**•** Herbicide use only when necessary, alternating herbicides belonging to different groups

The climatic factors more relevant to the persistence of the plants are: light, temperature, water,

The intensity, quality and duration of **ligh**t are important for determining the growth, reproduction and distribution of such plants. Light governs the photoperiodic response and determines the flowering time of seed maturation; therefore, it determines the latitudinal

The air and soil **temperature** and the duration of the frost, are important limits on the distri‐ bution of weeds. The soil temperature is directly related to the seed germination, and a drop in temperature will influence the same seed dormancy and survival of their underground

planting or use alternative herbicides not applicable in cereal crops.

**•** Employment of fallow and mechanical control practices.

**•** Using appropriate densities for a competitive cultivation.

*1.1.2. The climate influence in an agro system with a semi-arid environment*

weed infestation of plot.

tion and control of many methods as possible:

and harvesting equipment.

distribution limits of species.

according to their mode of action.

wind and seasonal characteristics of these factors:

**Table 1.** Herbicides used in cereal crops depending on the timing and type of weed.

infestations are often not distributed evenly throughout the field, it will also be useful to know the location of problematic weeds populations and if they are particularly aggressive species or found in very high densities. Pre-emergent herbicides act upon weed seeds, seedlings or form a barrier in the soil to prevent weed seed germination or establishment. These herbicides are usually used in the spring to prevent seeds establishing when the soil temperatures begin to warm up and a properly timed application can provide control for several months.

In the case of herbicides used in post-emergence (the most common use), it is desirable to perform the evaluation of the main weeds that are invading each field as soon as the cereal is established. This assessment should be made as soon as possible in order to plan and carry out early treatment, which is recommended due to their greater efficiency. Post-emergent herbi‐ cides work on actively growing weeds and can be further broken down into two categories:


After choosing the herbicide, it is necessary to decide the dose to apply. Typically there is a relatively large dose range according to what weeds dominate; what is the stage of develop‐ ment (the higher development, the greater the dose needed to control them) and what is the texture and the organic matter content of soil (in cases of pre-sowing applications or preemergence, the higher the content of clay and organic matter, the greater the dose).

Second, one must consider the costs of treatments considered. There are large differences between the costs of different products. For example, while the cost of treatment with hormonal herbicides (2.4-D, MCPA, etc.) for overall control of dicots is almost negligible, the use of specific herbicides against Galium spp. or Avena spp. may be a considerable investment.

Third, it is necessary to estimate the economic benefits of a treatment application. This involves estimating the expected yields in the crop (and its sale value) and the losses that would be avoided by such treatment. In this sense, while the application of herbicides in areas of high productivity (yields higher than 4 t/ha) is usually economically profitable in more marginal areas (with an income below 2 t/ha) these benefits are rather dubious. Similarly, in meteoro‐ logically favorable years higher investments in inputs may provide higher profits. In relation to avoidable losses, we should consider the competitiveness of the dominant species (it´s not the same having a plot infested by Avena spp. or it infested by Papaver spp.), and the level of weed infestation of plot.

Finally, we must consider the potential side effects arising from the application of such treatment. This section is not only to consider the effects on the environment (pollution of waterways, loss of biodiversity) but also the risk of resistance. The emergence of resistance as a result of poor practices is increasingly common. Continued application of the same product (or products belonging to the same chemical family or families with the same mode of action) over a certain period of time leads, sooner or later, to the emergence of resistant weed biotypes. The best strategy to prevent the emergence of weed resistance is the integrated use of preven‐ tion and control of many methods as possible:


infestations are often not distributed evenly throughout the field, it will also be useful to know the location of problematic weeds populations and if they are particularly aggressive species or found in very high densities. Pre-emergent herbicides act upon weed seeds, seedlings or form a barrier in the soil to prevent weed seed germination or establishment. These herbicides are usually used in the spring to prevent seeds establishing when the soil temperatures begin

**Timing of herbicide application Controlled weeds Active substances**

Dicotyledonous

Dicotyledonous

Grass

Grass isoproturon

Dicotyledonous clodinafop, tralkoxidim Grass Fenoxaprop-p-etil,

clortoluron, isoproturon, trifluralina,

bifenox,pendimetalina, triasulfuron.

2.4-D, MCPA, fluroxipir, bentazona, tifensulfuron-metil, tribenuron-metil.

clorsulfuron, linuron,

fenoxaprop-etil,

clortoluron, diclofop-metil,

iodosulfuron-metil-sodio, pendimetalina, tralkoxidim.

In the case of herbicides used in post-emergence (the most common use), it is desirable to perform the evaluation of the main weeds that are invading each field as soon as the cereal is established. This assessment should be made as soon as possible in order to plan and carry out early treatment, which is recommended due to their greater efficiency. Post-emergent herbi‐ cides work on actively growing weeds and can be further broken down into two categories: **•** Selective herbicides can be applied to an area and target weeds (i.e. dicots or monocots) while having little or no effect on the crop or non-target weeds. Some products may require

**•** Non-selective herbicides kill all susceptible plants they come into contact with. The most

After choosing the herbicide, it is necessary to decide the dose to apply. Typically there is a relatively large dose range according to what weeds dominate; what is the stage of develop‐ ment (the higher development, the greater the dose needed to control them) and what is the texture and the organic matter content of soil (in cases of pre-sowing applications or pre-

Second, one must consider the costs of treatments considered. There are large differences between the costs of different products. For example, while the cost of treatment with hormonal

emergence, the higher the content of clay and organic matter, the greater the dose).

to warm up and a properly timed application can provide control for several months.

**Table 1.** Herbicides used in cereal crops depending on the timing and type of weed.

repeated applications for effective control.

Pre emergence

140 Herbicides - Current Research and Case Studies in Use

Early post emergence

Late post emergence

used non-selective herbicide is glyphosate.


#### *1.1.2. The climate influence in an agro system with a semi-arid environment*

The climatic factors more relevant to the persistence of the plants are: light, temperature, water, wind and seasonal characteristics of these factors:

The intensity, quality and duration of **ligh**t are important for determining the growth, reproduction and distribution of such plants. Light governs the photoperiodic response and determines the flowering time of seed maturation; therefore, it determines the latitudinal distribution limits of species.

The air and soil **temperature** and the duration of the frost, are important limits on the distri‐ bution of weeds. The soil temperature is directly related to the seed germination, and a drop in temperature will influence the same seed dormancy and survival of their underground parts. Therefore, temperature is a critical factor for the persistence and adaptation of annual and perennial weeds.

Year J F M A My J Jy A S O N D Annual 18,4 33,4 9 18,2 76,8 10,8 3,6 0,8 40,6 52,2 26,8 4,6 295,2 18,2 28,4 0,8 10,8 33,4 56,2 7,6 3,0 11,4 5,6 45,2 71,2 291,8 84,6 24,2 16,4 9,4 94,4 5,8 1,4 6,8 12,4 4,0 58,4 86,6 404,4 76,2 2,8 0,0 63,2 47,2 6,8 23,6 32,0 28,0 17,6 145,4 53,4 496,2 28,5 45,1 15,9 27,6 14,2 1,6 2,0 0,4 0,2 1,0 29,6 20,0 186,1 36,0 14,8 18,0 55,3 65,8 22,6 4,0 3,0 57,0 96,7 45,8 0,0 419,0 64,0 0,8 30,0 103,8 69,6 25,0 10,0 0,0 18,2 26,5 70,0 129,7 547,6 108,8 20,7 60,1 26,5 37,7 7,2 4,3 7,0 14,0 79,4 4,9 0,0 370,6 54,0 5,7 46,2 41,5 76,7 12,3 12,0 5,1 32,5 50,6 86,0 48,2 470,8 51,2 52,3 39,5 57,2 22,2 1,8 1,2 2,6 9,4 99,5 67,6 35,5 440,0 4,2 74,7 55,1 43,7 102,2 5,3 46,1 18,5 5,4 99,4 20,4 17,8 492,8 0,0 15,5 11,3 7,2 7,1 1,3 0,0 3,2 12,2 86,3 66,0 27,0 237,1 40,2 45,5 20,0 37,0 14,0 34,8 1,5 5,7 15,9 84,6 98,3 20,5 418,0 7,8 43,8 13,6 104,5 95,7 37,0 0,0 6,8 10,3 30,8 30,3 4,0 384,6 27,6 32,6 2,1 80,1 106,0 36,9 0,0 0,0 22,2 56,7 25,2 42,0 431,4 38,7 43,9 11,2 31,4 8,0 17,3 0,0 20,7 8,6 23,2 12,8 111,7 327,5 70,2 84,8 51,3 47,5 33,5 58,1 17,5 2,8 40,6 31,0 41,0 62,9 541,2 44,0 30,0 46,7 65,0 168,5 24,5 1,0 24,0 1,6 33,2 49,5 6,0 494,0

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Average 42,92 33,28 24,84 46,11 59,61 20,29 7,54 7,91 18,92 48,79 51,29 41,17 402,68

**Figure 2.** Total number of plants recorded per sample (0,125 m2) and annual rainfall (mm) from 1995 to 2011.

**Table 2.** Annual distribution of rainfall (mm) and historical average during years object of study.

Historical

**Water** is the most important environmental factor in the habitat, with a marked morphological expression in the plant. The total water available in a location is related to both the initial supply with losses by runoff, evaporation and transpiration. The seasonal distribution of water is a key factor, since sometimes its scarcity at critical stages of the plant leads to lack of reproduction and survival.

The speed, **wind** direction and wind frequency defines the presence of all plants, including weeds. Also, it can produce transpiration losses of plants.

In summary, the weeds are primarily affected by the same factors as the crop: water, and the factors related to their availability (insolation and transpiration) and nutrients. If these parameters are not restricted, the weed growth will be higher than the crop.

On the other hand, when conditions are not suitable, the agronomic practices may be ineffec‐ tive in inducing seed germination. In this sense, one of chief limiting factor of crop yield in cereal agro systems with a semi-arid environment is the scarce irregular rainfall distribution. For this reason, we initiated a field experiment, at the experimental farm of INIA "La Canaleja", located in Alcala de Henares (Madrid). The field trials were located in a semi-arid agro system of central Spain, with an average total annual rainfall of 470 mm, and rainfall distribution registered over fifteen years were used to assess the effects of environmental conditions on weed community.

Our results showed that seasonal distribution of rainfall did restrict the effectiveness of the weed management practices and it affected the weed density. In 2000-2001 and 2010-2011, it we recorded higher annual rainfall than the average for this area, and in accordance with the increase of water availability, the weed density, measured by sampling (size of each sample of 0,125 m2 ), increased considerably. Between years 1995 and 2011 herbicides controlling dicotyledonous and / or against grass were used to control the weed community present in the field. In this situation, total weed density was maintained except in the 2009-2010 period, when weed density was large though the annual rainfall was below normal; this was mainly due to herbicides not being used in this period favoring the weed competition with the crops (Table 2 & Figure 2).

The community of weeds present in the field differed with the annual distribution of rainfall and may limit the effectiveness of the system used to control weeds, leading the specialization of some species under certain crop conditions. We observed in our field, that high rainfall occurring in the spring favored early-emergence weeds, such as *Papaver roheas* L. and high rainfall occurring in autumn favored late-emergence weeds such us *Lolium rigidum* Gaud. and *Hypecoum procumbens* L.; and weeds with extended patterns of emergence such as *Anacyclus clavatus* L. and Veronica spp.; or perennial weeds (Cardaria spp. and Convolvulus spp.) were favored by a general increase of annual rainfall in the area. Furthermore, increasing knowledge of how plants respond to different environmental conditions and the application of this knowledge allows more effective and efficient use of available tillage tools in combination with other weed control practices.

Weed Management in Cereals in Semi-Arid Environments: A Review http://dx.doi.org/10.5772/55970 143


**Table 2.** Annual distribution of rainfall (mm) and historical average during years object of study.

parts. Therefore, temperature is a critical factor for the persistence and adaptation of annual

**Water** is the most important environmental factor in the habitat, with a marked morphological expression in the plant. The total water available in a location is related to both the initial supply with losses by runoff, evaporation and transpiration. The seasonal distribution of water is a key factor, since sometimes its scarcity at critical stages of the plant leads to lack of reproduction

The speed, **wind** direction and wind frequency defines the presence of all plants, including

In summary, the weeds are primarily affected by the same factors as the crop: water, and the factors related to their availability (insolation and transpiration) and nutrients. If these

On the other hand, when conditions are not suitable, the agronomic practices may be ineffec‐ tive in inducing seed germination. In this sense, one of chief limiting factor of crop yield in cereal agro systems with a semi-arid environment is the scarce irregular rainfall distribution. For this reason, we initiated a field experiment, at the experimental farm of INIA "La Canaleja", located in Alcala de Henares (Madrid). The field trials were located in a semi-arid agro system of central Spain, with an average total annual rainfall of 470 mm, and rainfall distribution registered over fifteen years were used to assess the effects of environmental conditions on

Our results showed that seasonal distribution of rainfall did restrict the effectiveness of the weed management practices and it affected the weed density. In 2000-2001 and 2010-2011, it we recorded higher annual rainfall than the average for this area, and in accordance with the increase of water availability, the weed density, measured by sampling (size of each sample

dicotyledonous and / or against grass were used to control the weed community present in the field. In this situation, total weed density was maintained except in the 2009-2010 period, when weed density was large though the annual rainfall was below normal; this was mainly due to herbicides not being used in this period favoring the weed competition with the crops

The community of weeds present in the field differed with the annual distribution of rainfall and may limit the effectiveness of the system used to control weeds, leading the specialization of some species under certain crop conditions. We observed in our field, that high rainfall occurring in the spring favored early-emergence weeds, such as *Papaver roheas* L. and high rainfall occurring in autumn favored late-emergence weeds such us *Lolium rigidum* Gaud. and *Hypecoum procumbens* L.; and weeds with extended patterns of emergence such as *Anacyclus clavatus* L. and Veronica spp.; or perennial weeds (Cardaria spp. and Convolvulus spp.) were favored by a general increase of annual rainfall in the area. Furthermore, increasing knowledge of how plants respond to different environmental conditions and the application of this knowledge allows more effective and efficient use of available tillage tools in combination with

), increased considerably. Between years 1995 and 2011 herbicides controlling

parameters are not restricted, the weed growth will be higher than the crop.

weeds. Also, it can produce transpiration losses of plants.

and perennial weeds.

142 Herbicides - Current Research and Case Studies in Use

and survival.

weed community.

of 0,125 m2

(Table 2 & Figure 2).

other weed control practices.

**Figure 2.** Total number of plants recorded per sample (0,125 m2) and annual rainfall (mm) from 1995 to 2011.

### **2. The adoption of conservation tillage systems**

The European agricultural situation is modifying quickly due to the pressure of econom‐ ic factors and to the increased sensitivity of environmental problems. Nowadays, integrat‐ ed weed management could be a possible solution to rationalize the inputs of herbicides and to increase the use of complementary methods of weed control forming an integral component of sustainable agriculture [30]. However, the adoption of these practices have a considerable impact on communities of weeds, and therefore, their management should be different from that undertaken in a conventional system [31-33]. The benefits of conservation tillage include reducing soil erosion, increasing organic matter, improving soil structure, and reducing fuel consumption and some tillage machinery. As a result of these agronomic and economic incentives, direct seeding practices have been adopted in many regions. Weed control is often cited as the main challenge in minimum tillage systems and no-tillage, and often leads to increased herbicide use, so we must pay special attention to this system. Otherwise, conservation tillage systems are believed to worsen weed prob‐ lems with higher weed emergence promoted by higher concentrations of seed in the surface soil and shifts of the weed community towards increased abundance of troublesome species, e.g. grasses and perennials [34].

(a)

age system.

the unit area (1m2

).

(b)

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)

**Figure 3.** A) Field trials in the experimental farm "La Canaleja", and B) Weeds sampling scheme realized it in each till‐

The natural community of weeds present in the assay is comprised by dicotyledonous weed species and grasses, annual and perennial species typical of crop fields in the area (Table 3). Later, during all years of the study, weeds were counted by species with a similar methodology based on the selection of four random samples in the field with a quadrant of 0,125m2

in zigzag on the diagonal of a rectangle defined in each sub-plot (Figure 3B), which were identified and quantified in situ, the weed species present. Total density of weeds referred to

Adonis annua Chondrilia juncea Hordeum murimus Scabiosa spp. Amaranthus albus Chrozopera tinctoria Hypecoum pendulum Scorzonera laciniata Amaranthus blitoides Cichorium intybus Hypecoum procumbens Senecio vulagaris Amaranthus retroflexus Cirsium arvense Lactuca serriola Setaria viridis Anacyclus clavatus Cnicus benedictus Lamium amplexicaule Silybum marianum Anchusa azurea Convulvulus arvensis Lavatera spp. Sisymbrium iria Andryala integrifolia Conyza spp. Linaria micranha Sisymbrium orientale Asperugo procumbens Datura stramonium Lolium rigidum Solanun rigidum Avena spp. Descurania Sophia Medicago spp. Sonchus spp. Belladia trixago Diplotaxis erucoides Melilotus spp. Stellaria media Biscutella auriculata Echallium elaterium Papaver hybridum Torilis nodosa Bromus rigidus Echium spp. Papaver rhoeas Tragopogum psp. Bromus rubens Epilolium brachycarpum Plantago spp. Trifolium angustifolium Buglossoides arvensis Eruca vessicaria Polygonum aviculare Trigonella polyceratia Campanula erimus Eryngium spp. Portulaca aleracea Veccaria pyramidata Capsella burs-pastori Filago spp. Rapistrum rugosum Veronica hederifolia

Cardaria draba Fumaria officinalis Reseda phyteuma Vicia spp.

Chenopodium album Heliotroium europaeum Salsola kali

**Table 3.** Initial weed community in the farm "La Canaleja".

Centaurea aspera Galium murale Roemeria híbrida Xanthium spinosum

, taken

In summary, minimum tillage, particularly no-tillage, may favor a relative emergence of weeds over crops. Moreover, the increase of prior crop residues in these systems can alter the competitive ability of crops with weeds at early stages, increasing production losses thereof [35]. Thus, it appears that common tasks tend to select annual weeds and little work allows the dominance of perennial or biennial species. However, these predictions are strongly influenced by cultural practices and environmental conditions used in a specific area. Currently, insufficient information exists about the processes associated with changes in weed communities; such information is crucial in managing weeds. As a means of control it is necessary to assess the presence of weeds, setting thresholds for treatment of major species in crops and the adequate product selection, dose and time of applica‐ tion best suited among those authorized, while taking into account the environmental conditions.

Development of improved weed management systems requires more knowledge on how weed species respond to changing agronomic practices. In order to monitor weed development subjected to different agronomic practices, one experiment was conducted to determine weed population response to various tillage intensities in a cereal agro system in central Spain (Figure 3A). Field trials under a cold semi-arid environment were conducted in successive growing seasons from 1995 to 2011, to assess the effects of management practices on the weed community with three tillage systems: (1) conventional tillage (CT); (2) minimum tillage (MT) and (3) no-tillage system (NT). The experiment consisted of a field divided in four randomized complete blocks with three different tillage systems and four replications. To study the effectiveness of different managements, we performed a first identification of the flora present in the field where the experiment was developed.

**2. The adoption of conservation tillage systems**

144 Herbicides - Current Research and Case Studies in Use

species, e.g. grasses and perennials [34].

in the field where the experiment was developed.

conditions.

The European agricultural situation is modifying quickly due to the pressure of econom‐ ic factors and to the increased sensitivity of environmental problems. Nowadays, integrat‐ ed weed management could be a possible solution to rationalize the inputs of herbicides and to increase the use of complementary methods of weed control forming an integral component of sustainable agriculture [30]. However, the adoption of these practices have a considerable impact on communities of weeds, and therefore, their management should be different from that undertaken in a conventional system [31-33]. The benefits of conservation tillage include reducing soil erosion, increasing organic matter, improving soil structure, and reducing fuel consumption and some tillage machinery. As a result of these agronomic and economic incentives, direct seeding practices have been adopted in many regions. Weed control is often cited as the main challenge in minimum tillage systems and no-tillage, and often leads to increased herbicide use, so we must pay special attention to this system. Otherwise, conservation tillage systems are believed to worsen weed prob‐ lems with higher weed emergence promoted by higher concentrations of seed in the surface soil and shifts of the weed community towards increased abundance of troublesome

In summary, minimum tillage, particularly no-tillage, may favor a relative emergence of weeds over crops. Moreover, the increase of prior crop residues in these systems can alter the competitive ability of crops with weeds at early stages, increasing production losses thereof [35]. Thus, it appears that common tasks tend to select annual weeds and little work allows the dominance of perennial or biennial species. However, these predictions are strongly influenced by cultural practices and environmental conditions used in a specific area. Currently, insufficient information exists about the processes associated with changes in weed communities; such information is crucial in managing weeds. As a means of control it is necessary to assess the presence of weeds, setting thresholds for treatment of major species in crops and the adequate product selection, dose and time of applica‐ tion best suited among those authorized, while taking into account the environmental

Development of improved weed management systems requires more knowledge on how weed species respond to changing agronomic practices. In order to monitor weed development subjected to different agronomic practices, one experiment was conducted to determine weed population response to various tillage intensities in a cereal agro system in central Spain (Figure 3A). Field trials under a cold semi-arid environment were conducted in successive growing seasons from 1995 to 2011, to assess the effects of management practices on the weed community with three tillage systems: (1) conventional tillage (CT); (2) minimum tillage (MT) and (3) no-tillage system (NT). The experiment consisted of a field divided in four randomized complete blocks with three different tillage systems and four replications. To study the effectiveness of different managements, we performed a first identification of the flora present

**Figure 3.** A) Field trials in the experimental farm "La Canaleja", and B) Weeds sampling scheme realized it in each till‐ age system.

The natural community of weeds present in the assay is comprised by dicotyledonous weed species and grasses, annual and perennial species typical of crop fields in the area (Table 3). Later, during all years of the study, weeds were counted by species with a similar methodology based on the selection of four random samples in the field with a quadrant of 0,125m2 , taken in zigzag on the diagonal of a rectangle defined in each sub-plot (Figure 3B), which were identified and quantified in situ, the weed species present. Total density of weeds referred to the unit area (1m2 ).


**Table 3.** Initial weed community in the farm "La Canaleja".

The herbicides employed in the trials were post emergence against dicotyledonous weeds from 1994 to 2000; against dicotyledonous and grasses from 2004 to 2009; in 2009 we did not employ any herbicide and afterward, we used post emergence herbicide against dicotyledonous weeds. Also, in the NT system, the crops were seeded each year after an application of glyphosate at 2 l.ha-1. Within the time frame of this research, weed density and species composition were affected by year, which differs in environmental conditions, and by tillage intensity, indicating fluctuations in changes of weed community composition associated with changes in agronomic practices and environmental conditions are complex and difficult to predict, especially in semiarid regions with low and / or irregular rainfall.

Some species display greater capacity of infestation when the intensity of tillage is reduced [41-44].These species shifts generally resulted in the emergence of species tolerant to existing weed management practices [45, 46]. In this sense, Froud-Williams [47] also predicted that annual and perennial grasses, perennial dicotyledonous species, wind-disseminated species, and volunteer crops would increase and annual dicotyledonous weeds would decrease in association with MT systems; although, these predictions were strongly influenced by the agronomic practices employed within a specific study; and Liebman & Davis [48] suggested a possible solution for weed problems would be the combination of different soil tillage systems. Nevertheless, other authors have suggested that tillage did not produce any selective

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In this context, it is very important to identify which are the most troublesome weeds, because they are the most difficult to control. Also, we should follow those species maintained in the seed bank of soil without an initial risk because they present low density, but one change in the crop system and /or the environmental conditions can favor their propagation and convert them into a dominant species of the field. The specific objectives of the work reported here were to determine if decreasing tillage is accompanied by a predicted increase in the presence of annual and perennial grasses, perennial dicot species, wind disseminated species, and

In order to realize the following of several weed species along the year's object of study, we determined the relative weed density in the field each five years for representative species (Figure 5). In general, years with high rainfall in fall, 1995 and 2005, favoured later-emergence weeds and perennial species to escape suppression by the crops. Many weeds had patterns of emergence that peak in October and November such as *Fumaria officinalis* L.; *Lolium rigidum* Gaudin and *Hypecoum procumbens* L., as well as the perennial weed *Cardaria draba* L. Desraux, which increased the year where higher than average rainfall was received in fall. However, years with high rainfall occurring in April and May, 2000 and 2010, they favoured earlyemergence weeds such as Papaver spp. At the same time, in our experiment, we noted a reduction of dicotyledonous weeds *Cardaria draba* L. and *Fumaria officinalis* L., and the increase of Papaver spp., *Lamium amplexicaule* L. and Veronica spp. in sub-plots with NT system. Also, we could observe a clear tendency of increasing of *Lolium rigidum* L. and *Hypecoum procum‐ bens* L. density in MT sub-plots and another perennial species such as Cirsium spp. and Convolvulus spp., which we typically found in field margins, appeared frequently within NT

The decrease in soil water evaporation due to the residual cover in both NT and MT could have increased the soil water content compared with CT, and this could be one of the reasons for the increase in the density of weeds within these systems [50]. Also, the annual distribution of rainfall may limit the effectiveness of the system used to control weeds, predisposing the specialization of some species under certain crop conditions. Generally, no-till systems can be difficult to maintain over a long period of time without adequate weed management, and knowledge of the emergence process of weeds will increase the effectiveness of a postemergence herbicide, assuming an important qualitative advance in the integrated control of

effect in the composition of weed flora [49].

sub-plots.

weed populations.

volunteer crops, but a decrease in annual dicotyledonous weeds.

Specific research regarding the impact of crop production systems on weed communities is lacking and currently, there is not a common position among authors about which system produces the best weed control. Several researchers have described the effect of the tillage system on weed flora composition and valued the long term dependence on the crop system used and their studies showed changes in weed species composition as a consequence of tillage practices [36]. According with this idea, we observed that the community of weeds present in a field differs with the tillage system employed (Figure 4). Minimum tillage systems (MT) and no-tillage (NT) showed higher weed densities compared to conventional tillage (CT).

**Figure 4.** Percentage of total weeds observed in each tillage system studied from 1995 to 2011.

Other researchers have described the predominant weeds of different tillage systems, such as Lolium spp. in minimum tillage system [37]; Poligonum spp. in conventional tillage [38, 39], or *Fumaria officinalis* L. and *Lamium amplexicaule* L., also favored in conventional tillage [40].

Some species display greater capacity of infestation when the intensity of tillage is reduced [41-44].These species shifts generally resulted in the emergence of species tolerant to existing weed management practices [45, 46]. In this sense, Froud-Williams [47] also predicted that annual and perennial grasses, perennial dicotyledonous species, wind-disseminated species, and volunteer crops would increase and annual dicotyledonous weeds would decrease in association with MT systems; although, these predictions were strongly influenced by the agronomic practices employed within a specific study; and Liebman & Davis [48] suggested a possible solution for weed problems would be the combination of different soil tillage systems. Nevertheless, other authors have suggested that tillage did not produce any selective effect in the composition of weed flora [49].

The herbicides employed in the trials were post emergence against dicotyledonous weeds from 1994 to 2000; against dicotyledonous and grasses from 2004 to 2009; in 2009 we did not employ any herbicide and afterward, we used post emergence herbicide against dicotyledonous weeds. Also, in the NT system, the crops were seeded each year after an application of glyphosate at 2 l.ha-1. Within the time frame of this research, weed density and species composition were affected by year, which differs in environmental conditions, and by tillage intensity, indicating fluctuations in changes of weed community composition associated with changes in agronomic practices and environmental conditions are complex and difficult to

Specific research regarding the impact of crop production systems on weed communities is lacking and currently, there is not a common position among authors about which system produces the best weed control. Several researchers have described the effect of the tillage system on weed flora composition and valued the long term dependence on the crop system used and their studies showed changes in weed species composition as a consequence of tillage practices [36]. According with this idea, we observed that the community of weeds present in a field differs with the tillage system employed (Figure 4). Minimum tillage systems (MT) and

no-tillage (NT) showed higher weed densities compared to conventional tillage (CT).

**Figure 4.** Percentage of total weeds observed in each tillage system studied from 1995 to 2011.

Other researchers have described the predominant weeds of different tillage systems, such as Lolium spp. in minimum tillage system [37]; Poligonum spp. in conventional tillage [38, 39], or *Fumaria officinalis* L. and *Lamium amplexicaule* L., also favored in conventional tillage [40].

predict, especially in semiarid regions with low and / or irregular rainfall.

146 Herbicides - Current Research and Case Studies in Use

In this context, it is very important to identify which are the most troublesome weeds, because they are the most difficult to control. Also, we should follow those species maintained in the seed bank of soil without an initial risk because they present low density, but one change in the crop system and /or the environmental conditions can favor their propagation and convert them into a dominant species of the field. The specific objectives of the work reported here were to determine if decreasing tillage is accompanied by a predicted increase in the presence of annual and perennial grasses, perennial dicot species, wind disseminated species, and volunteer crops, but a decrease in annual dicotyledonous weeds.

In order to realize the following of several weed species along the year's object of study, we determined the relative weed density in the field each five years for representative species (Figure 5). In general, years with high rainfall in fall, 1995 and 2005, favoured later-emergence weeds and perennial species to escape suppression by the crops. Many weeds had patterns of emergence that peak in October and November such as *Fumaria officinalis* L.; *Lolium rigidum* Gaudin and *Hypecoum procumbens* L., as well as the perennial weed *Cardaria draba* L. Desraux, which increased the year where higher than average rainfall was received in fall. However, years with high rainfall occurring in April and May, 2000 and 2010, they favoured earlyemergence weeds such as Papaver spp. At the same time, in our experiment, we noted a reduction of dicotyledonous weeds *Cardaria draba* L. and *Fumaria officinalis* L., and the increase of Papaver spp., *Lamium amplexicaule* L. and Veronica spp. in sub-plots with NT system. Also, we could observe a clear tendency of increasing of *Lolium rigidum* L. and *Hypecoum procum‐ bens* L. density in MT sub-plots and another perennial species such as Cirsium spp. and Convolvulus spp., which we typically found in field margins, appeared frequently within NT sub-plots.

The decrease in soil water evaporation due to the residual cover in both NT and MT could have increased the soil water content compared with CT, and this could be one of the reasons for the increase in the density of weeds within these systems [50]. Also, the annual distribution of rainfall may limit the effectiveness of the system used to control weeds, predisposing the specialization of some species under certain crop conditions. Generally, no-till systems can be difficult to maintain over a long period of time without adequate weed management, and knowledge of the emergence process of weeds will increase the effectiveness of a postemergence herbicide, assuming an important qualitative advance in the integrated control of weed populations.

**Author details**

**References**

19 January (2012).

(2002). , 42, 417-428.

Inés Santín-Montanyá, Encarnación Zambrana-Quesada and José Luis Tenorio-Pasamón

Weed Management in Cereals in Semi-Arid Environments: A Review

http://dx.doi.org/10.5772/55970

149

[1] MARM (Ministerio de Agricultura. Alimentación y Medioambiente). Anuario de Es‐ tadística Agroalimentaria. http://www.marm.es/es/agricultura/estadisticasaccessed

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Current Contens/Agriculture Biology & Environmental Sciences (1984).

viability of weed seed in soil. Weed Science (1992). , 40(3), 429-433.

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bicide Resistance in Plants. John Wiley & Sons, New York. 1982. , 57-79.

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain

**Figure 5.** Relative density of weed species more representative in the field object of study.

### **3. Conclusion**

At the moment, sustainable agriculture is being promoted in Europe, and its industrialization using technologies that help to increase crop production should be designed in order to protect the environment. In this context, the increasing awareness of the farmers requires the adoption and adaptation of techniques that, without undermining the economic benefit of farms, could be also accepted by the environment.

Sometimes we ignore the ecological processes that occur in agro systems, and weed control problems associated with herbicide selectivity and changes occurring in weed communities within MT and NT systems have been reported by numerous authors. In this sense, changes in agricultural technologies, such as the employment of selective herbicides, require reevalu‐ ation of assumptions regarding the nature of weed communities in MT and NT systems and the information on the association of weeds species with tillage systems and herbicides are key in determining directions of future research in weed management.

### **Acknowledgements**

We appreciate the funds received from different Ministries for the realisation of this long-term experiment. This work has been funded by projects: INIA SC94-005-C2-2; SC94-003-C3-2; SC98-020-C4-2; MCYT-INIA RTA-02-058-C3-2; MEC-INIA RTA2006-00121-C03-02 and MICINN-INIA RTA2010-0006-C03-02.

We are grateful to all members of the experimental farm "La Canaleja" for helping managing the experiment.

### **Author details**

Inés Santín-Montanyá, Encarnación Zambrana-Quesada and José Luis Tenorio-Pasamón

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain

### **References**

**3. Conclusion**

be also accepted by the environment.

148 Herbicides - Current Research and Case Studies in Use

**Acknowledgements**

the experiment.

MICINN-INIA RTA2010-0006-C03-02.

At the moment, sustainable agriculture is being promoted in Europe, and its industrialization using technologies that help to increase crop production should be designed in order to protect the environment. In this context, the increasing awareness of the farmers requires the adoption and adaptation of techniques that, without undermining the economic benefit of farms, could

Sometimes we ignore the ecological processes that occur in agro systems, and weed control problems associated with herbicide selectivity and changes occurring in weed communities within MT and NT systems have been reported by numerous authors. In this sense, changes in agricultural technologies, such as the employment of selective herbicides, require reevalu‐ ation of assumptions regarding the nature of weed communities in MT and NT systems and the information on the association of weeds species with tillage systems and herbicides are

We appreciate the funds received from different Ministries for the realisation of this long-term experiment. This work has been funded by projects: INIA SC94-005-C2-2; SC94-003-C3-2; SC98-020-C4-2; MCYT-INIA RTA-02-058-C3-2; MEC-INIA RTA2006-00121-C03-02 and

We are grateful to all members of the experimental farm "La Canaleja" for helping managing

key in determining directions of future research in weed management.

**Figure 5.** Relative density of weed species more representative in the field object of study.


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**Chapter 7**

**The Use of Glyphosate in Sugarcane: A Brazilian**

In Brazil, sugarcane (*Saccharum* spp.) fields are renewed in intervals of five to six profitable crops on average. With each harvest, sugarcane displays a decrease in productivity due to diverse factors. Genetic, phytosanitary and edaphoclimatic issues are the main factors contributing to the degeneration that necessitates the renewal of sugarcane fields with more productive cultivars. After the last economical harvest, the ratoon crop is destroyed using mechanical or chemical processes or a combination of both. Chemical destruction is more practical and causes less impact on soil structure and quality due to less soil disturbance. Glyphosate is the most widely used non-selective herbicide in the chemical eradication of ratoon crops because there is a broad spectrum of plants susceptible to glyphosate. Glypho‐ sate's mechanism of action is through inhibition of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a precursor of the aromatic amino acids phenylalanine, tyrosine and tryptophan, which are essential for protein synthesis. The recommended dosage of glyphosate for the eradication of plants is 1440 to 2880 g acid equivalents (a. e.) ha-1 [1,2]. However, sugarcane cultivars present varying degrees of susceptibility and require different amounts of

In Brazil, sugarcane cultivars commercially released by genetic improvement programmes are not characterised in terms of their susceptibility to glyphosate. Nevertheless, knowledge of the degree of cultivar tolerance to glyphosate can generate savings for producers and benefit to the environment through the reduction of the quantity of applied herbicide. Literature studies of cultivar responses to herbicides, especially glyphosate, are supported solely by phytotech‐

> © 2013 Azania et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Azania et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Experience**

Carlos Alberto Mathias Azania,

http://dx.doi.org/10.5772/54958

**1. Introduction**

Luciana Rossini Pinto, Rodrigo Cabral Adriano, Dilermando Perecin and Andréa Padua Azania

Additional information is available at the end of the chapter

herbicide for the complete death of the plant.


## **The Use of Glyphosate in Sugarcane: A Brazilian Experience**

Carlos Alberto Mathias Azania, Luciana Rossini Pinto, Rodrigo Cabral Adriano, Dilermando Perecin and Andréa Padua Azania

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54958

### **1. Introduction**

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[45] Wrucke, M. A, & Arnold, W. E. Weed species distributions as influenced by tillage

[46] Radosevich, S. R, & Holt, J. S. Implications for vegetation management. John Wiley &

[47] Froud-williams, R. J, Chancellor, R. J, & Drennan, D. S. H. Potential changes in weed flora associated with reduce-tillage cultivation systems for cereal production in tem‐

[48] Liebman, M, & Davis, A. S. Integration of soil, crop and weed management in low-

[49] Roberts, H. A. Emergence and longevity in cultivated soil of seeds of some annual

[50] Santín-montanyá, I. Tenorio Pasamón, J.L. & García Baudín, J.M. Changes in Weed Community as affected by Tillage Systems in a Semi-Arid Environment. Italian Jour‐

external input farming systems. Weed Research (2000). , 40, 27-47.

Workshop S.E.E.P. Biodiversidad en pastos (2001). , 563-568.

Sons, New York (eds.) In: Weed ecology (1984). , 198-203.

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nal of Agronomy (2008).

18-26.

152 Herbicides - Current Research and Case Studies in Use

73-89.

42, 205-209.

33, 176-181.

In Brazil, sugarcane (*Saccharum* spp.) fields are renewed in intervals of five to six profitable crops on average. With each harvest, sugarcane displays a decrease in productivity due to diverse factors. Genetic, phytosanitary and edaphoclimatic issues are the main factors contributing to the degeneration that necessitates the renewal of sugarcane fields with more productive cultivars. After the last economical harvest, the ratoon crop is destroyed using mechanical or chemical processes or a combination of both. Chemical destruction is more practical and causes less impact on soil structure and quality due to less soil disturbance. Glyphosate is the most widely used non-selective herbicide in the chemical eradication of ratoon crops because there is a broad spectrum of plants susceptible to glyphosate. Glypho‐ sate's mechanism of action is through inhibition of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a precursor of the aromatic amino acids phenylalanine, tyrosine and tryptophan, which are essential for protein synthesis. The recommended dosage of glyphosate for the eradication of plants is 1440 to 2880 g acid equivalents (a. e.) ha-1 [1,2]. However, sugarcane cultivars present varying degrees of susceptibility and require different amounts of herbicide for the complete death of the plant.

In Brazil, sugarcane cultivars commercially released by genetic improvement programmes are not characterised in terms of their susceptibility to glyphosate. Nevertheless, knowledge of the degree of cultivar tolerance to glyphosate can generate savings for producers and benefit to the environment through the reduction of the quantity of applied herbicide. Literature studies of cultivar responses to herbicides, especially glyphosate, are supported solely by phytotech‐

© 2013 Azania et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Azania et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

nical observations, such as plant height, girth and mass gain. However, these characteristics are greatly affected by the environment and require longer periods of evaluation and data collection, such as the 12-month studies of [3] and [4].

growth period and a dry period during maturation, which favours increased sucrose accu‐

The Use of Glyphosate in Sugarcane: A Brazilian Experience

http://dx.doi.org/10.5772/54958

155

In Brazilian regions where it is traditional to grow sugarcane, planting may occur at different times of the year, as long as the producer possesses an irrigation system and cultivars that are adapted to each season [14]. Traditionally, in south-central Brazil, there are two cycles for planting: "cane of the year" and "year and a half". In "cane of the year", planting is performed between September and November, and the cane is harvested after 12 months [14]. This type of plantation addresses the demand for raw materials in the spring cycle (at the end of the harvest). In "year and a half" cane, planting is performed between January and April-May. In contrast to cane of year, this cycle allows for harvest during the autumn season (the beginning of harvest). Additionally, several producing units have practiced winter planting, particularly June through July, using rescue irrigation, and these units have obtained great productivity

Currently, Brazil is the largest producer of sugarcane in the world followed by India, China and Thailand [15]. The national production is estimated as 641.982 million tons with an average productivity of approximately 76.4 t ha-1 [16].The national sugar-energy industry sector accounted for 1,283,258 jobs up to 2008 with 37.5% occupied by plant growth, 44.8% in the production and refining of sugar and 17.7% in the production of ethanol. This sector also accounted for approximately 3.85 million people that are employed indirectly [17]. The production and processing of sugarcane is currently managed by the private sector in Brazil, which achieves the lowest cost for production worldwide for both sugar and ethanol, emerging

Glyphosate was commercially released in 1974 under the trade name Roundup initially in the USA for industrial purposes, in the United Kingdom for use in wheat crops and in Malaysia for use in rubber trees. Currently, the molecule is registered in more than 130 countries for the control of over 300 species of weeds in over 100 types of crops [19], making it the most widely used herbicide [20]. Worldwide, there are numerous registered trademarks of the herbicide, which, according to [50], number more than 150. In Brazil, glyphosate is also registered for the

The molecule belongs to the glycine-derived chemical group (Group G), and its mechanism of action consists of inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway [22], which is found only in microorganisms and plants [23]. The molecular formula of glyphosate is HO2CCH2NHCH2PO(OH)2. Glyphosate has a solubility in water of 15,700 mg L-1 at 25 °C and pH 7, a density of 1.74 g mL-1, a vapour pressure of 2.45 x 10-8 Pa (45 °C), pKa values of 2.6, 5.6 and 10.3 (acid) and a kow between 0.0006 and 0.0017. In the soil, glyphosate is strongly adsorbed to colloids, and its leachability is notably low. The compound has an average Koc of 24,000 mL g-1, and its volatilisation and photodegradation are negligible [22]. The half-life of the molecule in the environment depends on the surrounding

mulation [13].

compared to "year and a half" cane planting.

as a highly competitive segment in international markets [18].

**3. Characteristics of the herbicide glyphosate**

eradication of sugarcane ratoon crops [21].

The use of isoenzymatic markers allows for the prompt analysis of results with larger sample numbers while using a relatively simple and inexpensive technique that can substitute or reduce field experimentation. However, the choice of the correct enzymes to analyse is critical to the success of this technique and obtaining robust results.

### **2. Sugarcane crops**

Sugarcane probably originated in New Guinea, and from there, it was introduced to India, where the oldest evidence of its existence has been recorded [5]. Officially, Martins Afonso de Souza brought the first sugarcane plant to Brazil in 1532 and started its cultivation in the Captaincy of São Vicente (Capitania de São Vicente*)*. This transfer was the beginning of an industry that found in Brazil, among other nations that would later initiate production, its most fertile ground for rapid expansion and perpetuation for an almost uninterrupted 500 years. Starting in the 1970s, sugarcane farming became increasingly important for Brazil as the agro-industrial sector was tapped to contribute to a solution to the emerging energy crisis because of the potential for energy production from sugarcane as a renewable source [6]. Growth in the sugarcane-ethanol sector is important for the Brazilian economy in that the sector's growth entails both the creation of jobs and of 100% national renewable energy.

According to taxonomic classification, sugarcane belongs to the *Poaceae* family and the *Saccharum* genus. Sugarcane is a semi-perennial plant requiring a tropical or sub-tropical climate [7]. With a C4 metabolism, sugarcane is classified as having among the highest rates of photosynthetic efficiency and a high efficiency for water usage [8]. The sugarcane plant is divided into aerial (culm, leaves and inflorescences) and underground parts (roots and rhizomes). The culms are cylindrical and are composed of nodes and internodes; these parts are defined as the aboveground portion that supports the leaves and inflorescences [9]. According to [10], each node has one alternating bud and a root system. Inflorescences are panicles with a hermaphrodite flower containing one ovule; the pistils terminate in purple or reddish stigmae that characterise the flower's plumose panicle [9]. The root system is fascicu‐ lated and serves to support, as well as to absorb and transport water and nutrients [8]. Sugarcane tillering influences the sugarcane handling system because each tiller behaves as an independent plant with individual organs, such as roots, leaves and fruits [11].

The most appropriate agricultural conditions for sugarcane propagation are found between the 30° north and 30° south latitudes, which are characteristic of tropical and subtropical regions. Outside of these latitudes, lower temperatures limit the growth and development of the plant [12]. According to [10], the optimal temperature range for the growth of this crop is between 20 and 35 °C with an ideal photoperiod of 10 to 14 hours [12] and an annual rainfall ranging between 1,000 and 1,600 mm, preferentially with abundant rain during the vegetative growth period and a dry period during maturation, which favours increased sucrose accu‐ mulation [13].

In Brazilian regions where it is traditional to grow sugarcane, planting may occur at different times of the year, as long as the producer possesses an irrigation system and cultivars that are adapted to each season [14]. Traditionally, in south-central Brazil, there are two cycles for planting: "cane of the year" and "year and a half". In "cane of the year", planting is performed between September and November, and the cane is harvested after 12 months [14]. This type of plantation addresses the demand for raw materials in the spring cycle (at the end of the harvest). In "year and a half" cane, planting is performed between January and April-May. In contrast to cane of year, this cycle allows for harvest during the autumn season (the beginning of harvest). Additionally, several producing units have practiced winter planting, particularly June through July, using rescue irrigation, and these units have obtained great productivity compared to "year and a half" cane planting.

Currently, Brazil is the largest producer of sugarcane in the world followed by India, China and Thailand [15]. The national production is estimated as 641.982 million tons with an average productivity of approximately 76.4 t ha-1 [16].The national sugar-energy industry sector accounted for 1,283,258 jobs up to 2008 with 37.5% occupied by plant growth, 44.8% in the production and refining of sugar and 17.7% in the production of ethanol. This sector also accounted for approximately 3.85 million people that are employed indirectly [17]. The production and processing of sugarcane is currently managed by the private sector in Brazil, which achieves the lowest cost for production worldwide for both sugar and ethanol, emerging as a highly competitive segment in international markets [18].

### **3. Characteristics of the herbicide glyphosate**

nical observations, such as plant height, girth and mass gain. However, these characteristics are greatly affected by the environment and require longer periods of evaluation and data

The use of isoenzymatic markers allows for the prompt analysis of results with larger sample numbers while using a relatively simple and inexpensive technique that can substitute or reduce field experimentation. However, the choice of the correct enzymes to analyse is critical

Sugarcane probably originated in New Guinea, and from there, it was introduced to India, where the oldest evidence of its existence has been recorded [5]. Officially, Martins Afonso de Souza brought the first sugarcane plant to Brazil in 1532 and started its cultivation in the Captaincy of São Vicente (Capitania de São Vicente*)*. This transfer was the beginning of an industry that found in Brazil, among other nations that would later initiate production, its most fertile ground for rapid expansion and perpetuation for an almost uninterrupted 500 years. Starting in the 1970s, sugarcane farming became increasingly important for Brazil as the agro-industrial sector was tapped to contribute to a solution to the emerging energy crisis because of the potential for energy production from sugarcane as a renewable source [6]. Growth in the sugarcane-ethanol sector is important for the Brazilian economy in that the sector's growth entails both the creation of jobs and of 100% national renewable energy.

According to taxonomic classification, sugarcane belongs to the *Poaceae* family and the *Saccharum* genus. Sugarcane is a semi-perennial plant requiring a tropical or sub-tropical climate [7]. With a C4 metabolism, sugarcane is classified as having among the highest rates of photosynthetic efficiency and a high efficiency for water usage [8]. The sugarcane plant is divided into aerial (culm, leaves and inflorescences) and underground parts (roots and rhizomes). The culms are cylindrical and are composed of nodes and internodes; these parts are defined as the aboveground portion that supports the leaves and inflorescences [9]. According to [10], each node has one alternating bud and a root system. Inflorescences are panicles with a hermaphrodite flower containing one ovule; the pistils terminate in purple or reddish stigmae that characterise the flower's plumose panicle [9]. The root system is fascicu‐ lated and serves to support, as well as to absorb and transport water and nutrients [8]. Sugarcane tillering influences the sugarcane handling system because each tiller behaves as

an independent plant with individual organs, such as roots, leaves and fruits [11].

The most appropriate agricultural conditions for sugarcane propagation are found between the 30° north and 30° south latitudes, which are characteristic of tropical and subtropical regions. Outside of these latitudes, lower temperatures limit the growth and development of the plant [12]. According to [10], the optimal temperature range for the growth of this crop is between 20 and 35 °C with an ideal photoperiod of 10 to 14 hours [12] and an annual rainfall ranging between 1,000 and 1,600 mm, preferentially with abundant rain during the vegetative

collection, such as the 12-month studies of [3] and [4].

154 Herbicides - Current Research and Case Studies in Use

**2. Sugarcane crops**

to the success of this technique and obtaining robust results.

Glyphosate was commercially released in 1974 under the trade name Roundup initially in the USA for industrial purposes, in the United Kingdom for use in wheat crops and in Malaysia for use in rubber trees. Currently, the molecule is registered in more than 130 countries for the control of over 300 species of weeds in over 100 types of crops [19], making it the most widely used herbicide [20]. Worldwide, there are numerous registered trademarks of the herbicide, which, according to [50], number more than 150. In Brazil, glyphosate is also registered for the eradication of sugarcane ratoon crops [21].

The molecule belongs to the glycine-derived chemical group (Group G), and its mechanism of action consists of inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway [22], which is found only in microorganisms and plants [23]. The molecular formula of glyphosate is HO2CCH2NHCH2PO(OH)2. Glyphosate has a solubility in water of 15,700 mg L-1 at 25 °C and pH 7, a density of 1.74 g mL-1, a vapour pressure of 2.45 x 10-8 Pa (45 °C), pKa values of 2.6, 5.6 and 10.3 (acid) and a kow between 0.0006 and 0.0017. In the soil, glyphosate is strongly adsorbed to colloids, and its leachability is notably low. The compound has an average Koc of 24,000 mL g-1, and its volatilisation and photodegradation are negligible [22]. The half-life of the molecule in the environment depends on the surrounding soil texture and microbial activity and may vary from a few days to several years [24]. In roots, the absorption is slow due to the low diffusion and high adsorption to the soil, which also favours microbial action in the transformation of the molecule into its main metabolite, aminomethylphosphonic acid [25; 26]. Glyphosate is absorbed through leaf cuticles, and its translocation occurs mainly via the cellular symplast to the leaves and apical meristem, as well as to underground organs [22]. According to [27], glyphosate absorption depends on such factors as the age of the plant, environmental conditions, surfactants and herbicide concen‐ tration in the soil milieu.

requiring the renewal of the field. In the state of São Paulo, the average productivity of sugarcane fields is approximately 80 to 85 t ha-1, considering the longevity of ratoons to be between five and six cuts [40]. However, in the region of Ribeirão Preto, SP, ratoon crops after the sixth cut are no longer economically viable, and renewal of the sugarcane field is necessary [41]. Degeneration after successive years of production makes the renewal of sugarcane fields essential. The causes of degeneration are diverse and involve a combination of genetic, physiological, phytosanitary, edaphoclimatic and phytotechnical factors. The factors impact‐ ing degeneration may also be linked to characteristics of the growing environment, such as a decrease in soil fertility [42]. Another cause for degeneration can be soil compaction and consequent difficulties in root development, as proposed by [43]. The authors note that compacted soil still presents difficulties for root development, even if its humidity levels are

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[44] found that degeneration is linked to the health of the plants. Sugarcane-field longevity may also be affected by competition with weeds [45], nematode infestations [46] and uprooting

The fact that various cuts are performed from a single plantation allows for the formation of a significant number of root systems, which often make the elimination of the ratoon crop difficult, especially if the eradication is performed mechanically, which may also compromise the settlement of the next plantation. At the time of crop renewal, the ratoon crop is first eliminated through desiccating herbicides, specifically glyphosate, and after plant death,

Glyphosate is the most widely used herbicide for the chemical eradication of sugarcane ratoons due to its ease of use, low cost and absence of residual effects on the soil, which allows for repeated plantation in the same area, as is often practiced by farmers [49, 50]. Tolerance to glyphosate is highly prevalent in cultivars, and while certain cultivars are eradicated with a dose of 1080 g a. e. ha-1, others require a dose of 2520 g a. e. ha-1. According to [1,2], the minimal

The progression of symptoms caused by a glyphosate application occurs in a gradual fashion (Figure 2) until the eradication of the plants [23, 36]. The authors noted that glyphosate-induced damage develops slowly until complete death in contrast to the effects of other herbicides. According to these authors, molecular stability inside the plant allows for the occurrence of irreversible effects on processes that control both annual and perennial plants (Figures 3 and 4).

After the application of glyphosate in the eradication of sugarcane crops, there was a stunting of plant growth, with treated plants retaining the same size up to 45 days after herbicide application [51]. The negative effect on growth was evaluated by measuring the plant height, with the treated plants maintaining similar average height values throughout the evaluation period in contrast to controls, which were able to maintain vigorous vegetative growth. The growth stunting was due to the indirect influence of glyphosate on the regulators of plant growth, such as indole-3-acetic acid. This hormone is fundamental for cellular elongation, apical dominance and stem and root growth and is dependent on the shikimate pathway, being

eradication is later completed using mechanical destruction of the crop [48].

close to the soil's capacity.

of tufts during mechanical harvesting [47].

lethal dose for sugarcane is 1440 g a. e. ha-1.

inhibited when there is a disruption of EPSPS [36].

As an herbicide, glyphosate is among the less hazardous agro-toxins used in agriculture [28]. Glyphosate-based herbicides, when used according to their respective guidelines, display low toxicity and are safe to humans [30].

Often, the glyphosate molecule is not efficient in penetrating waxy cuticles. Therefore, commercial formulations contain surfactants capable of reducing surface tension in herbicide droplets, thus increasing their penetration in leaves [20]. However, these surfactants are more toxic than the glyphosate molecule [29]. For example, polyoxyethylene amine, the predomi‐ nant surfactant in Roundup® [30], has been classified as moderately to highly toxic in laboratory tests [31]. Glyphosate is a unique molecule, and although it is considered to be of low toxicity, its unrestrained use can affect the environment through direct or indirect effects on non-target organisms [32].

In plants, the EPSPS enzyme catalyses a reaction between shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to produce 5-enolpyruvylshikimate-3-phosphate and inorganic phosphate. Glyphosate binds to the catalytic site of EPSPS and to the S3P substrate to form the EPSP synthase-S3P-glyphosate complex [33]. The relevance of the shikimate pathway is such that approximately 35% of all plant mass is related to derivatives from this pathway; moreover, 20% of all of the carbon fixed during photosynthesis also travels through this metabolic pathway [34].

According [35], the inhibition of amino acids compromises the production of carotenoids and chlorophyll, thus causing irreversible cellular damage. Therefore, the translocation of the herbicide throughout the entirety of the plant causes plant death in a few days or weeks (Figure 1). The inhibition of EPSPS leads to the accumulation of high levels of shikimate in vacuoles, which is intensified by the loss of control of the carbon flow across this pathway [36]. Thus, there is an obstruction in the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan, which are essential for protein synthesis and serve as precursors for secondary metabolites that are important for plant growth [37], resulting in the slow development of symptoms [33].

### **4. Reforming sugarcane fields and the use of glyphosate**

With each harvest cut, sugarcane sprouts new tillers that develop into culms [38]. Nevertheless, ratoon-crop productivity gradually diminishes with an increasing number of cuts [39], thereby requiring the renewal of the field. In the state of São Paulo, the average productivity of sugarcane fields is approximately 80 to 85 t ha-1, considering the longevity of ratoons to be between five and six cuts [40]. However, in the region of Ribeirão Preto, SP, ratoon crops after the sixth cut are no longer economically viable, and renewal of the sugarcane field is necessary [41]. Degeneration after successive years of production makes the renewal of sugarcane fields essential. The causes of degeneration are diverse and involve a combination of genetic, physiological, phytosanitary, edaphoclimatic and phytotechnical factors. The factors impact‐ ing degeneration may also be linked to characteristics of the growing environment, such as a decrease in soil fertility [42]. Another cause for degeneration can be soil compaction and consequent difficulties in root development, as proposed by [43]. The authors note that compacted soil still presents difficulties for root development, even if its humidity levels are close to the soil's capacity.

soil texture and microbial activity and may vary from a few days to several years [24]. In roots, the absorption is slow due to the low diffusion and high adsorption to the soil, which also favours microbial action in the transformation of the molecule into its main metabolite, aminomethylphosphonic acid [25; 26]. Glyphosate is absorbed through leaf cuticles, and its translocation occurs mainly via the cellular symplast to the leaves and apical meristem, as well as to underground organs [22]. According to [27], glyphosate absorption depends on such factors as the age of the plant, environmental conditions, surfactants and herbicide concen‐

As an herbicide, glyphosate is among the less hazardous agro-toxins used in agriculture [28]. Glyphosate-based herbicides, when used according to their respective guidelines, display low

Often, the glyphosate molecule is not efficient in penetrating waxy cuticles. Therefore, commercial formulations contain surfactants capable of reducing surface tension in herbicide droplets, thus increasing their penetration in leaves [20]. However, these surfactants are more toxic than the glyphosate molecule [29]. For example, polyoxyethylene amine, the predomi‐ nant surfactant in Roundup® [30], has been classified as moderately to highly toxic in laboratory tests [31]. Glyphosate is a unique molecule, and although it is considered to be of low toxicity, its unrestrained use can affect the environment through direct or indirect effects

In plants, the EPSPS enzyme catalyses a reaction between shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to produce 5-enolpyruvylshikimate-3-phosphate and inorganic phosphate. Glyphosate binds to the catalytic site of EPSPS and to the S3P substrate to form the EPSP synthase-S3P-glyphosate complex [33]. The relevance of the shikimate pathway is such that approximately 35% of all plant mass is related to derivatives from this pathway; moreover, 20% of all of the carbon fixed during photosynthesis also travels through this metabolic

According [35], the inhibition of amino acids compromises the production of carotenoids and chlorophyll, thus causing irreversible cellular damage. Therefore, the translocation of the herbicide throughout the entirety of the plant causes plant death in a few days or weeks (Figure 1). The inhibition of EPSPS leads to the accumulation of high levels of shikimate in vacuoles, which is intensified by the loss of control of the carbon flow across this pathway [36]. Thus, there is an obstruction in the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan, which are essential for protein synthesis and serve as precursors for secondary metabolites that are important for plant growth [37], resulting in the slow development of

With each harvest cut, sugarcane sprouts new tillers that develop into culms [38]. Nevertheless, ratoon-crop productivity gradually diminishes with an increasing number of cuts [39], thereby

**4. Reforming sugarcane fields and the use of glyphosate**

tration in the soil milieu.

toxicity and are safe to humans [30].

156 Herbicides - Current Research and Case Studies in Use

on non-target organisms [32].

pathway [34].

symptoms [33].

[44] found that degeneration is linked to the health of the plants. Sugarcane-field longevity may also be affected by competition with weeds [45], nematode infestations [46] and uprooting of tufts during mechanical harvesting [47].

The fact that various cuts are performed from a single plantation allows for the formation of a significant number of root systems, which often make the elimination of the ratoon crop difficult, especially if the eradication is performed mechanically, which may also compromise the settlement of the next plantation. At the time of crop renewal, the ratoon crop is first eliminated through desiccating herbicides, specifically glyphosate, and after plant death, eradication is later completed using mechanical destruction of the crop [48].

Glyphosate is the most widely used herbicide for the chemical eradication of sugarcane ratoons due to its ease of use, low cost and absence of residual effects on the soil, which allows for repeated plantation in the same area, as is often practiced by farmers [49, 50]. Tolerance to glyphosate is highly prevalent in cultivars, and while certain cultivars are eradicated with a dose of 1080 g a. e. ha-1, others require a dose of 2520 g a. e. ha-1. According to [1,2], the minimal lethal dose for sugarcane is 1440 g a. e. ha-1.

The progression of symptoms caused by a glyphosate application occurs in a gradual fashion (Figure 2) until the eradication of the plants [23, 36]. The authors noted that glyphosate-induced damage develops slowly until complete death in contrast to the effects of other herbicides. According to these authors, molecular stability inside the plant allows for the occurrence of irreversible effects on processes that control both annual and perennial plants (Figures 3 and 4).

After the application of glyphosate in the eradication of sugarcane crops, there was a stunting of plant growth, with treated plants retaining the same size up to 45 days after herbicide application [51]. The negative effect on growth was evaluated by measuring the plant height, with the treated plants maintaining similar average height values throughout the evaluation period in contrast to controls, which were able to maintain vigorous vegetative growth. The growth stunting was due to the indirect influence of glyphosate on the regulators of plant growth, such as indole-3-acetic acid. This hormone is fundamental for cellular elongation, apical dominance and stem and root growth and is dependent on the shikimate pathway, being inhibited when there is a disruption of EPSPS [36].

**Figure 2.** Sugarcane plants ten days after the application of glyphosate (2880 g a.e. ha-1).

The varying tolerance of sugarcane cultivars to glyphosate was studied [52], who found different sensitivities among cultivars. The authors also classified the genotypes IAC86-2210, IAC83-1313, IAC82-2045, PO83-698 and IAC83-4157 as susceptible to glyphosate, IAC86-3154, IAC87-3184, RB72454 and SP80-1842 as of intermediate susceptibility, and IAC82-3092, IAC87-3396 and RB806043 as tolerant. Nevertheless, complete death, even in the less suscep‐ tible cultivars, occurred after 45 days following application. A plant's inherent tolerance is related to the plant's capability for absorption, translocation, metabolism and/or elimination of a herbicide [53]. In [54] also noted that differences in absorption depend primarily on morpho-anatomical characteristics of the species and that in the aerial parts of the plant, absorption is highly influenced by the presence or absence of cuticles. The physicochemical content of the leaf surface is another form of plant resistance to glyphosate [55]. According to these authors, leaves with flat cuticle surfaces and without large quantities of wax can better retain applied droplets. After penetration, the herbicide can then be metabolised into secon‐

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dary compounds without herbicidal activity, or its potency might be enhanced [56].

tolerant to the herbicide [58].

The plant's development stage is another factor that should be considered in the eradication of cultivars because plants must be 40 to 80 cm tall at the time of glyphosate application [2], and the total leaf area must be sufficient to intercept the herbicide. The inherent resistance to glyphosate is greater in taller plants [57]. After the formation of culms, plants become more

A relationship between plant size and glyphosate efficacy was also observed [59] while studying *Conyza bonariensis*. The authors observed that herbicidal efficacy was greater when

**Figure 1.** The shikimate pathway and the action of glyphosate on plants.

**Figure 2.** Sugarcane plants ten days after the application of glyphosate (2880 g a.e. ha-1).

The varying tolerance of sugarcane cultivars to glyphosate was studied [52], who found different sensitivities among cultivars. The authors also classified the genotypes IAC86-2210, IAC83-1313, IAC82-2045, PO83-698 and IAC83-4157 as susceptible to glyphosate, IAC86-3154, IAC87-3184, RB72454 and SP80-1842 as of intermediate susceptibility, and IAC82-3092, IAC87-3396 and RB806043 as tolerant. Nevertheless, complete death, even in the less suscep‐ tible cultivars, occurred after 45 days following application. A plant's inherent tolerance is related to the plant's capability for absorption, translocation, metabolism and/or elimination of a herbicide [53]. In [54] also noted that differences in absorption depend primarily on morpho-anatomical characteristics of the species and that in the aerial parts of the plant, absorption is highly influenced by the presence or absence of cuticles. The physicochemical content of the leaf surface is another form of plant resistance to glyphosate [55]. According to these authors, leaves with flat cuticle surfaces and without large quantities of wax can better retain applied droplets. After penetration, the herbicide can then be metabolised into secon‐ dary compounds without herbicidal activity, or its potency might be enhanced [56].

The plant's development stage is another factor that should be considered in the eradication of cultivars because plants must be 40 to 80 cm tall at the time of glyphosate application [2], and the total leaf area must be sufficient to intercept the herbicide. The inherent resistance to glyphosate is greater in taller plants [57]. After the formation of culms, plants become more tolerant to the herbicide [58].

A relationship between plant size and glyphosate efficacy was also observed [59] while studying *Conyza bonariensis*. The authors observed that herbicidal efficacy was greater when

**Figure 1.** The shikimate pathway and the action of glyphosate on plants.

158 Herbicides - Current Research and Case Studies in Use

the plants presented up to two pairs of leaves. Nevertheless, in more advanced stages of development, it was necessary to increase herbicide dosage by up to fivefold.

Glyphosate applied 40 days after the last harvest caused the highest percentage of dead tillers, and also, genotypes IAC87-3184, RB835489 and SP87-344 displayed high to intermediate sensitivity, while IAC91-5155 was considered tolerant to the herbicide [49]. The most effective application time for eradication was 65 days after cane harvesting [60]. The same authors reported that a dose of 960 g a. e. ha-1 eradicated the majority of cultivars, except for Co997, which needed 1920 g a. e. ha-1 of the herbicide.

The aggressive biological nature of hard to control weed species requires that handling start with the desiccation of the plants to optimise the use of glyphosate in the eradication of ratoon while also being able to introduce residual herbicides at higher doses. In these instances, the use of glyphosate in crop eradication serves the dual role desiccating the ratoons and control‐

**Figure 4.** Intoxication symptoms caused by glyphosate rates in sugarcane cultivars (IACSP94-2094-4004 and

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IACSP94-2191) to 45 days after application. Instituto Agronômico de Campinas - IAC, 2012.

In the time period following eradication but before planting sugarcane, the producer should formulate a strategy and opt for techniques that ensure higher sustainability of the system. These methods include such techniques as crop rotation or planting green-manure crops, although these may still compromise the techniques' sustainability if installed in fields that have been infested with "difficult-to-control weeds" or previously treated with residual herbicides for "disinfestation". The producer should carefully plan to use techniques that generate the most effective soil preparation and handling of weeds while simultaneously ensuring that after the treatments, the soil remains prepared for a new sugarcane plantation.

The shikimate pathway is found only in plants and microorganisms and is completely absent in mammals, birds, reptiles, fish and insects. These organisms extract the aromatic compounds necessary for survival and reproduction from their diet, while plants must produce such compounds because they do not have alternative means to obtain the compounds [23].The shikimate pathway is initiated with the reaction of PEP and erythrose 4-phosphate, a reaction

**5. The shikimate pathway and isoenzymatic markers**

ling problematic weeds.

**Figure 3.** Intoxication symptoms caused by glyphosate rates in sugarcane cultivars (IACSP94-2094-4004 and IACSP94-4004) to 27 days after application. Instituto Agronômico de Campinas -IAC, 2012.

The development of transgenic sugarcane plants, particularly those with tolerance to glyph‐ osate, will most certainly change the way in which sugarcane is eradicated. The use of glyphosate will reduce the costs associated with the control of weeds across cycles; however, during the period of cane eradication, the herbicide will now have a limited impact due to the tolerance introduced to the cultivars. In this case, eradication may have to be performed mechanically, which will have a negative impact on soil conservation and might stimulate weed germination from soil propagule banks. From this perspective, it is important to emphasise that research aimed at sugarcane plants tolerant to glyphosate should also consider the use of herbicides in the eradication of future cultivars.

In the eradication of ratoon crops, glyphosate is used to eradicate the crop and also to control emerged weeds. However, when a sugarcane field also possess weeds that are hard to control, such as *Cynodon dactylon* and *Cyperus rotundus*, the use of higher doses of residual herbicides after the application of glyphosate is adopted in a process known as "disinfestation".

the plants presented up to two pairs of leaves. Nevertheless, in more advanced stages of

Glyphosate applied 40 days after the last harvest caused the highest percentage of dead tillers, and also, genotypes IAC87-3184, RB835489 and SP87-344 displayed high to intermediate sensitivity, while IAC91-5155 was considered tolerant to the herbicide [49]. The most effective application time for eradication was 65 days after cane harvesting [60]. The same authors reported that a dose of 960 g a. e. ha-1 eradicated the majority of cultivars, except for Co997,

**Figure 3.** Intoxication symptoms caused by glyphosate rates in sugarcane cultivars (IACSP94-2094-4004 and

The development of transgenic sugarcane plants, particularly those with tolerance to glyph‐ osate, will most certainly change the way in which sugarcane is eradicated. The use of glyphosate will reduce the costs associated with the control of weeds across cycles; however, during the period of cane eradication, the herbicide will now have a limited impact due to the tolerance introduced to the cultivars. In this case, eradication may have to be performed mechanically, which will have a negative impact on soil conservation and might stimulate weed germination from soil propagule banks. From this perspective, it is important to emphasise that research aimed at sugarcane plants tolerant to glyphosate should also consider

In the eradication of ratoon crops, glyphosate is used to eradicate the crop and also to control emerged weeds. However, when a sugarcane field also possess weeds that are hard to control, such as *Cynodon dactylon* and *Cyperus rotundus*, the use of higher doses of residual herbicides

after the application of glyphosate is adopted in a process known as "disinfestation".

IACSP94-4004) to 27 days after application. Instituto Agronômico de Campinas -IAC, 2012.

the use of herbicides in the eradication of future cultivars.

development, it was necessary to increase herbicide dosage by up to fivefold.

which needed 1920 g a. e. ha-1 of the herbicide.

160 Herbicides - Current Research and Case Studies in Use

**Figure 4.** Intoxication symptoms caused by glyphosate rates in sugarcane cultivars (IACSP94-2094-4004 and IACSP94-2191) to 45 days after application. Instituto Agronômico de Campinas - IAC, 2012.

The aggressive biological nature of hard to control weed species requires that handling start with the desiccation of the plants to optimise the use of glyphosate in the eradication of ratoon while also being able to introduce residual herbicides at higher doses. In these instances, the use of glyphosate in crop eradication serves the dual role desiccating the ratoons and control‐ ling problematic weeds.

In the time period following eradication but before planting sugarcane, the producer should formulate a strategy and opt for techniques that ensure higher sustainability of the system. These methods include such techniques as crop rotation or planting green-manure crops, although these may still compromise the techniques' sustainability if installed in fields that have been infested with "difficult-to-control weeds" or previously treated with residual herbicides for "disinfestation". The producer should carefully plan to use techniques that generate the most effective soil preparation and handling of weeds while simultaneously ensuring that after the treatments, the soil remains prepared for a new sugarcane plantation.

### **5. The shikimate pathway and isoenzymatic markers**

The shikimate pathway is found only in plants and microorganisms and is completely absent in mammals, birds, reptiles, fish and insects. These organisms extract the aromatic compounds necessary for survival and reproduction from their diet, while plants must produce such compounds because they do not have alternative means to obtain the compounds [23].The shikimate pathway is initiated with the reaction of PEP and erythrose 4-phosphate, a reaction catalysed by the enzyme DAHP synthase (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [61]. The resulting product is the seven-carbon acyclic intermediate 3-deoxy-Darabino-heptulosonate-7-phosphate (DAHP).

**6. Esterase isoenzymes in abiotic stress**

polymorphisms.

RR salt stain [75].

catalysis of ester hydrolysis [74].

**and α-esterase in sugarcane**

Enzymatic activity is influenced by stress factors, such as non-optimal temperature or nutrient levels and infection by pathogens. These stress responses subsequently lead to gene activation and, as a consequence, to the emergence of several molecular forms [70]. Because of the involvement of isoenzymes in changes to metabolism and defence mechanisms in plants, studies involving isoenzymes can be used in cases of both biotic and abiotic stress [63]. The authors report that polymorphisms displayed by isoenzymes are intermediate products of gene expression and are closer to the final phenotypical expression than those of DNA

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Esterases are isoenzymes comprising a group of genetically distinct enzymes that are found across a large spectrum of living organisms and that play a large variety of roles; nevertheless, esterases display a common trait of catalysing the hydrolysis of esters, peptides, amides and halide bonds [71]. Esterases can be found as both monomers and dimers [72, 73]. Esterases are significantly linked to lipid metabolism, such as that of membrane phospholipids, due to

In polyacrylamide gel electrophoresis assays, the α-esterase isoenzyme is detected using naphthyl ester substrates and histochemical stains. Enzyme isoforms with an affinity to hydrolyse α-naphthyl acetate are identified on the gel as black bands derived from the precipitation of α-naphthol, which results from the hydrolysis of α-naphthyl with the Fast blue

Esterase isoenzymes have also been extensively explored in studies of genetic diversity due to their high rate of polymorphisms [76, 77]. In [78] the authors reported that sugarcane cultivars could be identified using esterase isoenzymes, and [79] in studying the parameters for sugarcane differentiation, observed that the electrophoretic profile of esterases is main‐ tained in plants of varying physiological ages, as long as the growth environment is controlled. Esterase isoenzymes are among the most widely used enzymes in the evaluation of enzymatic alteration in plants that are affected by parasitic nematodes across various pathosystems [80]. In [75] was studied esterase polymorphisms in 16 cultivars of soy that underwent or were spared treatment with glyphosate. The authors observed variation in the sensitivity to αesterase isoenzymes of the different cultivars and also found that sensitivity did not seem to

be connected with the homozygous RR status of the genetically modified plants.

**7. Practical results of the isoenzymatic profiles of shikimate dehydrogenase**

The tolerance of sugarcane cultivars to chemical eradication using varying doses of glyphosate was investigated [51] using phytotechnical parameters and isoenzymatic markers. The author hypothesised that the study of isoenzymatic profiles of shikimate dehydrogenase and αesterase could optimise phytotechnical fieldwork observations regarding herbicidal tolerance.

DAHP is converted to cyclic form through catalysis by 3-dehydroquinate synthase in the presence of NAD+ as a coenzyme. In this process, 3-dehydroshikimate dehydratase dehydrates the cyclic form of DAHP. Next, shikimate dehydrogenase, in the presence of NADP+ (oxidised NADPH), reduces the cyclic and dehydrated DAHP to shikimate. The molecule is later phosphorylated by the SP3 kinase, which converts a molecule of ATP to ADP. The phos‐ phorylated shikimate subsequently reacts with one molecule of PEP in the presence of 5 enolpyruvylshikimate-3-phosphate (EPSP) synthase, resulting in the production of EPSP. The shikimate pathway terminates with the production of chorismate (chorismic acid) through the dephosphorylation of EPSP by chorismate synthase.

Other metabolites essential to plant life may be produced from chorismate, including the amino acids tryptophan, phenylalanine and tyrosine, as well as vitamin K, ubiquinone and tetrahydrofolate [62, 23]. The amino acid phenylalanine is a precursor not only of proteins but also of other secondary products, such as phenolic compounds, anthocyanins, lignin and promoters and growth inhibitors. Tryptophan is also a precursor to indole-3-acetic acid, which is responsible for apical dominance and is vital for cellular growth and several other regulatory processes. Therefore, inhibitors of the shikimate metabolic pathway represent a strategic alternative in the development of herbicides with low environmental impact, such as glyph‐ osate [23]. In this context, it could be interesting to use protein electrophoresis as a tool to study the eradication of plants by glyphosate using the isoenzymes involved in the metabolic pathway of shikimic acid.

Isoenzymes are the multiple molecular forms of enzymes that perform the same or similar catalytic activities. These enzymes are coded by one or more genes and may play an important role in survival across diverse environments. Isoenzymes are directly affected by both biotic and abiotic stressors [63]. The band intensity and isoenzymatic profile are plant-, tissue- and development stage-specific [64]. Some factors that affect plant metabolism, such as mineral nutrition, low temperature and diseases, among others, influence the activity of isoenzymes, specifically, esterases, peroxidases, phosphatases and phenolases, which in turn generate different expression patterns and levels of activity.Isoenzymatic patterns were used as tools by [65] who concluded that the enzymatic system of malate dehydrogenase is an efficient marker for aerobic respiration in pepper seeds during the maturation period. In Serbia [66] (the Vojvodina region), observed that the shikimate dehydrogenase system is also an efficient isoenzymatic marker for the study of genetic variability and polymorphisms in different almond genotypes.

In [67] was used the same technique in soy cultivars and registered a difference in electro‐ phoretograms in terms of peroxidase activity. In [68] used isoenzymatic markers to identify species of lettuce nematodes, and [69] used the method "in vitro" in sugarcane to observe varietal differences among doses of glyphosate.

### **6. Esterase isoenzymes in abiotic stress**

catalysed by the enzyme DAHP synthase (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [61]. The resulting product is the seven-carbon acyclic intermediate 3-deoxy-D-

DAHP is converted to cyclic form through catalysis by 3-dehydroquinate synthase in the

the cyclic form of DAHP. Next, shikimate dehydrogenase, in the presence of NADP+ (oxidised NADPH), reduces the cyclic and dehydrated DAHP to shikimate. The molecule is later phosphorylated by the SP3 kinase, which converts a molecule of ATP to ADP. The phos‐ phorylated shikimate subsequently reacts with one molecule of PEP in the presence of 5 enolpyruvylshikimate-3-phosphate (EPSP) synthase, resulting in the production of EPSP. The shikimate pathway terminates with the production of chorismate (chorismic acid) through the

Other metabolites essential to plant life may be produced from chorismate, including the amino acids tryptophan, phenylalanine and tyrosine, as well as vitamin K, ubiquinone and tetrahydrofolate [62, 23]. The amino acid phenylalanine is a precursor not only of proteins but also of other secondary products, such as phenolic compounds, anthocyanins, lignin and promoters and growth inhibitors. Tryptophan is also a precursor to indole-3-acetic acid, which is responsible for apical dominance and is vital for cellular growth and several other regulatory processes. Therefore, inhibitors of the shikimate metabolic pathway represent a strategic alternative in the development of herbicides with low environmental impact, such as glyph‐ osate [23]. In this context, it could be interesting to use protein electrophoresis as a tool to study the eradication of plants by glyphosate using the isoenzymes involved in the metabolic

Isoenzymes are the multiple molecular forms of enzymes that perform the same or similar catalytic activities. These enzymes are coded by one or more genes and may play an important role in survival across diverse environments. Isoenzymes are directly affected by both biotic and abiotic stressors [63]. The band intensity and isoenzymatic profile are plant-, tissue- and development stage-specific [64]. Some factors that affect plant metabolism, such as mineral nutrition, low temperature and diseases, among others, influence the activity of isoenzymes, specifically, esterases, peroxidases, phosphatases and phenolases, which in turn generate different expression patterns and levels of activity.Isoenzymatic patterns were used as tools by [65] who concluded that the enzymatic system of malate dehydrogenase is an efficient marker for aerobic respiration in pepper seeds during the maturation period. In Serbia [66] (the Vojvodina region), observed that the shikimate dehydrogenase system is also an efficient isoenzymatic marker for the study of genetic variability and polymorphisms in different

In [67] was used the same technique in soy cultivars and registered a difference in electro‐ phoretograms in terms of peroxidase activity. In [68] used isoenzymatic markers to identify species of lettuce nematodes, and [69] used the method "in vitro" in sugarcane to observe

as a coenzyme. In this process, 3-dehydroshikimate dehydratase dehydrates

arabino-heptulosonate-7-phosphate (DAHP).

162 Herbicides - Current Research and Case Studies in Use

dephosphorylation of EPSP by chorismate synthase.

presence of NAD+

pathway of shikimic acid.

almond genotypes.

varietal differences among doses of glyphosate.

Enzymatic activity is influenced by stress factors, such as non-optimal temperature or nutrient levels and infection by pathogens. These stress responses subsequently lead to gene activation and, as a consequence, to the emergence of several molecular forms [70]. Because of the involvement of isoenzymes in changes to metabolism and defence mechanisms in plants, studies involving isoenzymes can be used in cases of both biotic and abiotic stress [63]. The authors report that polymorphisms displayed by isoenzymes are intermediate products of gene expression and are closer to the final phenotypical expression than those of DNA polymorphisms.

Esterases are isoenzymes comprising a group of genetically distinct enzymes that are found across a large spectrum of living organisms and that play a large variety of roles; nevertheless, esterases display a common trait of catalysing the hydrolysis of esters, peptides, amides and halide bonds [71]. Esterases can be found as both monomers and dimers [72, 73]. Esterases are significantly linked to lipid metabolism, such as that of membrane phospholipids, due to catalysis of ester hydrolysis [74].

In polyacrylamide gel electrophoresis assays, the α-esterase isoenzyme is detected using naphthyl ester substrates and histochemical stains. Enzyme isoforms with an affinity to hydrolyse α-naphthyl acetate are identified on the gel as black bands derived from the precipitation of α-naphthol, which results from the hydrolysis of α-naphthyl with the Fast blue RR salt stain [75].

Esterase isoenzymes have also been extensively explored in studies of genetic diversity due to their high rate of polymorphisms [76, 77]. In [78] the authors reported that sugarcane cultivars could be identified using esterase isoenzymes, and [79] in studying the parameters for sugarcane differentiation, observed that the electrophoretic profile of esterases is main‐ tained in plants of varying physiological ages, as long as the growth environment is controlled.

Esterase isoenzymes are among the most widely used enzymes in the evaluation of enzymatic alteration in plants that are affected by parasitic nematodes across various pathosystems [80]. In [75] was studied esterase polymorphisms in 16 cultivars of soy that underwent or were spared treatment with glyphosate. The authors observed variation in the sensitivity to αesterase isoenzymes of the different cultivars and also found that sensitivity did not seem to be connected with the homozygous RR status of the genetically modified plants.

### **7. Practical results of the isoenzymatic profiles of shikimate dehydrogenase and α-esterase in sugarcane**

The tolerance of sugarcane cultivars to chemical eradication using varying doses of glyphosate was investigated [51] using phytotechnical parameters and isoenzymatic markers. The author hypothesised that the study of isoenzymatic profiles of shikimate dehydrogenase and αesterase could optimise phytotechnical fieldwork observations regarding herbicidal tolerance. Shikimate dehydrogenase was selected because it is involved in the shikimic acid pathway, which is affected by glyphosate action, and α-esterase was chosen because it is associated with oxidative stress. The isoenzymatic profiles of shikimate dehydrogenase and α-esterase were studied in sugarcane cultivars IACSP94-2094, IACSP94-2101, IACSP93-3046, IACSP94-4004, IAC86-2480 and RB72454 at 8, 24, 48, 72 and 144 hours after the application of glyphosate at doses of 0, 1440, 2160, 2880, 3600 and 4320 g a. e. ha-1. The results showed that the bands for shikimate dehydrogenase tended to position near the cathode (at the top of the gel), while αesterases were positioned closer to the anode, due to a greater migration during the gel run. The enzymatic system of shikimate dehydrogenase presented bands that were less sharp and also had a lower number of bands (three). There were no observed polymorphisms among cultivars, regardless of whether the data were analysed according to herbicide dosages or in relation to controls. Therefore, the isoforms remained constant among the different cultivars and treatments. In [81] was studied 20 enzymatic systems in the identification of sugarcane cultivars and also did not obtain any promising results using shikimate dehydrogenase. In [82] was studied populations of *Stryphnodendron adstringens*, known in Brazil as *barbatimão,* and also did not find polymorphisms for shikimate dehydrogenase.

the isoenzymatic profiles of shikimate dehydrogenase and α-esterase did not constitute a

The Use of Glyphosate in Sugarcane: A Brazilian Experience

http://dx.doi.org/10.5772/54958

165

Maturation is one of the most important aspects of sugarcane crops because maturation is directly related to the optimal time-point for harvest/industrial transformation.For the plant to enter the maturation process naturally, one or more sources of stress are necessary, with a gradual reduction in photoperiod, temperature or precipitation being the most effective stimulants [85]. In Brazil, more specifically in the south-central region, the maturation process is initiated in April/May when the climate becomes colder and drier. However, even under favourable conditions, sugarcane maturation may also be induced in responsive cultivars as a strategy to produce high-quality raw material across all of the different phases of harvesting.

To guarantee that maturation is complete, uniform, early and programmed and to avoid undesirable flowering, the sugarcane-ethanol industry has been adopting the use of ripeners (growth regulators) in sugarcane. Ripeners are chemical compounds that induce the translo‐ cation and storage of sugars, mainly sucrose, in the culm. Therefore, the goal of the ripeners is both to advance and maintain natural maturation and to provide high-quality raw materials

The same authors state that to artificially induce maturation, growth regulators are applied by aircraft eight to ten months after the last harvest, that is, during the plants' vegetative state. In practice, the months of February and March or October are the periods during which farmers aim to apply the enhancers because they can anticipate the beginning or the end of the harvest.

There are two basic types of ripeners for sugarcane fields, "non-stressors" and "stressors". Non-stressor ripeners do not diminish the plants' growth rate, and their action induces the release of ethylene, the compound responsible for maturation that helps in the accumulation of sucrose in sugarcane culms. Stressor compounds, such as glyphosate, are growth inhibitors that markedly decrease the sugarcane growth rate, making the plants accumulate sucrose instead of expending it as an energy source for growth. This reduction in growth rate forces the plant to mature [85]. In [86] sugarcane plants with stagnated growth stop sprouting new leaves, and as a consequence, the reduced number of phytochromes in chloroplasts becomes insufficient to detect the photoperiod and thus stimulate the transition of the apical bud from

The effect of glyphosate, after it is applied to sugarcane, has a rapid onset, allowing for an increase of sucrose accumulation in 30 to 40 days after application. The glyphosate dose used is normally 0.3 to 0.4 l ha-1 and may reach a maximum of 0.6 – 0.8 l ha-1, leading to differences in maturation speed as a function of dosage. Harvest should be performed when the highest

for early industrial transformation, as well as to aid in the handling of cultivars [85].

**8. The use of glyphosate in sugarcane as a ripener**

reliable tool [51].

vegetative to reproductive.

levels of sucrose are reached [12].

The enzymatic system of α-esterase was specific for each studied cultivar, allowing for cultivar identification based on this biochemical marker [51]. This observation corroborates the findings [83], who created an analytical key for sugarcane cultivars and found a different pattern of α-esterase in each of the ten cultivars studied. A large number of bands of the αesterase enzymatic complex were found with varied band intensity and thickness. The characteristics of this complex can be related to the degree of ploidy of the plant species; sugarcane is polyploid [70, 84].

Cultivars of variety IACSP93-3046 and RB72454 did not present differences in terms of bands owing to the application of glyphosate. These cultivars were considered susceptible to glyphosate based on field studies reporting a percentage of tiller death of 93.16% and 94.25% respectively. Moreover, marked toxic effects were rated as high as 94% for IACSP93-3046 and 95.5 % for RB72454 [51].

Sugarcane cultivar IACSP94-4004 was the only cultivar to show an alteration in its band pattern due to the application of glyphosate. Across all of the treatments in which the herbicide was applied, there were two additional bands that were not present in the controls and that were present from the first assessment 8 hours after the application (HAA) of herbicide to the last assessment at 144 HAA. The appearance of additional bands may be due to the expression of genes from this enzymatic system in response to stress caused by glyphosate treatment, thereby demonstrating that the cultivar response to the herbicide is directly linked to the genotype of each cultivar variety. In fact, cultivar IACSP94-4004 was relatively tolerant to the field experiments, as it was the cultivar to show the lowest average (percentage) of tiller death at 45 HAA (80.15 %), the symptoms of toxicity in this cultivar were less pronounced with an average of 82.5 %, and only glyphosate doses of 3600 g a. e. ha-1 caused symptoms similar to those caused by the highest dose of 4320 g a. e. ha-1.

Evaluation of the isoenzymatic system of esterases has been used in other studies to characterise cultivar tolerance. Nevertheless, in the evaluation of sugarcane eradication, the isoenzymatic profiles of shikimate dehydrogenase and α-esterase did not constitute a reliable tool [51].

### **8. The use of glyphosate in sugarcane as a ripener**

Shikimate dehydrogenase was selected because it is involved in the shikimic acid pathway, which is affected by glyphosate action, and α-esterase was chosen because it is associated with oxidative stress. The isoenzymatic profiles of shikimate dehydrogenase and α-esterase were studied in sugarcane cultivars IACSP94-2094, IACSP94-2101, IACSP93-3046, IACSP94-4004, IAC86-2480 and RB72454 at 8, 24, 48, 72 and 144 hours after the application of glyphosate at doses of 0, 1440, 2160, 2880, 3600 and 4320 g a. e. ha-1. The results showed that the bands for shikimate dehydrogenase tended to position near the cathode (at the top of the gel), while αesterases were positioned closer to the anode, due to a greater migration during the gel run. The enzymatic system of shikimate dehydrogenase presented bands that were less sharp and also had a lower number of bands (three). There were no observed polymorphisms among cultivars, regardless of whether the data were analysed according to herbicide dosages or in relation to controls. Therefore, the isoforms remained constant among the different cultivars and treatments. In [81] was studied 20 enzymatic systems in the identification of sugarcane cultivars and also did not obtain any promising results using shikimate dehydrogenase. In [82] was studied populations of *Stryphnodendron adstringens*, known in Brazil as *barbatimão,* and

The enzymatic system of α-esterase was specific for each studied cultivar, allowing for cultivar identification based on this biochemical marker [51]. This observation corroborates the findings [83], who created an analytical key for sugarcane cultivars and found a different pattern of α-esterase in each of the ten cultivars studied. A large number of bands of the αesterase enzymatic complex were found with varied band intensity and thickness. The characteristics of this complex can be related to the degree of ploidy of the plant species;

Cultivars of variety IACSP93-3046 and RB72454 did not present differences in terms of bands owing to the application of glyphosate. These cultivars were considered susceptible to glyphosate based on field studies reporting a percentage of tiller death of 93.16% and 94.25% respectively. Moreover, marked toxic effects were rated as high as 94% for IACSP93-3046 and

Sugarcane cultivar IACSP94-4004 was the only cultivar to show an alteration in its band pattern due to the application of glyphosate. Across all of the treatments in which the herbicide was applied, there were two additional bands that were not present in the controls and that were present from the first assessment 8 hours after the application (HAA) of herbicide to the last assessment at 144 HAA. The appearance of additional bands may be due to the expression of genes from this enzymatic system in response to stress caused by glyphosate treatment, thereby demonstrating that the cultivar response to the herbicide is directly linked to the genotype of each cultivar variety. In fact, cultivar IACSP94-4004 was relatively tolerant to the field experiments, as it was the cultivar to show the lowest average (percentage) of tiller death at 45 HAA (80.15 %), the symptoms of toxicity in this cultivar were less pronounced with an average of 82.5 %, and only glyphosate doses of 3600 g a. e. ha-1 caused symptoms similar to

Evaluation of the isoenzymatic system of esterases has been used in other studies to characterise cultivar tolerance. Nevertheless, in the evaluation of sugarcane eradication,

also did not find polymorphisms for shikimate dehydrogenase.

sugarcane is polyploid [70, 84].

164 Herbicides - Current Research and Case Studies in Use

those caused by the highest dose of 4320 g a. e. ha-1.

95.5 % for RB72454 [51].

Maturation is one of the most important aspects of sugarcane crops because maturation is directly related to the optimal time-point for harvest/industrial transformation.For the plant to enter the maturation process naturally, one or more sources of stress are necessary, with a gradual reduction in photoperiod, temperature or precipitation being the most effective stimulants [85]. In Brazil, more specifically in the south-central region, the maturation process is initiated in April/May when the climate becomes colder and drier. However, even under favourable conditions, sugarcane maturation may also be induced in responsive cultivars as a strategy to produce high-quality raw material across all of the different phases of harvesting.

To guarantee that maturation is complete, uniform, early and programmed and to avoid undesirable flowering, the sugarcane-ethanol industry has been adopting the use of ripeners (growth regulators) in sugarcane. Ripeners are chemical compounds that induce the translo‐ cation and storage of sugars, mainly sucrose, in the culm. Therefore, the goal of the ripeners is both to advance and maintain natural maturation and to provide high-quality raw materials for early industrial transformation, as well as to aid in the handling of cultivars [85].

The same authors state that to artificially induce maturation, growth regulators are applied by aircraft eight to ten months after the last harvest, that is, during the plants' vegetative state. In practice, the months of February and March or October are the periods during which farmers aim to apply the enhancers because they can anticipate the beginning or the end of the harvest.

There are two basic types of ripeners for sugarcane fields, "non-stressors" and "stressors". Non-stressor ripeners do not diminish the plants' growth rate, and their action induces the release of ethylene, the compound responsible for maturation that helps in the accumulation of sucrose in sugarcane culms. Stressor compounds, such as glyphosate, are growth inhibitors that markedly decrease the sugarcane growth rate, making the plants accumulate sucrose instead of expending it as an energy source for growth. This reduction in growth rate forces the plant to mature [85]. In [86] sugarcane plants with stagnated growth stop sprouting new leaves, and as a consequence, the reduced number of phytochromes in chloroplasts becomes insufficient to detect the photoperiod and thus stimulate the transition of the apical bud from vegetative to reproductive.

The effect of glyphosate, after it is applied to sugarcane, has a rapid onset, allowing for an increase of sucrose accumulation in 30 to 40 days after application. The glyphosate dose used is normally 0.3 to 0.4 l ha-1 and may reach a maximum of 0.6 – 0.8 l ha-1, leading to differences in maturation speed as a function of dosage. Harvest should be performed when the highest levels of sucrose are reached [12].

### **9. Conclusions**

Glyphosate is a high-efficiency molecule in the sugar-ethanol industrial sector in Brazil. Glyphosate can be used as an herbicide in the control of weeds and the chemical eradication of sugarcane crops, and when applied in low dosages, glyphosate can be used as a ripener.

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Independently of where the product is used in the production system, the key factor is the adequacy of the dosage. If cultivar tolerance is known, it is possible to adjust the dose of glyphosate to eradicate sugarcane ratoon crops. Research has shown that a dose of 2 kg ha-1 (1440 g a. e. ha-1) of the commercial product Roundup WG eradicates 92.76% of the tillers of the IACSP94-2094 cultivar but only 40.3% of the IACSP94-4004 cultivar. For the most effective eradication of the IACSP94-4004 cultivar, 4 kg ha1 (2880 g a. e. ha-1) of chemical is needed. This finding demonstrates that knowledge of plant tolerance can be a valuable tool to adjust glyphosate doses to the appropriate concentrations for ratoon-crop eradication.

Reducing the quantity of applied glyphosate is possible, as long as the crop is sensitive. Environmental and economic benefits can also be obtained by applying lower quantities of the herbicide. Information on cultivar tolerance also allows producers to know when to use higher herbicide concentrations than those recommended in the literature. However, if the producer applies a lower dose than is necessary, the ratoon crop will not be completely eradicated. As a consequence, there will be additional expenditure on a additional herbicide application or on mechanical eradication, which is highly problematic because it causes the greatest disrup‐ tion to the soil and later leads to the presence of crop stubble in the new sugarcane field.

### **Acknowledgements**

The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the financial support (Grant # 2010/09016-9).

### **Author details**

Carlos Alberto Mathias Azania1\*, Luciana Rossini Pinto1 , Rodrigo Cabral Adriano1 , Dilermando Perecin2 and Andréa Padua Azania1

\*Address all correspondence to: azania@iac.sp.gov.br

1 Instituto Agronômico de Campinas - IAC, Centro de Cana, Ribeirão Preto, Brazil

2 Universidade Estadual Paulista – Unesp, Jaboticabal, Brazil

### **References**

**9. Conclusions**

166 Herbicides - Current Research and Case Studies in Use

**Acknowledgements**

**Author details**

Dilermando Perecin2

eradication of the IACSP94-4004 cultivar, 4 kg ha1

for the financial support (Grant # 2010/09016-9).

Carlos Alberto Mathias Azania1\*, Luciana Rossini Pinto1

\*Address all correspondence to: azania@iac.sp.gov.br

2 Universidade Estadual Paulista – Unesp, Jaboticabal, Brazil

and Andréa Padua Azania1

1 Instituto Agronômico de Campinas - IAC, Centro de Cana, Ribeirão Preto, Brazil

Glyphosate is a high-efficiency molecule in the sugar-ethanol industrial sector in Brazil. Glyphosate can be used as an herbicide in the control of weeds and the chemical eradication of sugarcane crops, and when applied in low dosages, glyphosate can be used as a ripener.

Independently of where the product is used in the production system, the key factor is the adequacy of the dosage. If cultivar tolerance is known, it is possible to adjust the dose of glyphosate to eradicate sugarcane ratoon crops. Research has shown that a dose of 2 kg ha-1 (1440 g a. e. ha-1) of the commercial product Roundup WG eradicates 92.76% of the tillers of the IACSP94-2094 cultivar but only 40.3% of the IACSP94-4004 cultivar. For the most effective

finding demonstrates that knowledge of plant tolerance can be a valuable tool to adjust

Reducing the quantity of applied glyphosate is possible, as long as the crop is sensitive. Environmental and economic benefits can also be obtained by applying lower quantities of the herbicide. Information on cultivar tolerance also allows producers to know when to use higher herbicide concentrations than those recommended in the literature. However, if the producer applies a lower dose than is necessary, the ratoon crop will not be completely eradicated. As a consequence, there will be additional expenditure on a additional herbicide application or on mechanical eradication, which is highly problematic because it causes the greatest disrup‐ tion to the soil and later leads to the presence of crop stubble in the new sugarcane field.

The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)

glyphosate doses to the appropriate concentrations for ratoon-crop eradication.

(2880 g a. e. ha-1) of chemical is needed. This

, Rodrigo Cabral Adriano1

,


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**Chapter 8**

**Herbicides Used in Tobacco**

Additional information is available at the end of the chapter

**1.1. Competitive effects of weeds on tobacco yield and quality**

industry as Non-Tobacco Related Material (NTRM).

Tobacco is a major and economically important crop in many countries worldwide, with 6.91 million tons produced annually, mainly in China, India, Brazil, Zimbabwe, Turkey, Indonesia, Russia, Malawi, nations of the European Union, and the United States [1]. In the United States alone, approximately 360,000 tons are produced annually at a value of more than \$USD 1.25 billion [2]. Although there are at least 14 different types of tobacco grown around the world, all are affected by pests. Disease and insect pests are of primary importance in tobacco production, but weeds are also a major focus of pest control in tobacco. Although weeds may not cause as much direct damage to tobacco as diseases and insects, weeds present in tobacco can influence tobacco yield and quality, cause harvest interference, and serve as hosts for disease and insects. Although tobacco is considered to be very competitive with weeds relative to other crops, use of herbicides, usually supplemented with cultivation, is still a primary component of weed control. The objective of the research presented here is to provide a more thorough understanding of the effects of weeds in tobacco and the characteristics of major herbicides available to control these weeds in tobacco production in the United States.

Weeds directly compete with tobacco for light, water, nutrients, carbon dioxide, and space and can negatively impact tobacco yield and quality. In addition, the quality of the final product may be further affected due to the presence of foreign plant material, referred to in the tobacco

The most direct impact of weed competition in tobacco is reduced leaf yield. Leaf quality can also be negatively affected if weeds physically damage tobacco before or during harvest. Contamination of the harvested tobacco crop by green weed vegetation or reproductive parts of weeds has the largest effect on tobacco quality [3, 4]. Chemical exudates from weedy species

> © 2013 Bailey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Bailey; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

William A. Bailey

**1. Introduction**

http://dx.doi.org/10.5772/56008

### **Chapter 8**

## **Herbicides Used in Tobacco**

William A. Bailey

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56008

### **1. Introduction**

Tobacco is a major and economically important crop in many countries worldwide, with 6.91 million tons produced annually, mainly in China, India, Brazil, Zimbabwe, Turkey, Indonesia, Russia, Malawi, nations of the European Union, and the United States [1]. In the United States alone, approximately 360,000 tons are produced annually at a value of more than \$USD 1.25 billion [2]. Although there are at least 14 different types of tobacco grown around the world, all are affected by pests. Disease and insect pests are of primary importance in tobacco production, but weeds are also a major focus of pest control in tobacco. Although weeds may not cause as much direct damage to tobacco as diseases and insects, weeds present in tobacco can influence tobacco yield and quality, cause harvest interference, and serve as hosts for disease and insects. Although tobacco is considered to be very competitive with weeds relative to other crops, use of herbicides, usually supplemented with cultivation, is still a primary component of weed control. The objective of the research presented here is to provide a more thorough understanding of the effects of weeds in tobacco and the characteristics of major herbicides available to control these weeds in tobacco production in the United States.

### **1.1. Competitive effects of weeds on tobacco yield and quality**

Weeds directly compete with tobacco for light, water, nutrients, carbon dioxide, and space and can negatively impact tobacco yield and quality. In addition, the quality of the final product may be further affected due to the presence of foreign plant material, referred to in the tobacco industry as Non-Tobacco Related Material (NTRM).

The most direct impact of weed competition in tobacco is reduced leaf yield. Leaf quality can also be negatively affected if weeds physically damage tobacco before or during harvest. Contamination of the harvested tobacco crop by green weed vegetation or reproductive parts of weeds has the largest effect on tobacco quality [3, 4]. Chemical exudates from weedy species

© 2013 Bailey; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Bailey; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

that contaminate tobacco leaves and remain until the tobacco is processed can also impact leaf chemical balance and resulting flavor of manufactured tobacco products.

(*Xanthium strumarium* L.) has the greatest root elongation rate and extracts the greatest amount of moisture per unit area of soil [9]. Under field conditions, the water requirements for various weed species vary from 150 to 1900 kg water per kg dry matter produced. Of the nutrients that weeds and tobacco compete for, nitrogen is often the first nutrient to come into short supply as a result of competition. Weeds are commonly better assimilators of nutrients than crop plants, normally possessing 50 to 100% more nitrogen than the crop plant based on a whole

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 177

Where water and nutrients are adequate, low light intensity that occurs from shading plays a major role in limiting plant growth. Plants compete for light by positioning their leaves to intercept available light more favorably than neighboring plants. Plants that exhibit more rapid early-season growth and have upright growth to grow taller than neighboring plants will be most successful in competition for light. Broadleaved crops such as tobacco have a distinct competitive advantage over grass plants or sedges that have narrow leaves. Tall, dense crops like tobacco successfully compete with shorter plants for light, particularly when weed emergence occurs later in the season after tobacco is well established and tobacco can easily

Aside from directly competing with tobacco to reduce marketable yield and quality, many weed species are troublesome with tobacco due to their ability to interfere with harvest operations. Tobacco crops that are heavily infested with weeds, even relatively non-competi‐ tive weeds, can have reduced yield through competition before harvest and even more during harvest. Weed species with twining or climbing growth habits such as morningglory species (*Ipomoea* spp.), honeyvine milkweed (*Ampelamus albidus* [Nutt.] Britt.), or common bindweed (*Convolvulus arvensis* L.) may not be very competitive with tobacco during the growing season, but can cause dramatic losses at harvest, even when weed densities are relatively low. A single climbing weed in a tobacco crop may become entangled in several tobacco plants and cause leaf damage and loss both prior to and during harvest. Infestations from weeds that become entwined around tobacco stalks are troublesome during hand harvest operations but even more troublesome for mechanical harvesting systems. Presence of morningglory at an average density of 1 plant per 10 m2 has caused a 5% reduction in harvested yield of dark tobacco in Kentucky USA due to damage and leaf loss during hand harvest (W.A. Bailey, unpublished data). Mechanical harvesters that encounter morningglory entwined in tobacco at similar densities would likely incur greater leaf losses as well as sustain extensive damage to the harvester itself. Parts of weedy plants that remain in the tobacco crop through curing are more likely to become NTRM, causing extensive reduction in price and likely reduction in marketing

Weeds can act as a major host site for other tobacco pests such as diseases, insects, and nematodes. Many weeds that commonly occur around tobacco fields can harbor other pests and result in increased infection on tobacco crops. Generally, weed species that have the closest botanical relationship to tobacco, such as solanaceous weed species, are most likely to harbor pests that can infest tobacco. However, many plant species with little botanical relationship to

impose a shading effect on newly emerged weed seedlings.

plant dry weight basis [10].

opportunities for future crops.

**1.2. Weeds as alternate hosts to other pests in tobacco**

The critical weed-free period is a phrase that is used to describe the period during crop production in which weeds are most likely to reduce crop growth and yield. This is the time period during which weed control efforts must be maintained to prevent crop yield loss. The significance of the critical weed-free period is that, if the crop is maintained weed-free for this period, it will be able to effectively compete with late-emerging weeds without sustaining yield loss. Critical weed-free periods are influenced by the competitiveness of the individual crop species and weed species. For most crops, the critical weed-free period for most weeds is 4 to 6 weeks after crop emergence. Since tobacco is transplanted in the field rather than seeded, it is inherently more competitive with weeds than direct-seeded crops. For this reason, the critical weed-free period for tobacco may be 1 to 2 weeks shorter than for direct seeded crops. In addition, the large leaves which most types of tobacco produce makes it more competitive than many other crops by having a greater ability to reduce photosynthetic ability of weeds growing under the tobacco canopy. Flue-cured tobacco maintained free of common ragweed (*Ambrosia artemesiifolia* L.) for two weeks following transplanting did not sustain significant yield losses from common ragweed that emerged later [4]. For most weed species, maintaining weed-free or near weed-free conditions for 6 weeks after transplanting allows tobacco to shade out weeds that emerge later in the season [5]. In Greece, yield of burley and oriental tobacco increased significantly with weed-free periods of 3 or 4 weeks and decreased when weeds were allowed to compete with tobacco for more than 3 to 4 weeks after transplanting. When yield was reduced due to weed competition, there were also differences in chemical composition of the tobacco [6]. Natural populations of weeds that were allowed to compete with dark tobacco for the entire season resulted in a 28% to 40% reduction in total yield compared to tobacco plots treated with herbicides [7, 8].

If weeds are allowed to compete with tobacco for the entire season, the level of competition that weeds impose is also influenced by the density of the weeds that are present in the crop. In general, crop yield decreases as weed density increases. Different weed species also have different competitive ability with tobacco and thus can effectively compete at lower densities than other species. In general, dicots (broadleaf weeds) are more competitive with tobacco than monocots (grass weeds). Within broadleaf and grass weeds, individual species can be more competitive with tobacco than others. For example, among broadleaf species, Eastern black nightshade (*Solanum ptycanthum* L.) has a more rapid growth, higher photosynthetic ability, and a more erect growth habit than black nightshade (*Solanum nigrum* L.), and is more competitive with tobacco. Among grass species, giant foxtail (*Setaria glauca* L.) is more competitive than either green (*Setaria viridis* L.) or yellow foxtail (*Setaria faberii* L.). Much of these differences in competitiveness can be attributed to differences in plant size among species. Perennial weed species are also generally more competitive and difficult to control in tobacco than annual weed species. Perennial species generally have a more extensive root system and extensive energy reserves than annual species.

Differences in root elongation rate also influence differences in competitiveness by affecting water and nutrient absorption potential. Among weedy broadleaf species, common cocklebur (*Xanthium strumarium* L.) has the greatest root elongation rate and extracts the greatest amount of moisture per unit area of soil [9]. Under field conditions, the water requirements for various weed species vary from 150 to 1900 kg water per kg dry matter produced. Of the nutrients that weeds and tobacco compete for, nitrogen is often the first nutrient to come into short supply as a result of competition. Weeds are commonly better assimilators of nutrients than crop plants, normally possessing 50 to 100% more nitrogen than the crop plant based on a whole plant dry weight basis [10].

that contaminate tobacco leaves and remain until the tobacco is processed can also impact leaf

The critical weed-free period is a phrase that is used to describe the period during crop production in which weeds are most likely to reduce crop growth and yield. This is the time period during which weed control efforts must be maintained to prevent crop yield loss. The significance of the critical weed-free period is that, if the crop is maintained weed-free for this period, it will be able to effectively compete with late-emerging weeds without sustaining yield loss. Critical weed-free periods are influenced by the competitiveness of the individual crop species and weed species. For most crops, the critical weed-free period for most weeds is 4 to 6 weeks after crop emergence. Since tobacco is transplanted in the field rather than seeded, it is inherently more competitive with weeds than direct-seeded crops. For this reason, the critical weed-free period for tobacco may be 1 to 2 weeks shorter than for direct seeded crops. In addition, the large leaves which most types of tobacco produce makes it more competitive than many other crops by having a greater ability to reduce photosynthetic ability of weeds growing under the tobacco canopy. Flue-cured tobacco maintained free of common ragweed (*Ambrosia artemesiifolia* L.) for two weeks following transplanting did not sustain significant yield losses from common ragweed that emerged later [4]. For most weed species, maintaining weed-free or near weed-free conditions for 6 weeks after transplanting allows tobacco to shade out weeds that emerge later in the season [5]. In Greece, yield of burley and oriental tobacco increased significantly with weed-free periods of 3 or 4 weeks and decreased when weeds were allowed to compete with tobacco for more than 3 to 4 weeks after transplanting. When yield was reduced due to weed competition, there were also differences in chemical composition of the tobacco [6]. Natural populations of weeds that were allowed to compete with dark tobacco for the entire season resulted in a 28% to 40% reduction in total yield compared to tobacco plots

If weeds are allowed to compete with tobacco for the entire season, the level of competition that weeds impose is also influenced by the density of the weeds that are present in the crop. In general, crop yield decreases as weed density increases. Different weed species also have different competitive ability with tobacco and thus can effectively compete at lower densities than other species. In general, dicots (broadleaf weeds) are more competitive with tobacco than monocots (grass weeds). Within broadleaf and grass weeds, individual species can be more competitive with tobacco than others. For example, among broadleaf species, Eastern black nightshade (*Solanum ptycanthum* L.) has a more rapid growth, higher photosynthetic ability, and a more erect growth habit than black nightshade (*Solanum nigrum* L.), and is more competitive with tobacco. Among grass species, giant foxtail (*Setaria glauca* L.) is more competitive than either green (*Setaria viridis* L.) or yellow foxtail (*Setaria faberii* L.). Much of these differences in competitiveness can be attributed to differences in plant size among species. Perennial weed species are also generally more competitive and difficult to control in tobacco than annual weed species. Perennial species generally have a more extensive root

Differences in root elongation rate also influence differences in competitiveness by affecting water and nutrient absorption potential. Among weedy broadleaf species, common cocklebur

chemical balance and resulting flavor of manufactured tobacco products.

176 Herbicides - Current Research and Case Studies in Use

treated with herbicides [7, 8].

system and extensive energy reserves than annual species.

Where water and nutrients are adequate, low light intensity that occurs from shading plays a major role in limiting plant growth. Plants compete for light by positioning their leaves to intercept available light more favorably than neighboring plants. Plants that exhibit more rapid early-season growth and have upright growth to grow taller than neighboring plants will be most successful in competition for light. Broadleaved crops such as tobacco have a distinct competitive advantage over grass plants or sedges that have narrow leaves. Tall, dense crops like tobacco successfully compete with shorter plants for light, particularly when weed emergence occurs later in the season after tobacco is well established and tobacco can easily impose a shading effect on newly emerged weed seedlings.

Aside from directly competing with tobacco to reduce marketable yield and quality, many weed species are troublesome with tobacco due to their ability to interfere with harvest operations. Tobacco crops that are heavily infested with weeds, even relatively non-competi‐ tive weeds, can have reduced yield through competition before harvest and even more during harvest. Weed species with twining or climbing growth habits such as morningglory species (*Ipomoea* spp.), honeyvine milkweed (*Ampelamus albidus* [Nutt.] Britt.), or common bindweed (*Convolvulus arvensis* L.) may not be very competitive with tobacco during the growing season, but can cause dramatic losses at harvest, even when weed densities are relatively low. A single climbing weed in a tobacco crop may become entangled in several tobacco plants and cause leaf damage and loss both prior to and during harvest. Infestations from weeds that become entwined around tobacco stalks are troublesome during hand harvest operations but even more troublesome for mechanical harvesting systems. Presence of morningglory at an average density of 1 plant per 10 m2 has caused a 5% reduction in harvested yield of dark tobacco in Kentucky USA due to damage and leaf loss during hand harvest (W.A. Bailey, unpublished data). Mechanical harvesters that encounter morningglory entwined in tobacco at similar densities would likely incur greater leaf losses as well as sustain extensive damage to the harvester itself. Parts of weedy plants that remain in the tobacco crop through curing are more likely to become NTRM, causing extensive reduction in price and likely reduction in marketing opportunities for future crops.

#### **1.2. Weeds as alternate hosts to other pests in tobacco**

Weeds can act as a major host site for other tobacco pests such as diseases, insects, and nematodes. Many weeds that commonly occur around tobacco fields can harbor other pests and result in increased infection on tobacco crops. Generally, weed species that have the closest botanical relationship to tobacco, such as solanaceous weed species, are most likely to harbor pests that can infest tobacco. However, many plant species with little botanical relationship to tobacco can also serve as hosts. For example, *Datura* species such as Jimsonweed are common alternate hosts to at least 12 tobacco diseases, at least one nematodes species, and at least 3 major insect pests of tobacco. *Nicandra* species such as Apple-of-Peru are common alternate hosts to at least 4 major tobacco diseases including blue mold, brown spot, bushy top virus, and vein banding virus.

**Disease Causal Agent Hosts**

Tomato Spotted Wilt Virus (TSWV) **Species**

Stolbur Mycoplasma 65 24 Field Bindweed

Various 166 34 Dandelion

Aster yellows Mycoplasma 175 52 Dodder

**Plant Families Common Weedy Hosts**

Groundcherry species

Nicandra physaloides (L.) Pers.

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 179

(*Convolvulus arvensis* L.)

(*Taraxacum officinale* L.) Spiny amaranth (*Amaranthus spinosus* L.)

(*Datura stramonium* L.) Clasping coneflower

(Rudbeckia amplexicaulis Vahl.)

(Verbena brasiliensis Velloso) Mouseear chickweed (Cerastium vulgatum) Prickly lettuce (Lactuca scariola) Carpetweed (Mollugo verticillata) Blackseed plantain (Plantago rugelii) Hairy buttercup (Ranunculus sardous) Spiny sowthistle (Sonchus asper) Common chickweed (Stellaria media) Hairy bittercress (Cardamine hirsuta)

(*Physalis* sp.) Apple of Peru

(*Cuscuta* sp.)

Jimsonweed

Brazilian vervain

Dogfennel

(Eupatorium capillifolium) Carolina geranium (Geranium carolinianum) Purple cudweed

(Gnaphalium purpureum)

#### **1.3. Diseases**

Table 1 lists weed species that commonly act as alternate hosts for tobacco diseases. Many diseases have an extremely wide host range and so only the number of species, families, genera, or most common host species are listed. Reference materials [11-14] were used to construct Tables 1, 2. and 3.



tobacco can also serve as hosts. For example, *Datura* species such as Jimsonweed are common alternate hosts to at least 12 tobacco diseases, at least one nematodes species, and at least 3 major insect pests of tobacco. *Nicandra* species such as Apple-of-Peru are common alternate hosts to at least 4 major tobacco diseases including blue mold, brown spot, bushy top virus,

Table 1 lists weed species that commonly act as alternate hosts for tobacco diseases. Many diseases have an extremely wide host range and so only the number of species, families, genera, or most common host species are listed. Reference materials [11-14] were used to construct

**Plant Families Common Weedy Hosts**

*Solanum* sp.

Jimsonweed

(*Datura stramonium* L.) Smartweed species (*Polygonum* sp.) Shepards-purse

(*Capsella bursa-pastoris* L.) Black nightshade (*Solanum nigrum* L.) Barnyardgrass

(*Echinochloa crus-galli* L.)

(*Solanum carolinense* L.)

(*Datura stramonium* L.)

(*Taraxacum officinale* Weber)

Dandelion

Horsenettle

Ground cherry (*Physalis angulata* L.) Jimsonweed

(*Solanum* sp.) *Chenopodium* sp.

(*Ambrosia art*emisiifolia L.) Pennsylvania smartweed (*Polygonum pennsylvanicum* L*.*)

197 33 Common ragweed

Brassicaceae Cucurbitaceae

Most common: Solanaceae Compositae Hydrophyllaceae Scrophulariaceae

*Solanaceae*

Many Most common: *Solanaceae*

and vein banding virus.

178 Herbicides - Current Research and Case Studies in Use

**1.3. Diseases**

Tables 1, 2. and 3.

Wildfire / Angular leafspot

Tobacco Mosaic Virus (TMV)

Vein Banding Virus

**Disease Causal Agent Hosts**

*solanacearum*

*Pseudomonas syringae* pv. tabaci

Hollow stalk *Erwinia* sp. 120 Solanaceae

Various 350 29

Various many Most common:

Bacterial Wilt *Pseudomonas*

**Species**


**Disease Causal Agent Hosts**

Tobacco Leaf Curl Virus (TLCV)

Beet Curly Top Virus (BCTV)

Tobacco Rattle Virus (TRV)

Tobacco Ringspot Virus (TRSV)

**Species**

Various Many 14

Various Many Many

**Plant Families Common Weedy Hosts**

*Datura* sp. *Physalis* sp. *Solanum* sp. *Sida* sp. Least snoutbean

Henbit

(*Rhynchosia minima* [L.] DC)

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 181

(*Acanthospermum hispidum* L.)

(*Capsella bursa-pastoris* L.) Black nightshade (*Solanum nigrum* L.) Common chickweed (*Stellaria media* L.)

(*Lamium amplexicaule* L.) Redroot pigweed

(*Amaranthus retroflexus* L.)

Spiny sowthistle (*Sonchus asper* [L.] All.)

(*Descurainia sophia* L.) Redstem filaree (*Erodium cicutarium* L.)

Common ragweed (*Ambrosia artemisiifolia* L.)

(*Taraxacum officinale* L.)

(*Solanum carolinense* L.)

(*Datura stramonium* L.)

Wild carrot (*Daucus carota* L.) Dandelion

Horsenettle

Groundcherry (*Physalis* sp.) Common pokeweed (*Phytolacca americana* L.)

Jimsonweed

Flixweed

Most common: Malvaceae Euphorbiaceae Fabaceae Solanaceae

Various 244 Bristly starbur

Various 380 Many Shepards-purse

Most common: Solanaceae Compositae Cucurbitaceae Scrophulariaceae


**Disease Causal Agent Hosts**

180 Herbicides - Current Research and Case Studies in Use

Cucumber Mosaic Virus (CMV)

Tobacco Etch Virus (TEV)

Tobacco Vein Mottle Virus (TVMV)

Peanut Stunt Virus

Alfalfa Mosaic Virus (AMV)

(PSV)

**Species**

Various many 36 dicot families

Various 69 11 *Solanum* sp.

Various *Solanaceae* Horsenettle

Solanaceae

Various 305 47 Jimsonweed

Bushy Top Virus Various *Solanaceae* Jimsonweed

Various Fabaceae

4 monocot families

**Plant Families Common Weedy Hosts**

Blue toadflax (Linaria canadensis) Carolina desert-chicory (Pyrrhopappus carolinianus)

Wild radish

Dayflower

(Raphanus raphanistrum) Venus' looking-glass (Triodanis perfoliata)

Carolina geranium (*Geranium carolineanum* L.) Cutleaf groundcherry (*Physalis angulata* L.)

(*Commelina nudiflora* L.) American pokeweed

Caulkins & Wyatt Common Chickweed (*Stellaria media* L.) Jimsonweed

(*Datura stramonium* L.) Chenopodium sp.

(*Datura stramonium* L.)

(*Solanum carolinense* L.) Cutleaf groundcherry (*Physalis angulata* L.)

(*Datura stramonium* L.) Apple of Peru

(*Datura strumonium* L.)

(*Datura stramonium* L.)

Kudzu

Jimsonweed

(*Nicandra physaloides* [L.] Scop.)

(*Pueraria thumbergiana* [Sieb. & Succ.] Benth.)

Jimsonweed

(*Phytolacca americana* [L.] *var. rigida* [Small]


**Disease Causal Agent Hosts**

*basicola* (Berk. And Br.) Ferraris

*seoli* (Maubl.)

Brown Spot *Alternaria alternata* 56 19

*cichoracearum* DC

*nicotianae* Ellis. & Everhart

nicotianae Boning

tabacina D. B. Adam

Black Root Rot *Thielaviopsis*

Charcoal Rot *Macrophominapha*

Blue Mold Peronospora

Powdery mildew *Erysiphe*

Frogeye leafspot *Cercospora*

Anthracnose Colletotrichum

**Species**

>300

Mainly *Nicotiana*

137 33

Most common: Fabacae Solanaceae Cucurbitaceae

Most common: *Solanaceae*

Main families: Cucurbitaceae Compositae

Many Many Some grasses

Many 115 genera

28 16

**Table 1.** Common weeds that serve as alternate hosts for tobacco diseases.

**Plant Families Common Weedy Hosts**

*Solanaceae* Poorman's orchid

White poplar (*Populus alba* L.)

(*Schizanthus pinnatus* Ruiz & Pav.

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 183

Egyptian henbane) (*Hyoscyamus muticus* L.) Lanceleaf groiundcherry (*Physalis lancifolia* L.)

(*Atropa belladonna* L.) Apple of Peru

(*Datura stramonium* L.) Apple of Peru

Common pokeweed (*Phytolacca americana* L.)

Geranium (*Geranium* sp.) Lettuce (*Lactuca* sp.)

*Nicandra physalodes* (L.) Scop.)

(*Nicandra physalodes* [L.] Scop.)

Belladonna

Jimsonweed


**Table 1.** Common weeds that serve as alternate hosts for tobacco diseases.

**Disease Causal Agent Hosts**

182 Herbicides - Current Research and Case Studies in Use

*Olpidium brassicae* (Wor.) Dang

*Olpidium brassicae* (Wor.) Dang

Tobacco Streak Virus (TSV)

Tobacco necrosis virus (TNV)

Tobacco stunt virus (TSV)

Potato Virus Y (PVY)

Damping off Stem/root rot

Southern Stem/ Root Rot

Fusarium wilt *Fusarium*

Verticillium wilt *Verticillium*

Olpidium seedling

blight

Sore shin *Rhizoctonia solani* Kuhn

Sacc.

*oxysporum* (Schlecht) Wr. f. *nicotianae* Johnson

*Sclerotium rolfsii*

*alboatrum* Reinke and Berth

*Olpidium brassicae* (Wor.) Dang

**Species**

88 37

Various *Solanaceae* (most

*Pythium* sp. At least 270 genera

230 66

Many

189 *Compositae*

250 Dicots

Many Most common: Cruciferae Graminae Brassicacae

common), also *Amaranthaceae, Chenopodiaceae, Compositae, Fabaceae*

Various Many 31 Common burdock

**Plant Families Common Weedy Hosts**

(*Arctium minus* [Hill] Bernh.)

(*Datura stramonium* L.)

*Chenopodium* sp.

Shepards-purse

(*Capsella bursa-pastoris* [L.]Medik)

Common lambsquarters (*Chenopodium album* L.)

Field bindweed (*Convolvulus arvensis* L.)

Plantain (*Plantago* sp.) White clover (*Trifolium repens* L.) *Crotalaria* sp. Jimsonweed

#### *Nematodes*

Table 2 lists weed species that act as alternate hosts to nematodes that may infect tobacco.

*Insects*

**Insect Genus**

Green peach aphid Red tobacco aphid

Myzus persicae

Myzus nicotianae Many

Western Flower Thrips *Frankliniella* sp. Many Many

Flea beetle *Epitrix* sp. Many Many

Cabbage looper *Trichoplusia ni* Many

Table 3 lists weeds species that serve as alternate hosts for insects that may attack tobacco.

**Number of host species**

**Number of host**

Many

Most common: Solanaceae Amaranthaceae, Chenopodaceae, Compositae, Fabaceae Brassicacae

Solanaceae Brassicaceae

**families Common Weedy Hosts**

*Solanum* sp. *Chenopodium* sp. Groundcherry

Tansymustard (*Descurainia pinnata* L.)

Curly dock (*Rumex crispus* L.) Jimsonweed

Kudzu

Dandelion

Jimsonweed

Horsenettle

(*Physalis virginiana* Mill.) Virginia pepperweed (*Lepidium virginicum* L.)

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 185

(*Datura stramonium* L.) Common chickweed (*Stellaria media* L.) Dayflower (*Commelina* sp.)

(*Pueraria lobata* L.) Common ragweed (*Ambrosia artemisiifolia* L.)

(*Taraxacum officinale* L.) Spiny amaranth (*Amaranthus spinosus* L.)

(*Datura stramonium* L.)

(*Solanum carolinense* L.) Morningglory sp. (*Ipomoea* sp.)

Black nightshade (*Solanum nigrum* L.) Wild mustard (*Brassica napus* L.) Peppergrass (*Lepidium* spp.)


**Table 2.** Common weeds that serve as hosts for nematodes.

*Insects*

*Nematodes*

**Nematode species**

Root knot

Tobacco cyst nematode

Brown root rot (Lesion nematodes)

Stem-break (Stem and Bulb nematode)

Stubby root nematode

**Genus**

184 Herbicides - Current Research and Case Studies in Use

nematode *Meloidogyne* sp. >3,000

*Globodera* sp. At least 45

*Pratylenchus* sp. >500

*Trichodorus* sp. At least 51

**Table 2.** Common weeds that serve as hosts for nematodes.

*Ditylenchus dipsaci* [Kuhn] Filipjev

Stunt nematode *Tylenchorhynchus* sp. Many

Table 2 lists weed species that act as alternate hosts to nematodes that may infect tobacco.

>400 44

**Number of Plant**

Most major plant families.

Most common: *Solanaceae*

Most common: Graminae Fabaceae Solanaceae Compositae

Many

15

Most common: Fabaceae Graminae Euphorbiaceae

Common families: Graminae Solanaceae

Dicots and monocots.

**Families Common weedy hosts**

Large crabgrass (*Digitaria sanguinalis* L.)

(*Solanum carolinense* L.)

Horsenettle

Fescue (*Festuca* sp.) Lettuce (*Lactuca* sp.) Vetch (*Vicia* sp.) Wild onion

(*Allium canadense* L.)

(*Datura stramonium* L.)

Lespedeza (*Lespedeza* sp.) Showy crotalaria (*Crotalaria spectabilis* L.)

Jimsonweed

**Number of Hosts Species** Table 3 lists weeds species that serve as alternate hosts for insects that may attack tobacco.



**Insect Genus**

*Broadleaf seed species:*

*Grass weed species:*

**Table 3.** Common weeds that serve as hosts for insects.

**1.4. Most common and troublesome weeds in tobacco**

the world based on the 2006 survey of tobacco growing regions.

**Table 4.** Most common and troublesome weeds in tobacco worldwide.

**Number of host species**

It is not the intention here to list every possible weed problem that exists in tobacco. Some species can be found in numerous tobacco growing regions while others are region specific. However, several plant families do have species that are common and problematic in many tobacco production regions. According to a weed survey conducted across several tobaccogrowing regions of the world in 2006 (W. A. Bailey, unpublished data), the five most common and troublesome weed genera in tobacco are: *Amaranthus*, *Cyperus*, *Digitaria*, *Chenopodium*, and *Ipomoea.* Descriptions of each genera are adapted from references [15, 16, 17]. Table 4 lists the most common and troublesome weeds in the most prevalent tobacco growing regions around

**Weed Species Plant Family Scientific Name**

Redroot pigweed *Amaranthaceae* (pigweed family) *Amaranthus retroflexus* Yellow nutsedge *Cyperaceae* (sedge family) *Cyperus esculentus* Ivyleaf morningglory *Convolvulaceae* (morningglory family) *Ipomoea hederacea* Common lambsquarters *Chenopodiaceae* (Goosefoot family) *Chenopodium album* Common ragweed *Asteraceae* (sunflower family) *Ambrosia artemisiifolia* Horsenettle *Solanaceae* (nightshade family) *Solanum carolinense*

Large crabgrass *Poaceae* (grass family) *Digitaria sanguinalis* Goosegrass *Poaceae* (grass family) *Eleusine indica* Fall panicum *Poaceae* (grass family) *Panicum dichotomiflorum*

Giant foxtail *Poaceae* (grass family) *Setaria faberi* Johnsongrass *Poaceae* (grass family) *Sorghum halepense*

**Number of host**

**families Common Weedy Hosts**

(*Nuttallanthus* c*anadensis*

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 187

[L.] D.A. Sutton) Velvetleaf

Medik.)

(*Abutilon theophrasti*


**Table 3.** Common weeds that serve as hosts for insects.

**Insect Genus**

186 Herbicides - Current Research and Case Studies in Use

Cutworms *Lepidoptera* sp. Many Many

Budworm *Heliothis virescens* F. Many Many

Hornworm *Manduca sexta Solanaceae* only

**Number of host species**

**Number of host**

**families Common Weedy Hosts**

Field bindweed (*Convolvulus arvensis* L.)

Canada thistle (*Cirsium arvense* L.)

Horsenettle

Jimsonweed

Beardstongue (*Penstemon laevigatus*

Black medic

Cranesbill

Deergrass (*Rhexia* spp.) Dock (*Rumex* spp.) Groundcherry (*Physalis* spp.) Japanese honeysuckle (*Lonicera japonica* Thunb.)

Lupine (*Lupinus* spp.) Morningglory (*Ipomoea* spp.) Passionflower (*Passiflora* spp.) Prickly sida (*Sida spinosa* L.) Sunflower (*Helianthus* spp.) Toadflax

(*Medicago lupulina* L.)

(*Geranium dissectum* L.)

Aiton) Beggarweed (*Desmodium* spp.) Bicolor lespedeza (*Lespedeza bicolor* Turcz.)

(*Solanum carolinense* L.)

(*Datura stramonium* L.) Nightshade species

#### **1.4. Most common and troublesome weeds in tobacco**

It is not the intention here to list every possible weed problem that exists in tobacco. Some species can be found in numerous tobacco growing regions while others are region specific. However, several plant families do have species that are common and problematic in many tobacco production regions. According to a weed survey conducted across several tobaccogrowing regions of the world in 2006 (W. A. Bailey, unpublished data), the five most common and troublesome weed genera in tobacco are: *Amaranthus*, *Cyperus*, *Digitaria*, *Chenopodium*, and *Ipomoea.* Descriptions of each genera are adapted from references [15, 16, 17]. Table 4 lists the most common and troublesome weeds in the most prevalent tobacco growing regions around the world based on the 2006 survey of tobacco growing regions.


**Table 4.** Most common and troublesome weeds in tobacco worldwide.

### **2. Cultural practices for weed control in tobacco**

#### **2.1. Site selection, rotation, and scouting**

Integrated weed management involves using practices that reduce weed infestations but does not necessarily eliminate all weeds. Weed control can range from poor to excellent, depending on the characteristics of the weed species involved and the effectiveness of the control practices used. A small number of weeds with relatively lower competitive ability than tobacco can be allowed to remain in the crop without negatively influencing yield, quality, or harvest efficiency. Weed control practices available for tobacco can be placed into four general groups: 1) preventative; 2) cultural; 3) mechanical or physical; and 4) chemical.

tions, cultivation, or hand weeding. Scouting allows for timely operations that will be more

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 189

Where conservation tillage (no-tillage or strip-tillage) practices are not imposed, primary tillage with moldboard plowing, chisel plowing, and disking are the major methods used in field preparation for tobacco in the United States. Primary tillage is the major method of destroying weeds and preparing the ground for tobacco transplanting. Moldboard plowing is the primary means of turning under residue to allow decomposition and is most necessary with grass crops or annual grass weeds, while chisel plowing and disking are secondary tillage practices that aid in destruction of residue and help level the ground in preparation for tobacco transplanting. Field cultivators or mechanical rotary tillers are also used as a finishing tool just

Mechanical cultivation is still a necessary supplemental weed control practice in conventional tillage tobacco production because herbicides generally do not control all weeds that occur in tobacco production. Cultivation can also aid in soil aeration when soil crusting occurs, but also contributes to soil erosion and soil drying near the surface. No more than two cultivations are necessary for tobacco. Excessive or late cultivation can injure tobacco root systems, causing problems with water and nutrient uptake while also potentially increasing problems with tobacco mosaic virus, black shank (*Phytophthora nicotianae* Breda de Haan), and Granville wilt (*Pseudomonas solanacearum* E. F. Smith). Cultivation should be made shallow in the top 5 cm of soil so that tobacco roots are not injured and weed seed present below the herbicide treated

Herbicides play an important role in weed control, particularly in commercial tobacco production in more developed countries. Of all the pesticides used in tobacco production, herbicides make up the smallest percentage, approximately 10.4% [18]. The number of herbicides registered for use in tobacco has remained constant for several years and exhibits little signs of growth. There are approximately 50 different chemicals registered for use as herbicides for tobacco worldwide and they take on many different trade names and formula‐ tions depending on which regions they are used in. Recently, the presence of generic manu‐ facturers has played an increasing role with many of these products having varying compositions and labels that may differ significantly from the original manufacturer's specifications. Although several herbicides are registered for control of weed species in tobacco, certain herbicides are not registered in all countries or regions. Readers should refer to herbicide registrations for the specific country or region of interest, and follow use instruc‐

Similar to common names of weeds, trade names of herbicides vary around the world depending on the company marketing the product, local regulations, and regulatory param‐

effective than attempting to control weeds after they become more mature.

**2.2. Field preparation and cultivation**

area are not disturbed and allowed to germinate.

tions given on all product labels.

**3. Herbicides used for weed control in tobacco**

prior to transplanting.

Preventative weed control involves taking measures to prevent the introduction, establish‐ ment, or spread of weed species into areas that are not currently infested with these species. Preventative weed control practices for tobacco can include measures such as using weed-free seed and weed-free transplants, weed-free animal manures if manures are used as a nutrient source, weed-free transplanting and tillage equipment, and elimination of weed infestations in areas bordering tobacco fields. Preventative weed control can also include manually eradicating weeds in and around fields before they can mature and produce seed to proliferate their infestation.

Choosing sites for tobacco production that have low weed populations is also a major means of preventative weed control. Many sites may have good production characteristics, such as well-drained, fertile soil, with minimal potential for erosion or loss from disease, but may contain heavy populations of highly competitive weeds that can limit tobacco production. Some fields may become so infested with heavy populations of troublesome weeds that it is no longer feasible to grow tobacco in those fields, even when the most appropriate herbicides are used correctly. Sites chosen for tobacco production should have relatively low weed populations and, ideally, should not contain weed species that cannot be controlled by herbicides registered for use in tobacco.

Proper site selection for tobacco involves planning, observation, and knowledge of weed populations in fields several seasons prior to growing tobacco in those fields. Entire fields or portions of fields that contain particularly noxious or troublesome weeds should be avoided. Fields being considered for tobacco production should be observed while they are fallow and while they are in production of other crops for at least 2 seasons in order to get an idea of the weed species that are present. Having knowledge of the weed species that will occur in a field and where the heaviest infestations occur will help the grower plan the best choice of herbicide system, application rate and method, and total weed management system.

Once a site is chosen and tobacco is transplanted, scouting during the production season is also an important means of cultural weed control. Scouting involves intensively observing the crop on a weekly basis in at least four random areas of each hectare in the field. Weekly scouting is important to reveal the status of emerging weed problems in the field, but also to observe any potential insect and disease problems that may be developing. Knowing the status of weeds in the field allows for planning of any needed control measures of herbicide applica‐ tions, cultivation, or hand weeding. Scouting allows for timely operations that will be more effective than attempting to control weeds after they become more mature.

#### **2.2. Field preparation and cultivation**

**2. Cultural practices for weed control in tobacco**

1) preventative; 2) cultural; 3) mechanical or physical; and 4) chemical.

Integrated weed management involves using practices that reduce weed infestations but does not necessarily eliminate all weeds. Weed control can range from poor to excellent, depending on the characteristics of the weed species involved and the effectiveness of the control practices used. A small number of weeds with relatively lower competitive ability than tobacco can be allowed to remain in the crop without negatively influencing yield, quality, or harvest efficiency. Weed control practices available for tobacco can be placed into four general groups:

Preventative weed control involves taking measures to prevent the introduction, establish‐ ment, or spread of weed species into areas that are not currently infested with these species. Preventative weed control practices for tobacco can include measures such as using weed-free seed and weed-free transplants, weed-free animal manures if manures are used as a nutrient source, weed-free transplanting and tillage equipment, and elimination of weed infestations in areas bordering tobacco fields. Preventative weed control can also include manually eradicating weeds in and around fields before they can mature and produce seed to proliferate

Choosing sites for tobacco production that have low weed populations is also a major means of preventative weed control. Many sites may have good production characteristics, such as well-drained, fertile soil, with minimal potential for erosion or loss from disease, but may contain heavy populations of highly competitive weeds that can limit tobacco production. Some fields may become so infested with heavy populations of troublesome weeds that it is no longer feasible to grow tobacco in those fields, even when the most appropriate herbicides are used correctly. Sites chosen for tobacco production should have relatively low weed populations and, ideally, should not contain weed species that cannot be controlled by

Proper site selection for tobacco involves planning, observation, and knowledge of weed populations in fields several seasons prior to growing tobacco in those fields. Entire fields or portions of fields that contain particularly noxious or troublesome weeds should be avoided. Fields being considered for tobacco production should be observed while they are fallow and while they are in production of other crops for at least 2 seasons in order to get an idea of the weed species that are present. Having knowledge of the weed species that will occur in a field and where the heaviest infestations occur will help the grower plan the best choice of herbicide

Once a site is chosen and tobacco is transplanted, scouting during the production season is also an important means of cultural weed control. Scouting involves intensively observing the crop on a weekly basis in at least four random areas of each hectare in the field. Weekly scouting is important to reveal the status of emerging weed problems in the field, but also to observe any potential insect and disease problems that may be developing. Knowing the status of weeds in the field allows for planning of any needed control measures of herbicide applica‐

system, application rate and method, and total weed management system.

**2.1. Site selection, rotation, and scouting**

188 Herbicides - Current Research and Case Studies in Use

herbicides registered for use in tobacco.

their infestation.

Where conservation tillage (no-tillage or strip-tillage) practices are not imposed, primary tillage with moldboard plowing, chisel plowing, and disking are the major methods used in field preparation for tobacco in the United States. Primary tillage is the major method of destroying weeds and preparing the ground for tobacco transplanting. Moldboard plowing is the primary means of turning under residue to allow decomposition and is most necessary with grass crops or annual grass weeds, while chisel plowing and disking are secondary tillage practices that aid in destruction of residue and help level the ground in preparation for tobacco transplanting. Field cultivators or mechanical rotary tillers are also used as a finishing tool just prior to transplanting.

Mechanical cultivation is still a necessary supplemental weed control practice in conventional tillage tobacco production because herbicides generally do not control all weeds that occur in tobacco production. Cultivation can also aid in soil aeration when soil crusting occurs, but also contributes to soil erosion and soil drying near the surface. No more than two cultivations are necessary for tobacco. Excessive or late cultivation can injure tobacco root systems, causing problems with water and nutrient uptake while also potentially increasing problems with tobacco mosaic virus, black shank (*Phytophthora nicotianae* Breda de Haan), and Granville wilt (*Pseudomonas solanacearum* E. F. Smith). Cultivation should be made shallow in the top 5 cm of soil so that tobacco roots are not injured and weed seed present below the herbicide treated area are not disturbed and allowed to germinate.

### **3. Herbicides used for weed control in tobacco**

Herbicides play an important role in weed control, particularly in commercial tobacco production in more developed countries. Of all the pesticides used in tobacco production, herbicides make up the smallest percentage, approximately 10.4% [18]. The number of herbicides registered for use in tobacco has remained constant for several years and exhibits little signs of growth. There are approximately 50 different chemicals registered for use as herbicides for tobacco worldwide and they take on many different trade names and formula‐ tions depending on which regions they are used in. Recently, the presence of generic manu‐ facturers has played an increasing role with many of these products having varying compositions and labels that may differ significantly from the original manufacturer's specifications. Although several herbicides are registered for control of weed species in tobacco, certain herbicides are not registered in all countries or regions. Readers should refer to herbicide registrations for the specific country or region of interest, and follow use instruc‐ tions given on all product labels.

Similar to common names of weeds, trade names of herbicides vary around the world depending on the company marketing the product, local regulations, and regulatory param‐ eters. With any pesticide application, it is essential that the correct product be selected for the identified target weed species and that the product has a legal registration for use on tobacco in a given country. There may also be cases where a product has a legal registration for use on tobacco in that country but the tobacco manufacturers do not want the product applied to the crop due to leaf residue issues or other concerns. Over the past two decades, analytical techniques have allowed manufacturing companies to accurately evaluate residue levels of tobacco pesticides on cured leaves. In some cases, these residue levels have prompted companies to discourage the use of some products.

liquid formulation and the normal use rate is approximately 2.2 to 3.4 kg ai/ha. NOTE: Alachlor is a restricted use herbicide due to oncogenicity (tumor causing potential in laboratory animals) and alachlor has also been identified as having the potential to leach through the soil into ground water, particularly where soils are coarse and groundwater is near the surface [19, 20].

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 191

Clomazone is a carotenoid and chlorophyll inhibitor that causing bleaching/whitening in susceptibleplants.Clomazonecontrolsseveralcommonannualgrassesspeciessuchascrabgrass (*Digitaria* spp.), *Panicum* spp., and foxtails (*Setaria* spp.). In addition to grass control, cloma‐ zone also controls jimsonweed (*Datura stramonium* L.), common lambsquarters (*Chenopodium album*L.),hairygalinsoga (*Galinsoga quadriradiata*Cav.), commonragweed(*Ambrosia artemisiifo‐ lia*L.*),*andvelvetleaf(*Abutilontheophrasti*Medik.).Clomazoneisnormallyappliedasasoilsurface PRETR application, but can also be applied over-the-top of tobacco within 7 days of transplant‐ ing as tobacco shows good tolerance to this herbicide. Although clomazone is usually applied to the soil surface with no incorporation, it can be incorporated into the soil surface provided that caution is taken not to incorporate deeper than 5 cm. Clomazone is available in liquid

Metolachlor is a chloroacetamide herbicide similar to alachlor that has the same mode of action and same basic spectrum of weed activity, controlling numerous annual grass weeds and yellow nutsedge (*Cyperus esculentus* L.), but has limited activity against broadleaf weeds. Metolachlor applications for tobacco are normally applied prior to transplanting and shallowly incorporated in the top 2.5 to 5 cm of soil, but may also be applied pretransplant without incorporation. Metolachlor is normally a liquid formulation and the use rate is approximately

Napropamide is an acid amide herbicide that inhibits several metabolic processes including lipid biosynthesis and the synthesis of proteins and gibberellins. Napropamide is used primarily for the control of annual grasses such as crabgrass (*Digitaria* spp.), *Panicum* spp., and foxtails (*Setaria* spp.). Napropamide also provides some control of small-seeded broadleaf weeds such as pigweed (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). Napropamide is highly volatile and should be mechanically incorporated immediately after application, and preferably in the same operation as the application. Application of napropamide is normally made prior to transplanting. Napropamide is available in dry and

Pebulate is a thiocarbamate herbicide that inhibits lipid formation in sensitive plants. Pebulate controls annual grasses such as crabgrass (*Digitaria* spp.) and foxtails (*Setaria* spp.) as well as

liquid formulations and the normal use rate is approximately 1.1 kg ai/ha [19, 20].

formulations and the normal use rate is approximately 0.84 to 1.1 kg ai/ha [19, 20].

*3.1.2. Clomazone*

*3.1.3. Metolachlor*

1.1 to 2.1 kg ai/ha [19, 20].

*3.1.4. Napropamide*

*3.1.5. Pebulate*

Herbicides may be applied in many different ways, but most herbicides for use in tobacco are applied to the soil prior to weed emergence, and many must be applied prior to tobacco transplanting. Some of these herbicides are applied as pretransplant surface (PRETR) appli‐ cations and others are applied as pretransplant incorporated (PTI) applications where the herbicide is mechanically incorporated into top 2.5 to 5 cm of soil. Seed of most annual weed species occur in this depth of soil and therefore it is advantageous to keep herbicides at this depth. All soil-applied herbicides need adequate soil moisture in order to be effective, and incorporation increases the availability of moisture for herbicide activation and prevents loss of the herbicide through volatilization into the atmosphere. Only a limited number of herbi‐ cides are registered for use in tobacco and none control all weeds that may occur. Therefore, much attention should be given to planning weed control strategies [19, 4].

Spray applicators should always remember to follow application instructions given on the label and also insure that the herbicide is registered for use in tobacco in the area where it is to be applied. The following is a listing and description of herbicides currently used in tobacco in various parts of the world for control of grasses, sedges, and broadleaf weeds. The general application guidelines described and weed spectrum of control are based on the use of these herbicides in tobacco within the United States. Consult the product labels of these herbicides for additional information.

#### **3.1. Herbicides commonly used in tobacco**

On a worldwide basis, the most commonly used herbicides for tobacco include alachlor, clomazone, metolachlor, napropamide, pebulate, pendimethalin, sethoxydim, and sulfentra‐ zone. The following are descriptions of the weed control properties and basic use patterns.

#### *3.1.1. Alachlor*

Alachlor is a chloroacetamide herbicide that inhibits lipid biosynthesis and the synthesis of proteins, gibberellins, lignin, and anthocyanin production in susceptible plants. Alachlor controls many common annual grasses such as crabgrass (*Digitaria* sp.), foxtail (*Setaria* sp.), goosegrass (*Eleusine indica* [L.] Gaertn.), fall panicum (*Panicum dichotomiflorum* Michx.), and barnyardgrass (*Echinochloa crus-galli* [L.] P. Beauv.); as well as yellow nutsedge (*Cyperus esculentus* L.), but is of limited value for control of broadleaf weeds. Alachlor applications for tobacco are normally applied prior to transplanting and shallowly incorporated in the top 2.5 to 5 cm of soil, but may also be applied pretransplant without incorporation. Alachlor is a liquid formulation and the normal use rate is approximately 2.2 to 3.4 kg ai/ha. NOTE: Alachlor is a restricted use herbicide due to oncogenicity (tumor causing potential in laboratory animals) and alachlor has also been identified as having the potential to leach through the soil into ground water, particularly where soils are coarse and groundwater is near the surface [19, 20].

### *3.1.2. Clomazone*

eters. With any pesticide application, it is essential that the correct product be selected for the identified target weed species and that the product has a legal registration for use on tobacco in a given country. There may also be cases where a product has a legal registration for use on tobacco in that country but the tobacco manufacturers do not want the product applied to the crop due to leaf residue issues or other concerns. Over the past two decades, analytical techniques have allowed manufacturing companies to accurately evaluate residue levels of tobacco pesticides on cured leaves. In some cases, these residue levels have prompted

Herbicides may be applied in many different ways, but most herbicides for use in tobacco are applied to the soil prior to weed emergence, and many must be applied prior to tobacco transplanting. Some of these herbicides are applied as pretransplant surface (PRETR) appli‐ cations and others are applied as pretransplant incorporated (PTI) applications where the herbicide is mechanically incorporated into top 2.5 to 5 cm of soil. Seed of most annual weed species occur in this depth of soil and therefore it is advantageous to keep herbicides at this depth. All soil-applied herbicides need adequate soil moisture in order to be effective, and incorporation increases the availability of moisture for herbicide activation and prevents loss of the herbicide through volatilization into the atmosphere. Only a limited number of herbi‐ cides are registered for use in tobacco and none control all weeds that may occur. Therefore,

Spray applicators should always remember to follow application instructions given on the label and also insure that the herbicide is registered for use in tobacco in the area where it is to be applied. The following is a listing and description of herbicides currently used in tobacco in various parts of the world for control of grasses, sedges, and broadleaf weeds. The general application guidelines described and weed spectrum of control are based on the use of these herbicides in tobacco within the United States. Consult the product labels of these herbicides

On a worldwide basis, the most commonly used herbicides for tobacco include alachlor, clomazone, metolachlor, napropamide, pebulate, pendimethalin, sethoxydim, and sulfentra‐ zone. The following are descriptions of the weed control properties and basic use patterns.

Alachlor is a chloroacetamide herbicide that inhibits lipid biosynthesis and the synthesis of proteins, gibberellins, lignin, and anthocyanin production in susceptible plants. Alachlor controls many common annual grasses such as crabgrass (*Digitaria* sp.), foxtail (*Setaria* sp.), goosegrass (*Eleusine indica* [L.] Gaertn.), fall panicum (*Panicum dichotomiflorum* Michx.), and barnyardgrass (*Echinochloa crus-galli* [L.] P. Beauv.); as well as yellow nutsedge (*Cyperus esculentus* L.), but is of limited value for control of broadleaf weeds. Alachlor applications for tobacco are normally applied prior to transplanting and shallowly incorporated in the top 2.5 to 5 cm of soil, but may also be applied pretransplant without incorporation. Alachlor is a

much attention should be given to planning weed control strategies [19, 4].

companies to discourage the use of some products.

190 Herbicides - Current Research and Case Studies in Use

for additional information.

*3.1.1. Alachlor*

**3.1. Herbicides commonly used in tobacco**

Clomazone is a carotenoid and chlorophyll inhibitor that causing bleaching/whitening in susceptibleplants.Clomazonecontrolsseveralcommonannualgrassesspeciessuchascrabgrass (*Digitaria* spp.), *Panicum* spp., and foxtails (*Setaria* spp.). In addition to grass control, cloma‐ zone also controls jimsonweed (*Datura stramonium* L.), common lambsquarters (*Chenopodium album*L.),hairy galinsoga (*Galinsoga quadriradiata*Cav.), commonragweed(*Ambrosia artemisiifo‐ lia*L.*),*andvelvetleaf(*Abutilontheophrasti*Medik.).Clomazoneisnormallyappliedasasoilsurface PRETR application, but can also be applied over-the-top of tobacco within 7 days of transplant‐ ing as tobacco shows good tolerance to this herbicide. Although clomazone is usually applied to the soil surface with no incorporation, it can be incorporated into the soil surface provided that caution is taken not to incorporate deeper than 5 cm. Clomazone is available in liquid formulations and the normal use rate is approximately 0.84 to 1.1 kg ai/ha [19, 20].

#### *3.1.3. Metolachlor*

Metolachlor is a chloroacetamide herbicide similar to alachlor that has the same mode of action and same basic spectrum of weed activity, controlling numerous annual grass weeds and yellow nutsedge (*Cyperus esculentus* L.), but has limited activity against broadleaf weeds. Metolachlor applications for tobacco are normally applied prior to transplanting and shallowly incorporated in the top 2.5 to 5 cm of soil, but may also be applied pretransplant without incorporation. Metolachlor is normally a liquid formulation and the use rate is approximately 1.1 to 2.1 kg ai/ha [19, 20].

### *3.1.4. Napropamide*

Napropamide is an acid amide herbicide that inhibits several metabolic processes including lipid biosynthesis and the synthesis of proteins and gibberellins. Napropamide is used primarily for the control of annual grasses such as crabgrass (*Digitaria* spp.), *Panicum* spp., and foxtails (*Setaria* spp.). Napropamide also provides some control of small-seeded broadleaf weeds such as pigweed (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). Napropamide is highly volatile and should be mechanically incorporated immediately after application, and preferably in the same operation as the application. Application of napropamide is normally made prior to transplanting. Napropamide is available in dry and liquid formulations and the normal use rate is approximately 1.1 kg ai/ha [19, 20].

#### *3.1.5. Pebulate*

Pebulate is a thiocarbamate herbicide that inhibits lipid formation in sensitive plants. Pebulate controls annual grasses such as crabgrass (*Digitaria* spp.) and foxtails (*Setaria* spp.) as well as suppression of certain small-seeded broadleaf weeds such as pigweeds (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). In addition, pebulate is one of the few herbicides available for use in tobacco that provides good suppression of nutsedge sp. (*Cyperus* spp.). Similar to napropamide, pebulate is highly volatile and should be incorporated immediately after application, preferably in the same operation. Pebulate is applied prior to tobacco transplanting at a use rate of approximately 4.5 kg ai/ha [19, 20].

species (*Polygonum* spp.), pigweed species (*Amaranthus* spp.), and common lambsquarters (*Chenopodium album* L.). Sulfentrazone must be applied prior to transplanting tobacco and should be applied to the soil surface without incorporation. If incorporation is used, it must not be deeper than 5 cm from the soil surface. Currently, sulfentrazone is also marketed in the United States in a prepackaged combination with carfentrazone. Carfentrazone is a postemer‐ gence burn down herbicide designed for broadleaf weed control prior to transplanting. Sulfentrazone is available as a liquid formulation and normal use rate is approximately 0.28

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 193

No-tillage and strip-tillage tobacco production requires that any existing vegetation, whether it be weed growth or cover crop, be killed prior to transplanting tobacco without using extensive tillage as in conventional tillage tobacco production. Paraquat is a common herbicide that is used as a burndown prior to tobacco transplanting in no-tillage tobacco in the United States. Paraquat should be applied as a broadcast application to actively growing weeds or cover crops no larger than approximately 15 cm in height. Use rates for paraquat for burndown prior to tobacco transplanting are approximately 0.7 to 1.1 kg ai/ha. Glyphosate may also be used to burndown existing vegetation prior to tobacco transplanting as a broadcast application at approximately 0.28 kg ai/ha. Glyphosate should be applied 30 days or more prior to tobacco transplanting and paraquat should be applied several days prior to tobacco transplanting. Carfentrazone may also be used in conservation tillage tobacco prior to transplanting at use rates up to 0.027 kg ai/ha. Carfentrazone has generally not been as effective as paraquat or

Although there are a limited number of herbicides registered for tobacco relative to other crops that occupy more total area, the herbicides available for use in tobacco generally provide adequate weed control, particularly when supplemented with cultivation in conventional

The following are results from herbicide experiments conducted in dark tobacco in western Kentucky USA from 2005 to 2007. Treatments included all residual herbicides that were currently registered for use in tobacco. Soil type was a Grenada silt loam (fine-silty, mixed, thermic Oxyaquic Fraglossudalf) with 1.8% organic matter and pH of 6.4. Tobacco plots were prepared by conventional tillage with moldboard plowing and disking. Final field preparation and incorporation of herbicide treatments that required incorporation was done with a field cultivator. Fertilization and other crop production practices were according to standard recommendations [21]. Experiments were arranged in a randomized complete block design with 4 replications and plots were 4 rows, 4.1 m wide by 12.2 m long. Herbicide treatments were applied one day prior to transplanting as broadcast applications using CO2-pressurized sprayers with flat fan nozzles calibrated to deliver 187 L/ha at 120 kPa. 'Narrowleaf Madole' dark tobacco was then transplanted on 1-m row spacing and 81-cm plant spacing within rows. Crop injury and weed control was evaluated using a 0 to 100% scale where 0 = no plant injury

*3.1.9. Burndown of weeds or cover crops in conservation tillage production systems*

glyphosate for pretransplant burndown in conservation tillage tobacco [19].

**3.2. Weed control expected from herbicides used in tobacco**

to 0.42 kg ai/ha [19, 20].

tillage production systems.

#### *3.1.6. Pendimethalin*

Pendimethalin is a dinitroanaline herbicide that inhibits mitosis in susceptible plants. Pendi‐ methalin provides excellent control of annual grasses and certain small-seeded broadleaf weeds. Pendimethalin provides excellent control of crabgrass species (*Digitaria* spp.), foxtail species (*Setaria* spp.), *Panicum* species, and goosegrass (*Eleusine indica* [L.] Gaertn.), and also provides some control of broadleaf species such as pigweed (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). Pendimethalin is normally applied as a PTI application to a well-prepared soil surface up to 60 days prior to transplanting tobacco. Pendimethalin should be incorporated into the top 2.5 to 5 cm of soil within 7 days after application. Pendi‐ methalin is available as liquid formulations and normal use rate is approximately 1.4 to 1.7 kg ai/ha [19, 20].

#### *3.1.7. Sethoxydim*

Sethoxydim is a cyclohexanedione herbicide that inhibits lipid biosynthesis in susceptible grass species. Sethoxydim only controls grasses, so it is totally safe to broadleaf crops such as tobacco. Sethoxydim has no soil residual activity and is the only true postemergence herbicide that can be applied over-the-top of tobacco later than 7 days after transplanting. Sethoxydim may be applied up to 42 days prior to tobacco harvest. Sethoxydim is effective on annual grass species such as crabgrass (*Digitaria* spp.), *Panicum* species, and foxtails (*Setaria* spp.), and also controls perennial grasses such as shattercane (*Sorghum bicolor* L.) and Johnsongrass (*Sorghum halepense* L.). Application must be made to emerged, actively growing grasses to be effective. For perennial shattercane and Johnsongrass, sethoxydim is most effective if grass plants are allowed to get 45 to 60 cm tall before application. Do not cultivate within 5 days before application or 7 days after application. Crop oil concentrate at 1% of the spray volume per hectare is recommended with sethoxydim application. Recommended rates of sethoxydim are approximately 0.3 kg ai/ha. For spot treatment by hand, prepare 1 to 1.5% sethoxydim solution with 1% crop oil concentrate and spray grass plants until wetted [19, 20].

#### *3.1.8. Sulfentrazone*

Sulfentrazone is an aryl triazolinone herbicide that inhibits photosynthesis by inhibiting the enzyme protoporphyrinogen oxidase. Sulfentrazone provides partial control and suppression of annual grasses such as crabgrass (*Digitaria* spp.), *Panicum* sp., foxtails (*Setaria* spp.), and goosegrass (*Eleusine indica* L.). However, its main attribute is control of nutsedge species (*Cyperus* spp.) and troublesome broadleaf weed species such as nightshade species (*Solanum* spp.), groundcherry species (*Physalis* spp.), morningglory species (*Ipomoea* spp.), smartweed species (*Polygonum* spp.), pigweed species (*Amaranthus* spp.), and common lambsquarters (*Chenopodium album* L.). Sulfentrazone must be applied prior to transplanting tobacco and should be applied to the soil surface without incorporation. If incorporation is used, it must not be deeper than 5 cm from the soil surface. Currently, sulfentrazone is also marketed in the United States in a prepackaged combination with carfentrazone. Carfentrazone is a postemer‐ gence burn down herbicide designed for broadleaf weed control prior to transplanting. Sulfentrazone is available as a liquid formulation and normal use rate is approximately 0.28 to 0.42 kg ai/ha [19, 20].

#### *3.1.9. Burndown of weeds or cover crops in conservation tillage production systems*

suppression of certain small-seeded broadleaf weeds such as pigweeds (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). In addition, pebulate is one of the few herbicides available for use in tobacco that provides good suppression of nutsedge sp. (*Cyperus* spp.). Similar to napropamide, pebulate is highly volatile and should be incorporated immediately after application, preferably in the same operation. Pebulate is applied prior to

Pendimethalin is a dinitroanaline herbicide that inhibits mitosis in susceptible plants. Pendi‐ methalin provides excellent control of annual grasses and certain small-seeded broadleaf weeds. Pendimethalin provides excellent control of crabgrass species (*Digitaria* spp.), foxtail species (*Setaria* spp.), *Panicum* species, and goosegrass (*Eleusine indica* [L.] Gaertn.), and also provides some control of broadleaf species such as pigweed (*Amaranthus* spp.) and common lambsquarters (*Chenopodium album* L.). Pendimethalin is normally applied as a PTI application to a well-prepared soil surface up to 60 days prior to transplanting tobacco. Pendimethalin should be incorporated into the top 2.5 to 5 cm of soil within 7 days after application. Pendi‐ methalin is available as liquid formulations and normal use rate is approximately 1.4 to 1.7 kg

Sethoxydim is a cyclohexanedione herbicide that inhibits lipid biosynthesis in susceptible grass species. Sethoxydim only controls grasses, so it is totally safe to broadleaf crops such as tobacco. Sethoxydim has no soil residual activity and is the only true postemergence herbicide that can be applied over-the-top of tobacco later than 7 days after transplanting. Sethoxydim may be applied up to 42 days prior to tobacco harvest. Sethoxydim is effective on annual grass species such as crabgrass (*Digitaria* spp.), *Panicum* species, and foxtails (*Setaria* spp.), and also controls perennial grasses such as shattercane (*Sorghum bicolor* L.) and Johnsongrass (*Sorghum halepense* L.). Application must be made to emerged, actively growing grasses to be effective. For perennial shattercane and Johnsongrass, sethoxydim is most effective if grass plants are allowed to get 45 to 60 cm tall before application. Do not cultivate within 5 days before application or 7 days after application. Crop oil concentrate at 1% of the spray volume per hectare is recommended with sethoxydim application. Recommended rates of sethoxydim are approximately 0.3 kg ai/ha. For spot treatment by hand, prepare 1 to 1.5% sethoxydim solution

Sulfentrazone is an aryl triazolinone herbicide that inhibits photosynthesis by inhibiting the enzyme protoporphyrinogen oxidase. Sulfentrazone provides partial control and suppression of annual grasses such as crabgrass (*Digitaria* spp.), *Panicum* sp., foxtails (*Setaria* spp.), and goosegrass (*Eleusine indica* L.). However, its main attribute is control of nutsedge species (*Cyperus* spp.) and troublesome broadleaf weed species such as nightshade species (*Solanum* spp.), groundcherry species (*Physalis* spp.), morningglory species (*Ipomoea* spp.), smartweed

with 1% crop oil concentrate and spray grass plants until wetted [19, 20].

tobacco transplanting at a use rate of approximately 4.5 kg ai/ha [19, 20].

*3.1.6. Pendimethalin*

192 Herbicides - Current Research and Case Studies in Use

ai/ha [19, 20].

*3.1.7. Sethoxydim*

*3.1.8. Sulfentrazone*

No-tillage and strip-tillage tobacco production requires that any existing vegetation, whether it be weed growth or cover crop, be killed prior to transplanting tobacco without using extensive tillage as in conventional tillage tobacco production. Paraquat is a common herbicide that is used as a burndown prior to tobacco transplanting in no-tillage tobacco in the United States. Paraquat should be applied as a broadcast application to actively growing weeds or cover crops no larger than approximately 15 cm in height. Use rates for paraquat for burndown prior to tobacco transplanting are approximately 0.7 to 1.1 kg ai/ha. Glyphosate may also be used to burndown existing vegetation prior to tobacco transplanting as a broadcast application at approximately 0.28 kg ai/ha. Glyphosate should be applied 30 days or more prior to tobacco transplanting and paraquat should be applied several days prior to tobacco transplanting. Carfentrazone may also be used in conservation tillage tobacco prior to transplanting at use rates up to 0.027 kg ai/ha. Carfentrazone has generally not been as effective as paraquat or glyphosate for pretransplant burndown in conservation tillage tobacco [19].

#### **3.2. Weed control expected from herbicides used in tobacco**

Although there are a limited number of herbicides registered for tobacco relative to other crops that occupy more total area, the herbicides available for use in tobacco generally provide adequate weed control, particularly when supplemented with cultivation in conventional tillage production systems.

The following are results from herbicide experiments conducted in dark tobacco in western Kentucky USA from 2005 to 2007. Treatments included all residual herbicides that were currently registered for use in tobacco. Soil type was a Grenada silt loam (fine-silty, mixed, thermic Oxyaquic Fraglossudalf) with 1.8% organic matter and pH of 6.4. Tobacco plots were prepared by conventional tillage with moldboard plowing and disking. Final field preparation and incorporation of herbicide treatments that required incorporation was done with a field cultivator. Fertilization and other crop production practices were according to standard recommendations [21]. Experiments were arranged in a randomized complete block design with 4 replications and plots were 4 rows, 4.1 m wide by 12.2 m long. Herbicide treatments were applied one day prior to transplanting as broadcast applications using CO2-pressurized sprayers with flat fan nozzles calibrated to deliver 187 L/ha at 120 kPa. 'Narrowleaf Madole' dark tobacco was then transplanted on 1-m row spacing and 81-cm plant spacing within rows. Crop injury and weed control was evaluated using a 0 to 100% scale where 0 = no plant injury and 100 = plant death [22]. Tobacco injury data shown in Table 5 is from 2 weeks following transplanting while weed control data shown in Table 6 is from one week prior to harvest. Dark tobacco was fire-cured using standard practices [21] and yield and quality data are shown in Table 7.

In addition, weeds can more indirectly affect tobacco by harboring several major tobacco diseases, insects, and nematodes. Weed control practices for tobacco include field site selection, rotation, scouting, and many fields receive intensive tillage prior to transplanting and cultivation following transplanting. In many areas of the world, weed control for tobacco is almost exclusively a manual task using hand weeding and animal-drawn cultivation imple‐ ments. Although tobacco is not a food crop, the high value of tobacco relative to other crops

In more developed regions, however, the use of herbicides is the main component of weed control practices in tobacco. Mechanical cultivation is still used to supplement herbicides in most fields, as no-tillage or reduced tillage production systems have not been adopted as readily in tobacco as in other crops like corn, soybean, and small grains. Although only a limited number of herbicides are available for use in tobacco compared to grain crops, the herbicides that are available have generally provided adequate weed control, particularly when supplemented with cultivation. Of the herbicides that are available, combinations of two herbicides are generally more effective than a single herbicide and some herbicide combina‐ tions are more effective than others. Data presented here indicate that sulfentrazone plus clomazone or pendimethalin followed by sulfentrazone were the most effective herbicide

**Herbicide Treatment Application Timing Application Rate 2005 2006 2007**

Sulfentrazone PRETRb 0.42 2 bc 3 bc 0 b Clomazone PRETR 1.12 1 bc 0 c 0 b Sulfentrazone + Clomazone PRETR 0.42 + 1.12 3 bc 4 b 0 b Pendimethalin PTIa 1.66 5 b 11 a 2 a

Pebulate PTI 4.48 2 bc 3 bc 0 b Napropamide PTI 2.24 1 bc 2 bc 0 b Pebulate + Napropamide PTI 4.48 + 2.24 2 bc 5 b 0 b Untreated Control - - 0 bc 0 c 0 b

Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007. Injury data presented by

Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD

b Abbreviations: fb = followed by; PRETR = pretransplant; PTI = pretransplant incorporated.

**Table 5.** Early-season tobacco injury observed from herbicide treatments.

**Tobacco Injuryc**

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008 195


PTI fb PRETRb 1.66 + 0.42 10 a 5 b 2 a

makes manual weed management practices economically feasible in some regions.

programs for weed control in dark tobacco.

Pendimethalin fba Sulfentrazone

a

c

year.

at P=0.05.

Herbicide treatments evaluated included sulfentrazone, clomazone, sulfentrazone plus clomazone, pendimethalin, pendimethalin followed by sulfentrazone, pebulate, napropa‐ mide, and pebulate plus napropamide. All herbicide treatments were applied using maximum use rates allowed on U.S. labels. Sulfentrazone and clomazone treatments were applied as pretransplant applications to the soil surface while pendimethalin, pebulate, and napropamide treatments were incorporated immediately after application. Tobacco was cultivated twice early in the season following transplanting as is the standard practice.

As these data illustrate, there is potential to observe mild crop injury under some conditions following application of these tobacco herbicides (Table 5). Greatest potential for injury occurred following sulfentrazone and pendimethalin applications, although injury was never greater than 11% in any year and tobacco recovered quickly.

These data also illustrate that combinations of two tobacco herbicides provide more effective control of a broader spectrum of weeds than any one tobacco herbicide (Table 6). Sulfentra‐ zone applied alone effectively controlled yellow nutsedge and ivyleaf morningglory, but was not as effective on large crabgrass and common ragweed. Conversely, clomazone was effec‐ tiveonlarge crabgrassandcommonragweedbutnotas effectiveonyellownutsedgeandivyleaf morningglory. The most effective herbicide treatment evaluated across these four weed species was sulfentrazone and clomazone applied together. Pendimethalin followed by sulfentrazone was also a very effective treatment, but did not control common ragweed as well as sulfentra‐ zoneplus clomazone.Pebulateplusnapropamide alsoprovidedbetterweedcontrolthaneither herbicide applied alone, but this combination was still not as effective as sulfentrazone plus clomazone or pendimethalin followed by sulfentrazone on the weed species evaluated here.

Although obvious differences in weed control were seen, these differences did not always translate to yield, quality, or gross revenue differences (Table 7). Total yield of dark tobacco treated with herbicides ranged from 2,765 kg/ha with pendimethalin alone to 3,051 kg/ha with pendimethalin followed by sulfentrazone with minimal differences in total yield between treatments. Herbicide treatments increased total yield by at least 359 kg/ha compared to tobacco that was only cultivated without herbicide treatment. Differences in quality grade index were also few, ranging from 61.9 to 70.1 across all treatments. There were no differences is gross revenue between herbicide treatments, with gross revenue ranging from 11,163 to 12,911 \$USD/ha with herbicide treated tobacco, compared to 9,377 \$USD/ha with tobacco that was only cultivated with no herbicide treatment.

### **4. Conclusion**

Although tobacco is considered a very competitive crop, weeds can directly impact tobacco by limiting yield and quality, and causing interference of harvest and other field operations. In addition, weeds can more indirectly affect tobacco by harboring several major tobacco diseases, insects, and nematodes. Weed control practices for tobacco include field site selection, rotation, scouting, and many fields receive intensive tillage prior to transplanting and cultivation following transplanting. In many areas of the world, weed control for tobacco is almost exclusively a manual task using hand weeding and animal-drawn cultivation imple‐ ments. Although tobacco is not a food crop, the high value of tobacco relative to other crops makes manual weed management practices economically feasible in some regions.

and 100 = plant death [22]. Tobacco injury data shown in Table 5 is from 2 weeks following transplanting while weed control data shown in Table 6 is from one week prior to harvest. Dark tobacco was fire-cured using standard practices [21] and yield and quality data are shown

Herbicide treatments evaluated included sulfentrazone, clomazone, sulfentrazone plus clomazone, pendimethalin, pendimethalin followed by sulfentrazone, pebulate, napropa‐ mide, and pebulate plus napropamide. All herbicide treatments were applied using maximum use rates allowed on U.S. labels. Sulfentrazone and clomazone treatments were applied as pretransplant applications to the soil surface while pendimethalin, pebulate, and napropamide treatments were incorporated immediately after application. Tobacco was cultivated twice

As these data illustrate, there is potential to observe mild crop injury under some conditions following application of these tobacco herbicides (Table 5). Greatest potential for injury occurred following sulfentrazone and pendimethalin applications, although injury was never

These data also illustrate that combinations of two tobacco herbicides provide more effective control of a broader spectrum of weeds than any one tobacco herbicide (Table 6). Sulfentra‐ zone applied alone effectively controlled yellow nutsedge and ivyleaf morningglory, but was not as effective on large crabgrass and common ragweed. Conversely, clomazone was effec‐ tiveonlarge crabgrassandcommonragweedbutnotas effectiveonyellownutsedgeandivyleaf morningglory. The most effective herbicide treatment evaluated across these four weed species was sulfentrazone and clomazone applied together. Pendimethalin followed by sulfentrazone was also a very effective treatment, but did not control common ragweed as well as sulfentra‐ zoneplus clomazone.Pebulateplusnapropamide alsoprovidedbetterweedcontrolthaneither herbicide applied alone, but this combination was still not as effective as sulfentrazone plus clomazone or pendimethalin followed by sulfentrazone on the weed species evaluated here. Although obvious differences in weed control were seen, these differences did not always translate to yield, quality, or gross revenue differences (Table 7). Total yield of dark tobacco treated with herbicides ranged from 2,765 kg/ha with pendimethalin alone to 3,051 kg/ha with pendimethalin followed by sulfentrazone with minimal differences in total yield between treatments. Herbicide treatments increased total yield by at least 359 kg/ha compared to tobacco that was only cultivated without herbicide treatment. Differences in quality grade index were also few, ranging from 61.9 to 70.1 across all treatments. There were no differences is gross revenue between herbicide treatments, with gross revenue ranging from 11,163 to 12,911 \$USD/ha with herbicide treated tobacco, compared to 9,377 \$USD/ha with tobacco that

Although tobacco is considered a very competitive crop, weeds can directly impact tobacco by limiting yield and quality, and causing interference of harvest and other field operations.

early in the season following transplanting as is the standard practice.

greater than 11% in any year and tobacco recovered quickly.

was only cultivated with no herbicide treatment.

**4. Conclusion**

in Table 7.

194 Herbicides - Current Research and Case Studies in Use

In more developed regions, however, the use of herbicides is the main component of weed control practices in tobacco. Mechanical cultivation is still used to supplement herbicides in most fields, as no-tillage or reduced tillage production systems have not been adopted as readily in tobacco as in other crops like corn, soybean, and small grains. Although only a limited number of herbicides are available for use in tobacco compared to grain crops, the herbicides that are available have generally provided adequate weed control, particularly when supplemented with cultivation. Of the herbicides that are available, combinations of two herbicides are generally more effective than a single herbicide and some herbicide combina‐ tions are more effective than others. Data presented here indicate that sulfentrazone plus clomazone or pendimethalin followed by sulfentrazone were the most effective herbicide programs for weed control in dark tobacco.


a Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007. Injury data presented by year.

b Abbreviations: fb = followed by; PRETR = pretransplant; PTI = pretransplant incorporated.

c Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD at P=0.05.

**Table 5.** Early-season tobacco injury observed from herbicide treatments.


**Stalk Positionab Quality**

2877 ab

2828 ab

3004 ab

kg ai/ha -------------kg/ha-------------- 0-100 \$/ha

**Revenue Herbicide <sup>d</sup>**

PTI 4.48 + 2.24 370 ab 603 a 2031 a

Untreated Control - - 314 b 499 b 1592 a 2406 c 66.2 ab 9,377 b

Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007. Tobacco yield data pooled

Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD

bTobacco leaves removed by stalk position following fire-curing. Lug corresponds to lower stalk leaves, second from

Quality grade index is a numerical representation of Federal quality grade received for tobacco and is a weighted average

Department of Plant & Soil Sciences, University of Kentucky, Research and Education Cen‐

[1] United Nations Food and Agriculture OrganizationFood and Agriculture Organiza‐ tion Statistical Yearbook 2010. Food and Agriculture Organization Statistical Divison.

dGross revenue is the total gross value of tobacco (in \$USD) based on Federal grade and price support values.

**Table 7.** Effect of herbicide treatment on dark-fired tobacco yield, quality grade index, and gross revenuea

Abbreviations: fb = followed by; PRETR = pretransplant; PTI = pretransplant incorporated.

**Rate Lug Second Leaf Total**

**Treatment**

Pebulate + Napropamide

over years.

at P=0.05.

midstalk, and leaf from upper stalk.

of grade index for all stalk positions.

**Author details**

William A. Bailey

ter, Princeton, KY, USA

**References**

Address all correspondence to: abailey@uky.edu

a

a

c

e

**Application Timing**

**Application**

Pebulate PTI 4.48 351 ab 569 ab 1958 a

Napropamide PTI 2.24 355 ab 594 a 1879 a

**Grade Indexc** **Gross**

197

63.6 ab 11,779 a

Herbicides Used in Tobacco http://dx.doi.org/10.5772/56008

66.7 ab 12,067 a

65.9 ab 12,430 a

.

a Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007, weed control data pooled over years.

bAbbreviations: fb = followed by; PRETR = pretransplant surface application; PTI = pretransplant incorporated application.

c Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD at P=0.05.

**Table 6.** Late-season weed control from herbicides and herbicide systems currently used in dark tobacco production in the U.S.a



a Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007. Tobacco yield data pooled over years.

a Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD at P=0.05.

bTobacco leaves removed by stalk position following fire-curing. Lug corresponds to lower stalk leaves, second from midstalk, and leaf from upper stalk.

c Quality grade index is a numerical representation of Federal quality grade received for tobacco and is a weighted average of grade index for all stalk positions.

dGross revenue is the total gross value of tobacco (in \$USD) based on Federal grade and price support values.

e Abbreviations: fb = followed by; PRETR = pretransplant; PTI = pretransplant incorporated.

**Table 7.** Effect of herbicide treatment on dark-fired tobacco yield, quality grade index, and gross revenuea .

### **Author details**

**Weed Controlc**

**Common ragweed**

> **Quality Grade Indexc**

**Gross**

64.9 ab 12,497 a

70.1 a 12,911 a

64.4 ab 12,598 a

**Ivyleaf morningglor y**

**Yellow nutsedge**

**Herbicide Treatment Application**

196 Herbicides - Current Research and Case Studies in Use

Sulfentrazone + Clomazone

Pendimethalin fba Sulfentrazone

Pebulate + Napropamide

over years.

at P=0.05.

in the U.S.a

**Treatment**

Sulfentrazone + Clomazone

Pendimethalin fbe Sulfentrazone

**Application Timing**

**Application**

Sulfentrazone PRETR 0.42 405 a 580 ab 1992 a

Clomazone PRETR 1.12 355 ab 579 ab 2010 a

a

c

**Timing Application Rate Large**

Sulfentrazone PRETRb 0.42 61 c 91 a 31 e 90 b Clomazone PRETR 1.12 86 a 17 c 83 a 62 c

Pendimethalin PTIa 1.66 89 a 23 c 42 d 73 b

Pebulate PTI 4.48 54 c 77 b 53 c 35 de Napropamide PTI 2.24 72 b 22 c 68 b 31 e

Untreated Control - - 0 d 0 d 0 f 0 f

Data collected from herbicide trials conducted near Murray, KY USA in 2005, 2006, and 2007, weed control data pooled

bAbbreviations: fb = followed by; PRETR = pretransplant surface application; PTI = pretransplant incorporated application.

Means within a column followed by the same letter are not significantly different according to Fisher's Protected LSD

**Table 6.** Late-season weed control from herbicides and herbicide systems currently used in dark tobacco production

**Revenue Herbicide <sup>d</sup>**

PRETR 0.42 + 1.12 394 a 595 a 2028 a

Pendimethalin PTI 1.66 351 ab 565 ab 1843 a 2765 b 61.9 ab 11,163 ab

**Stalk Positionab**

PTI fb PRETR 1.66 + 0.42 375 ab 617 a 2059 a 3051 a 63.4 ab 11,883 a

kg ai/ha -------------kg/ha-------------- 0-100 \$/ha

2977 ab

2943 ab

3017 ab

**Rate Lug Second Leaf Total**

**crabgrass**


PRETR 0.42 + 1.12 89 a 96 a 85 a 97 a

PTI fb PRETRb 1.66 + 0.42 96 a 93 a 54 c 94 ab

PTI 4.48 + 2.24 75 b 78 b 71 b 39 d

William A. Bailey

Address all correspondence to: abailey@uky.edu

Department of Plant & Soil Sciences, University of Kentucky, Research and Education Cen‐ ter, Princeton, KY, USA

### **References**

[1] United Nations Food and Agriculture OrganizationFood and Agriculture Organiza‐ tion Statistical Yearbook 2010. Food and Agriculture Organization Statistical Divison. http://www.fao.org/docrep/015/am081m/PDF/am081m00b.pdfaccessed 29 September (2012).

[16] Elmore, C. D. editor. Southern Weed Science Society Weed Identification Guide.

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[17] Radford, A. E, Ahles, H. E, & Bell, C. R. Manual of the Vascular Flora of the Caroli‐

[18] Sigma Database Agrochemical Use on Tobacco. Product Studies Research, Newbury

[19] Seebold, K, Green, J. D, & Townsend, L. Pest Management. *In* 2007 Kentucky Tobac‐ co Production Guide. Lexington, KY: Kentucky Cooperative Extension Service.

[20] Anderson, W. P. Weed Science: Principles and Applications. New York: West Pub‐

[21] Seebold, K. editor. Kentucky & Tennessee Tobacco Production Guide. Lexington, KY:

[22] Frans, R, Talbert, R, Marx, D, & Crowley, H. Experimental design and techniques for measuring and analyzing plant responses to weed control practices. *in* N. D. Camper, ed. Research Methods in Weed Science. 3rd ed. Champaign, IL: Southern Weed Sci‐

Champaign, IL: Southern Weed Science Society. (1999).

Berks, UK. (1999).

lishing Company. (1996).

ence Society. (1986). , 29-46.

(2007).

nas. Chapel Hill: University of North Carolina Press. (1968).

Kentucky Cooperative Extension Service. (2011). , 2011-2012.


[16] Elmore, C. D. editor. Southern Weed Science Society Weed Identification Guide. Champaign, IL: Southern Weed Science Society. (1999).

http://www.fao.org/docrep/015/am081m/PDF/am081m00b.pdfaccessed 29 September

[2] National Agriculture Statistics ServiceAgricultural Statistics Annual Report. http:// www.nass.usda.gov/Publications/Ag\_Statistics/2011/Chapter02.pdfAccessed 29 Sep‐

[3] Collins, W. K, & Hawks, S. N. Jr. Cultivation and weed management. *In* W. K. Col‐ lins and S. N. Hawks (eds.) Principles of Flue-Cured Tobacco Production. Raleigh:

[4] Parker, R. G, Fisher, L. R, & Whitley, D. S. Weed management in conventional and no-till burley tobacco. *In* 2007 Burley Tobacco Information. Raleigh: North Carolina

[5] Palmer, G. K, & Pearce, R. C. Light air-cured tobacco. *In* D. L. Davis and M. T. Niel‐ sen (eds.) Tobacco: Production, Chemistry, and Technology. Oxford: Blackwell Sci‐

[6] Lolas, P. C. Weed community interference in burley oriental tobacco (*Nicotiana taba‐*

[7] Bailey, W. A. Comparison of herbicide systems for dark tobacco. Proc. South. Weed

[8] Bailey, W. A. Dark tobacco (*Nicotiana tabacum*) tolerance to trifloxysulfuron and halo‐

[9] Davis, R. G, Weise, A. F, & Pafford, J. L. (1965). Root moisture extraction profiles of

[11] Daub, M. E, Echandi, E, Gooding, G. V, Jr, K. J, Jones, G. B, Lucas, C. E, Main, N. T, Powell, S. M, Schneider, H. D, Shew, P. B, & Shoemaker, H. W. Spurr, Jr. H. D. Shew and G. B. Lucas (eds.) Compendium of Tobacco Diseases. St. Paul: American Phyto‐

[12] Groves, R. L, Walgenbach, J. F, Moyer, J. W, & Kennedy, G. G. The role of weed hosts and tobacco thrips, *Frankliniella fusca*, in the epidemiology of *tomato spotted wilt virus.*

[13] Wisler, G. C, & Norris, R. F. Interactions between weeds and cultivated plants as re‐ lated to management of plant pathogens. Weed Sci. 2005;(2005). , 53, 914-917.

[14] Lucas, G. B. Diseases of Tobacco. Raleigh: Biological Consulting Associates. (1975).

[15] Flora of North America Editorial Committeeeds. Flora of North America North of

[10] Greer, H. A. L. Weeds: costly competitors for nutrients. Plant Food Rev. (1966).

(2012).

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ence. (1999).

Sci. Soc. 60:18. (2007).

North Carolina State University. (1993).

Cooperative Extension Service. (2007).

*cum*). Weed Res. (1986). , 26(1), 1-8.

sulfuron. Weed Technol. (2007). , 21, 1016-1022.

various weeds. Weeds 1965;, 13, 98-102.

Plant Dis. 2002;(2002). , 86(6), 573-582.

Mexico. 12 vols. New York and Oxford. (1993).

pathological Society. (1991).


**Section 2**

**Natural Areas, Aquatic, and Turf Case Studies**

**Natural Areas, Aquatic, and Turf Case Studies**

**Chapter 9**

**Herbicides for Natural Area Weed Management**

Natural areas represent a significant resource for many countries. In the U.S. natural areas can be defined as conservation lands set aside for preservation or restoration, such as city or county park, private woods, state or national park, Bureau of Land Management (BLM) lands, or other areas [1,2]. In many cases these areas are utilized for recreation, ecosystem services or other non-agricultural purposes [3,4]. Given this broad definition, natural areas encompass a huge portion of the land mass of the United States and represent incredible biological diversity. According to the U.S. National Vegetation Classification in 2012 there are 8 major classifica‐ tions in the U.S. with 430 groupings and over 6100 associations [5]. Some of the more common ecological communities include deciduous temperate forests, temperate coniferous forests,

Many natural areas are managed to some degree for a variety of uses, but due to the complexity of many natural area systems, the management techniques developed for, and utilized in these areas is diverse. Some areas are managed exclusively for recreation and include water attractions, hiking and biking trails, horse trails, or camping. In these cases, user satisfaction, human health and safety are the primary goals, with ecological community diversity being a secondary, but often equally important, goal [6]. Other areas that are managed for conservation (including hunting), preservation or restoration may not require as intense or frequent

Vegetation management in natural areas is performed for a variety of purposes but falls broadly into two primary categories: 1) maintaining the existing vegetation at desirable levels and species composition or 2) restoring the ecosystem to a desirable state. With the latter category, restoration can include reintroduction of naturally occurring species,

> © 2013 MacDonald et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 MacDonald et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

grasslands, and wetlands such as swamps, tidal marshes, and riparian zones.

Gregory E. MacDonald, Lyn A. Gettys, Jason A. Ferrell and Brent A. Sellers

http://dx.doi.org/10.5772/56183

**1. Introduction**

management [7].

Additional information is available at the end of the chapter

## **Herbicides for Natural Area Weed Management**

Gregory E. MacDonald, Lyn A. Gettys, Jason A. Ferrell and Brent A. Sellers

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56183

### **1. Introduction**

Natural areas represent a significant resource for many countries. In the U.S. natural areas can be defined as conservation lands set aside for preservation or restoration, such as city or county park, private woods, state or national park, Bureau of Land Management (BLM) lands, or other areas [1,2]. In many cases these areas are utilized for recreation, ecosystem services or other non-agricultural purposes [3,4]. Given this broad definition, natural areas encompass a huge portion of the land mass of the United States and represent incredible biological diversity. According to the U.S. National Vegetation Classification in 2012 there are 8 major classifica‐ tions in the U.S. with 430 groupings and over 6100 associations [5]. Some of the more common ecological communities include deciduous temperate forests, temperate coniferous forests, grasslands, and wetlands such as swamps, tidal marshes, and riparian zones.

Many natural areas are managed to some degree for a variety of uses, but due to the complexity of many natural area systems, the management techniques developed for, and utilized in these areas is diverse. Some areas are managed exclusively for recreation and include water attractions, hiking and biking trails, horse trails, or camping. In these cases, user satisfaction, human health and safety are the primary goals, with ecological community diversity being a secondary, but often equally important, goal [6]. Other areas that are managed for conservation (including hunting), preservation or restoration may not require as intense or frequent management [7].

Vegetation management in natural areas is performed for a variety of purposes but falls broadly into two primary categories: 1) maintaining the existing vegetation at desirable levels and species composition or 2) restoring the ecosystem to a desirable state. With the latter category, restoration can include reintroduction of naturally occurring species,

reintroduction of a natural ecological process such as fire or water fluctuations, and/or providing an environment that allows for natural reintroduction/colonization of native species [8].

**Herbicide (common name)** **Mechanism of Action2**

**Rate range kg/ha3**

**Application Methods**<sup>4</sup>

2,4-D O (4) 0.56-3.8 F, B, I, CS Microbial (7-10) POST – annual, perennial broadleaves Diclorprop O (4) 4.1-10.4 F, S, I, CS (10) PRE, POST – annual, perennial BL's, brush Dicamba O (4) 0.28-2.2 F, S, B, CS Microbial (4-14) PRE, POST – annual, perennial BL's, brush Picloram O (4) 0.14-1.12 F,S, B, I, CS Microbial (90-300) PRE, POST – perennial BL's, brush, trees Triclopyr O (4) 0.56-9.0 F, B, I, CS Microbial (30) POST – perennial BL's, brush, trees Fluroxypyr O (4) 0.14-0.56 F Microbial (38) POST – annual, perennial BL's, brush Clopyralid O (4) 0.14-0.56 F, S Microbial (40) PRE, POST – annual, perennial BL's, brush Aminopyralid O (4) 0.09-0.25 F, S, I Microbial (35) PRE, POST – annual, perennial BL's, brush Aminocyclopyrachlor O (4) 0.06-0.28 F, S, B, I Microbial (60) PRE, POST – annual, perennial BL's, brush Simazine C1 (5) 2.2-8.9 S Microbial (70-90) PRE – annuals, perennials Diuron C2 (7) 4.5-18 S Microbial (90) PRE – annuals, perennials Tebuthiuron C2 (7) 0.84-4.48 S Microbial (400) PRE – perennial herbs, brush, trees Hexazinone C1 (5) 2.5-7.5 S Microbial (90) PRE – perennial grass, brush, trees Bromacil C1 (5) 1.8-13.4 S Microbial (60) PRE – annual, perennial, brush Prometon C1 (5) 8.9-36 S Microbial (450) PRE – perennial grass, brush, trees Glyphosate G (9) 1.1-5.6 F Irreversibly bound POST – annuals, perennials, brush Fosamine Z (27) 2.24-26.9 F Microbial (8) POST – woody brush, trees Glufosinate H (10) 0.32-1.56 F Irreversibly bound POST – annuals, limited perennials Paraquat D (22) 0.71-1.14 F Irreversibly bound POST – annual species, no soil activity Sethoxydim A(1) 0.31 - 0.53 F Microbial (4-11) POST - annual grasses only Clethodim A(1) 0.11 – 0.28 F Microbial (3) POST - annual and perennial grasses only Fluazifop-p-butyl A(1) 0.13 – 0.42 F (7-21) POST - annual and perennial grasses only Imazapyr B (2) 0.56 – 1.70 F, S, B, I, CS Microbial (25-140) PRE, POST – perennial grass, brush, trees Imazapic B (2) 0.05 – 0.21 F, S Microbial (60-120) PRE, POST – annuals, perennial grasses Imazamox B (2) 0.14 – 0.56 F Microbial (20-30) POST – annuals, brush, trees Chlorsulfuron B (2) 0.018-0.15 F, S Hydrolysis (40) PRE, POST - rangeland annual/perennials Metsulfuron-methyl B (2) 0.012-0.17 F, S Hydrolysis (30) PRE, POST – annuals, perennials, brush Sulfometuron-methyl B (2) 0.065-0.4 F,S Hydrolysis (20-28) PRE, POST – annual, perennials, brush Flumioxazin E (14) 0.28-0.42 S Microbial (12-18) PRE – annual species Oxyfluorfen E (14) 0.56-2.24 S Photolysis (35) PRE- annual species Isoxaben L (21) 0.56-1.12 S Microbial (50-120) PRE – seedling annual species Pendimethalin K1 (3) 0.84-3.36 S Photolysis (44) PRE – seedling annual species

**Environmental Dissipation5**

**\*Common Application Methods and General Spectrum of Control6**

http://dx.doi.org/10.5772/56183

205

Herbicides for Natural Area Weed Management

Within the past 2 decades, vegetation management in natural areas has intensified due to issues with invasive species. Invasive weedy species represent one of the biggest threats to the diversity and utility of many natural areas [9]. Moreover, invasive species are considered to be a major threat to endangered species, second only to habitat loss [10]. Currently there are over 400 invasive non-native plants impacting approximately 133 million acres in the U.S. alone and it is estimated that invasive species are spread‐ ing at the rate of 1.7 million acres annually [11]. In 1999, a mandated executive order specifically addressed invasive species and their impacts, leading to the formation of the National Invasive Species Council (NISC) and the Invasive Species Advisory Commit‐ tee (ISAC) [12]. These organizations and many more at the regional, state and local level dedicated to invasive species management has greatly influenced natural area vegeta‐ tion management.

This chapter will provide an overview of the types of management practices used in a range of natural area systems and detail those herbicides used in natural areas. Weed management in aquatic systems will not be discussed in this chapter.

### **2. Herbicide registration and regulation for use in natural areas**

Herbicides are labeled for use on a specific crop or site as defined by the U.S. Environ‐ mental Protection Agency [13]. Many herbicides can be used in natural areas, but labeling may be restricted to only specific uses within the broader context of a 'natu‐ ral area'. In addition, many states, agencies, and/or local governments may prohibit or restrict usage of a particular product or compound. It is not the intent of this chapter to list those specific sites where a particular herbicide could be used, but rather provide details of how the herbicide is applied, its mode of action, its spectrum of activity and environmental considerations associated with use.

### **3. Overview of natural area herbicides and their mechanisms/modes-ofaction**

This section will provide background of those herbicides used in natural areas and will include information on chemistry, formulations, mode-of-action and selectivity. Specific details to each herbicide are listed in Table 1.


reintroduction of a natural ecological process such as fire or water fluctuations, and/or providing an environment that allows for natural reintroduction/colonization of native

Within the past 2 decades, vegetation management in natural areas has intensified due to issues with invasive species. Invasive weedy species represent one of the biggest threats to the diversity and utility of many natural areas [9]. Moreover, invasive species are considered to be a major threat to endangered species, second only to habitat loss [10]. Currently there are over 400 invasive non-native plants impacting approximately 133 million acres in the U.S. alone and it is estimated that invasive species are spread‐ ing at the rate of 1.7 million acres annually [11]. In 1999, a mandated executive order specifically addressed invasive species and their impacts, leading to the formation of the National Invasive Species Council (NISC) and the Invasive Species Advisory Commit‐ tee (ISAC) [12]. These organizations and many more at the regional, state and local level dedicated to invasive species management has greatly influenced natural area vegeta‐

This chapter will provide an overview of the types of management practices used in a range of natural area systems and detail those herbicides used in natural areas. Weed management

Herbicides are labeled for use on a specific crop or site as defined by the U.S. Environ‐ mental Protection Agency [13]. Many herbicides can be used in natural areas, but labeling may be restricted to only specific uses within the broader context of a 'natu‐ ral area'. In addition, many states, agencies, and/or local governments may prohibit or restrict usage of a particular product or compound. It is not the intent of this chapter to list those specific sites where a particular herbicide could be used, but rather provide details of how the herbicide is applied, its mode of action, its spectrum of activity and

**3. Overview of natural area herbicides and their mechanisms/modes-of-**

This section will provide background of those herbicides used in natural areas and will include information on chemistry, formulations, mode-of-action and selectivity. Specific details to each

**2. Herbicide registration and regulation for use in natural areas**

in aquatic systems will not be discussed in this chapter.

environmental considerations associated with use.

species [8].

204 Herbicides - Current Research and Case Studies in Use

tion management.

**action**

herbicide are listed in Table 1.


In addition to 2,4-D, there have been several other phenoxycarboxylic acid herbicides devel‐ oped that are used in natural areas. These include MCPA, diclorprop, and mecoprop. Once again several formulations of each have been developed, including salt and ester forms. Of the three, diclorprop is used most extensively in natural areas [18], while MCPA and mecoprop are mainly used in grass crop and turf situations for annual and perennial broadleaf weed control [19]. As of 2007, the parent acid and the dimethylamine salt and ethylhexyl ester formulations of dichlorprop are registered for use by the US EPA. This herbicide has better activity on woody brush and trees, compared to 2,4-D. The ester formulation is often used alone or in oil-based carriers for spot specific plant treatments such as fencerows and rights of

Herbicides for Natural Area Weed Management

http://dx.doi.org/10.5772/56183

207

For many years, the phenoxy herbicide 2,4,5-T was the standard treatment for woody brush and tree control in pastures and rangeland [20]. This herbicide was highly active on several species and possessed considerable soil persistence, which contributed to its effectiveness. 2,4,5-T was cancelled for use by the U.S. EPA in the early 1980's due to concerns from the contaminant dioxin during certain manufacturing processes. Dioxin has been demonstrated to be a known carcinogen and was present in considerable quantities of 2,4,5-T used during the Vietnam war [21]. The herbicide known as 'Agent Orange' was actually a combination of 2,4,5-T and 2,4-D used for widespread aerial-applied jungle defoliation [22]. However, the levels of dioxin in commercially produced 2,4,5-T after the war were very low, but continuing

The benzoic acid herbicide chemical family contains only one currently available herbicide for use in natural areas, dicamba. Dicamba is formulated only as a salt, with the following salts registered for use by the US EPA: dimethylamine (DMA) salt, sodium (NA) salt, isopropyla‐ mine (IPA) salt, diglycolamine (DGA) salt, and potassium (K) salt [14]. Interestingly, this herbicide can volatilize and move off target, despite being formulated as a salt. Dicamba is highly effective on many weeds in crops and is widely used in pasture/rangeland situations for perennial weed management [24]. It is considered to have superior perennial broadleaf weed control compared to many of the phenoxy herbicides, while still providing selectivity towards crops (primarily corn and sorghum). Dicamba also possesses greater soil persistence

The pyridine or picolinic acid herbicide chemical family comprises several herbicides that are widely used for natural area weed control. In general these herbicides are more potent compared to equivalent rates of phenoxy herbicides, and many possess considerable soil residual activity. The first picolinic acid herbicide developed was picloram in 1963 by Dow Chemical [14]. Similar to 2,4-D, picloram is formulated as salts (triisopropanolamine and potassium) and ester (ethylhexyl/isooctyl). Picloram is used in a wide range of natural areas, particularly open rangeland, for woody brush control [18]. Several formulations are also used in permanent pasture situations for perennial broadleaf weed control. The use of picloram is limited in certain areas over potential groundwater contamination concerns due to high water solubility and relatively long soil half-life. Moreover, many crops are highly sensitive to picloram at very low rates (<ppb), which also limits use in tolerant crops due to rotational

concerns and public outcry lead to the cancellation of this herbicide [23].

than phenoxys, which also contributes to its control [25].

ways.

concerns [26].

1 Information presented derived from sources including, but not limited to: 2007 Herbicide Handbook, Weed Science Society of America, Lawrence, KS. 458p; ExToxNet - The EXtension TOXicology NETwork, http://extoxnet.orst.edu/; Crop Data Management Systems, Inc., http://www.cdms.net/.

2 Mode of action classification based on Herbicide Resistance Action Committee (HRAC) – [letters and subscript numbers] and the Weed Science Society of America (parentheses). HRAC http://www.hracglobal.com/ WSSA http:// www.wssa.net/Weeds/Resistance/WSSA-Mechanism-of-Action.pdf

3 Rate range based on current label guidelines for control in natural areas or non-cropland sites. Rate expressed in kilograms of active ingredient per hectare.

4 Application methods include: F - foliar, S – soil, B – basal bark, I – stem injection, CS – cut stump

5 Environmental dissipation includes the major means of breakdown and half-life range in days in soil. In some cases, the mechanism of breakdown is not available.

6 abbreviations: POST – postemergence activity/application; PRE – preemergence soil activity; BL's – broadleaf species.

\*General application information only – refer to product label and local/state recommendations for specifics on use rates, application methods and timing, species controlled and restrictions for use.

**Table 1.** Properties and application methods of commonly used herbicides used in natural areas1.

#### **3.1. Synthetic auxins or growth regulators**

The growth regulator herbicides represent the oldest and possibly the most widely used of the herbicides used in natural areas. These materials are mechanistically classified as synthetic auxins [14] and include herbicides in the phenoxycarboxylic acids, benzoic acid and pyridine carboxylic acid (picolinic acid) chemical groups.

2,4-D is the principle herbicide in the chemical group phenoxycarboxylic acids and has been used for broadleaf weed control since the late 1940's. This compound was first noted to have growth regulator properties in 1942, and registered as an herbicide after World War II [15]. There have been 28 different chemical formulations registered for 2,4-D, including the parent acid, amine salts and esters [14]. Salt formulations are characterized by fairly high water solubility and low volatility, while esters are more prone to volatility and more soluble in liquid fertilizers [16]. Ester formulations show greater phytotoxicity per acid equivalent basis due to greater cuticle penetration and foliar uptake. Short chain esters are highly prone to volatiliza‐ tion, and no longer registered for use. As of 2005, there were 9 formulations of 2,4-D supported for reregistration by the United States Environmental Protection Agency [17]. These include the parent acid, the sodium, diethanolamine, dimethylamine, isopropylamine, and triisopro‐ panolamine salts, and the 2-butoxyethyl, 2-ethylhexyl, and isopropyl esters. In general salts are formulated as wettable powders, granules or soluble concentrates, while the waterinsoluble esters are formulated as emulsifiable concentrates or mixed with oils or liquid fertilizers.

In addition to 2,4-D, there have been several other phenoxycarboxylic acid herbicides devel‐ oped that are used in natural areas. These include MCPA, diclorprop, and mecoprop. Once again several formulations of each have been developed, including salt and ester forms. Of the three, diclorprop is used most extensively in natural areas [18], while MCPA and mecoprop are mainly used in grass crop and turf situations for annual and perennial broadleaf weed control [19]. As of 2007, the parent acid and the dimethylamine salt and ethylhexyl ester formulations of dichlorprop are registered for use by the US EPA. This herbicide has better activity on woody brush and trees, compared to 2,4-D. The ester formulation is often used alone or in oil-based carriers for spot specific plant treatments such as fencerows and rights of ways.

**Herbicide (common name)** **Mechanism of Action2**

206 Herbicides - Current Research and Case Studies in Use

Data Management Systems, Inc., http://www.cdms.net/.

kilograms of active ingredient per hectare.

mechanism of breakdown is not available.

fertilizers.

**3.1. Synthetic auxins or growth regulators**

carboxylic acid (picolinic acid) chemical groups.

www.wssa.net/Weeds/Resistance/WSSA-Mechanism-of-Action.pdf

application methods and timing, species controlled and restrictions for use.

**Rate range kg/ha3**

**Application Methods**<sup>4</sup>

Oryzalin K1 (3) 2.24-6.72 S Photolysis (20-90) PRE – seedling annual species Diclobenil L (20) 4.5-22.4 S Microbial (60) PRE – seedling annual species, nutsedge S-Metolachlor K3 (15) 1.4-2.8 S Microbial (67) PRE – seedling annual species, nutsedge 1 Information presented derived from sources including, but not limited to: 2007 Herbicide Handbook, Weed Science Society of America, Lawrence, KS. 458p; ExToxNet - The EXtension TOXicology NETwork, http://extoxnet.orst.edu/; Crop

2 Mode of action classification based on Herbicide Resistance Action Committee (HRAC) – [letters and subscript numbers] and the Weed Science Society of America (parentheses). HRAC http://www.hracglobal.com/ WSSA http://

3 Rate range based on current label guidelines for control in natural areas or non-cropland sites. Rate expressed in

5 Environmental dissipation includes the major means of breakdown and half-life range in days in soil. In some cases, the

6 abbreviations: POST – postemergence activity/application; PRE – preemergence soil activity; BL's – broadleaf species. \*General application information only – refer to product label and local/state recommendations for specifics on use rates,

The growth regulator herbicides represent the oldest and possibly the most widely used of the herbicides used in natural areas. These materials are mechanistically classified as synthetic auxins [14] and include herbicides in the phenoxycarboxylic acids, benzoic acid and pyridine

2,4-D is the principle herbicide in the chemical group phenoxycarboxylic acids and has been used for broadleaf weed control since the late 1940's. This compound was first noted to have growth regulator properties in 1942, and registered as an herbicide after World War II [15]. There have been 28 different chemical formulations registered for 2,4-D, including the parent acid, amine salts and esters [14]. Salt formulations are characterized by fairly high water solubility and low volatility, while esters are more prone to volatility and more soluble in liquid fertilizers [16]. Ester formulations show greater phytotoxicity per acid equivalent basis due to greater cuticle penetration and foliar uptake. Short chain esters are highly prone to volatiliza‐ tion, and no longer registered for use. As of 2005, there were 9 formulations of 2,4-D supported for reregistration by the United States Environmental Protection Agency [17]. These include the parent acid, the sodium, diethanolamine, dimethylamine, isopropylamine, and triisopro‐ panolamine salts, and the 2-butoxyethyl, 2-ethylhexyl, and isopropyl esters. In general salts are formulated as wettable powders, granules or soluble concentrates, while the waterinsoluble esters are formulated as emulsifiable concentrates or mixed with oils or liquid

4 Application methods include: F - foliar, S – soil, B – basal bark, I – stem injection, CS – cut stump

**Table 1.** Properties and application methods of commonly used herbicides used in natural areas1.

**Environmental Dissipation5**

**\*Common Application Methods and General Spectrum of Control6**

> For many years, the phenoxy herbicide 2,4,5-T was the standard treatment for woody brush and tree control in pastures and rangeland [20]. This herbicide was highly active on several species and possessed considerable soil persistence, which contributed to its effectiveness. 2,4,5-T was cancelled for use by the U.S. EPA in the early 1980's due to concerns from the contaminant dioxin during certain manufacturing processes. Dioxin has been demonstrated to be a known carcinogen and was present in considerable quantities of 2,4,5-T used during the Vietnam war [21]. The herbicide known as 'Agent Orange' was actually a combination of 2,4,5-T and 2,4-D used for widespread aerial-applied jungle defoliation [22]. However, the levels of dioxin in commercially produced 2,4,5-T after the war were very low, but continuing concerns and public outcry lead to the cancellation of this herbicide [23].

> The benzoic acid herbicide chemical family contains only one currently available herbicide for use in natural areas, dicamba. Dicamba is formulated only as a salt, with the following salts registered for use by the US EPA: dimethylamine (DMA) salt, sodium (NA) salt, isopropyla‐ mine (IPA) salt, diglycolamine (DGA) salt, and potassium (K) salt [14]. Interestingly, this herbicide can volatilize and move off target, despite being formulated as a salt. Dicamba is highly effective on many weeds in crops and is widely used in pasture/rangeland situations for perennial weed management [24]. It is considered to have superior perennial broadleaf weed control compared to many of the phenoxy herbicides, while still providing selectivity towards crops (primarily corn and sorghum). Dicamba also possesses greater soil persistence than phenoxys, which also contributes to its control [25].

> The pyridine or picolinic acid herbicide chemical family comprises several herbicides that are widely used for natural area weed control. In general these herbicides are more potent compared to equivalent rates of phenoxy herbicides, and many possess considerable soil residual activity. The first picolinic acid herbicide developed was picloram in 1963 by Dow Chemical [14]. Similar to 2,4-D, picloram is formulated as salts (triisopropanolamine and potassium) and ester (ethylhexyl/isooctyl). Picloram is used in a wide range of natural areas, particularly open rangeland, for woody brush control [18]. Several formulations are also used in permanent pasture situations for perennial broadleaf weed control. The use of picloram is limited in certain areas over potential groundwater contamination concerns due to high water solubility and relatively long soil half-life. Moreover, many crops are highly sensitive to picloram at very low rates (<ppb), which also limits use in tolerant crops due to rotational concerns [26].

Triclopyr is probably the most widely used picolinic acid herbicide in natural areas, especially for woody brush species [27]. This herbicide is formulated as the triethylamine salt and the butoxyethyl ester, both of which are used across a wide range of natural, forest and pasture/rangeland situations. It possesses good activity on many annual and perenni‐ al broadleaf weeds and brush, but at rates slightly higher when compared to picloram [20]. However, unlike picloram, triclopyr has limited soil activity and is generally considered to be non-soil active [14].

normal regulation of plant responses to growth, development and environmental stimuli [39]. Auxin is regulated through two processes; metabolism via biosynthesis, conjugation, deconjugation and degradation or transport and distribution within and between cells. The distribution of auxin, including directional flow, is regulated by the presence and activity of auxin transporters in the plasma membrane. Because auxins are weak acids, they are dissoci‐ ated in the presence of neutral cellular pH (7.0) and trapped as anions within the cell. Thus transport out of the cell can be mediated through plasma membrane located facilitators specific

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Herbicides within this classification are considered auxin mimics, and are thought to act like auxin within plant tissues. Earlier research suggested that these herbicides acted to acidify the cell wall by activating a membrane bound ATPase proton pump and this acidification induced cell elongation [40]. Other work showed an increase in RNA polymerase, leading to increases in cell division and uncontrolled growth. Ethylene generation has also been reported, likely to counteract the stimulatory effect of auxin [41]. However, recent work has shown 2,4-D to be transported by influx carriers into the cell [42] and also through efflux carriers [43]. Due to limited metabolism, the auxin-effect of these herbicides presumably causes rapid cell division in some cells and a complete cessation of growth in other cells. This unregulated growth results in stem twisting, leaf strapping, puckering, and a plethora of other symptoms associated with

Synthetic auxin herbicides are chemically weak acids, and although some possess soil activity, these herbicides are applied to the foliage of plants. Once applied these herbicides are rapidly absorbed by leaf tissue and remobilized, similar to carbohydrate movement, to areas of meristematic growth via the phloem [14]. They possess the similar anion trapping mechanism as natural auxins, and this likely contributes to their effectiveness in herbicidal activity. Soil uptake of these herbicides occurs through the xylem where upward movement to shoots and leaves takes place. However, once diffusing from the xylem into leaf tissues, the herbicide is

transported, in a similar manner to carbohydrates, to regions of meristematic growth.

The ability to metabolize is the primary selectivity mechanism for tolerant plant species. In most cases, grasses are moderately to highly tolerant to growth regulator herbicides through the ability to conjugate these herbicides with amino acids or sugars [25]. Most of these herbicides are slowly degraded regardless of plant species, but grasses appear to have the ability to shunt the herbicide conjugate to the vacuole, where it is either sequestered from sites of action, and/or slowly degraded. Many picolinic acid herbicides such as picloram, amino‐ pyralid and clopryralid are sequestered in the vacuole of tolerant plants, but the compound remains intact and thus herbicidally active [44]. This has lead to many issues with off-target damage due to removal of the herbicide sequestering plant tissue and subsequent release of

This phenomenon was first observed with picloram, and later with clopyralid and aminopyr‐ alid. In the case of picloram, animals grazing on treated forage grasses were observed to have the ability to transfer the herbicide through urination or defecation. Concentrating of the herbicide, coupled with soil persistence lead to problems with sensitive crops planted in fields after grazing. Dried hay, either degraded as plant biomass or via manure, transferred from

for auxin.

growth regulator herbicides.

the herbicide in the environment.

The picolinic acid herbicide fluroxypyr also has limited soil activity, and is used primarily for broadleaf weed control in cereals, fallow cropland and pastures. It is formulated as a meptyl and butometyl ester and is often combined with other growth regulator herbicides to broaden weed control spectrum [28]. The use of fluroxypyr in natural areas is limited, primarily rights of ways, mainly due to superior weed control spectrum from other picolinic acid herbicides and labeling restrictions.

Clopyralid is another picolinic herbicide with moderate utility in natural areas. This herbicide was discovered in 1961 by Dow Chemical Company but was not registered for herbicidal use in the U.S. until 1987 [14]. It is mainly formulated as the monoethanolamine salt, but ester formations are also available. Clopyralid has moderate soil persistence and may cause problems with sensitive crops planted after clopyralid use in the previous crop [28]. This herbicide has broadleaf weed activity, similar to the picolinic acid herbicides as a whole, but has greater specificity and therefore selectivity towards many legume, solanaceous and composite type weeds [29,30,31].

Aminopyralid is a relatively new picolinic acid herbicide registered for use in pastures/ rangeland, forestry and natural areas [14]. Aminopyralid is only formulated as the potassium salt. It has moderate soil persistence, and like clopyralid, has specificity towards legume, composite and solanaceous weeds [32]. In fact, one of the primary registrations for this herbicide is for the control of tropical soda apple (*Solanum viarum*) in southeastern U.S. pastures [33]. In other areas of the U.S. the primary target species is composites such as thistles (*Cirsium spp.*) and species of knapweeds (*Centaurea spp.*) [34]. It is formulated as a salt and often combined with other herbicides to increase weed spectrum.

Aminocyclopyrachlor is the most recent herbicide to be registered for use in natural areas [35]. This herbicide possesses the typical growth regulator mode of action, but does not fit within the chemical classifications listed above. The uses of this compound are still being developed, but like aminopyralid and clopyralid, it has remarkable specificity at low use rates [36]. Aminocyclopyrachlor is primarily formulated as a salt, but ester formulations have been tested for basal bark applications in oil carriers. This herbicide is very active on a range of broadleaf species, but also possesses considerable activity on certain grasses, including many perennial grasses [37].

The mode of action of the synthetic auxin herbicides is not well understood, but appears to disrupt the normal cellular and tissue response to auxin. Auxin is present in plants at very small concentrations (nanomolar) and acts as a signaling molecular for a wide range of cellular functions and responses [38]. Auxin levels must be precisely controlled within the plant for normal regulation of plant responses to growth, development and environmental stimuli [39]. Auxin is regulated through two processes; metabolism via biosynthesis, conjugation, deconjugation and degradation or transport and distribution within and between cells. The distribution of auxin, including directional flow, is regulated by the presence and activity of auxin transporters in the plasma membrane. Because auxins are weak acids, they are dissoci‐ ated in the presence of neutral cellular pH (7.0) and trapped as anions within the cell. Thus transport out of the cell can be mediated through plasma membrane located facilitators specific for auxin.

Triclopyr is probably the most widely used picolinic acid herbicide in natural areas, especially for woody brush species [27]. This herbicide is formulated as the triethylamine salt and the butoxyethyl ester, both of which are used across a wide range of natural, forest and pasture/rangeland situations. It possesses good activity on many annual and perenni‐ al broadleaf weeds and brush, but at rates slightly higher when compared to picloram [20]. However, unlike picloram, triclopyr has limited soil activity and is generally considered

The picolinic acid herbicide fluroxypyr also has limited soil activity, and is used primarily for broadleaf weed control in cereals, fallow cropland and pastures. It is formulated as a meptyl and butometyl ester and is often combined with other growth regulator herbicides to broaden weed control spectrum [28]. The use of fluroxypyr in natural areas is limited, primarily rights of ways, mainly due to superior weed control spectrum from other picolinic acid herbicides

Clopyralid is another picolinic herbicide with moderate utility in natural areas. This herbicide was discovered in 1961 by Dow Chemical Company but was not registered for herbicidal use in the U.S. until 1987 [14]. It is mainly formulated as the monoethanolamine salt, but ester formations are also available. Clopyralid has moderate soil persistence and may cause problems with sensitive crops planted after clopyralid use in the previous crop [28]. This herbicide has broadleaf weed activity, similar to the picolinic acid herbicides as a whole, but has greater specificity and therefore selectivity towards many legume, solanaceous and

Aminopyralid is a relatively new picolinic acid herbicide registered for use in pastures/ rangeland, forestry and natural areas [14]. Aminopyralid is only formulated as the potassium salt. It has moderate soil persistence, and like clopyralid, has specificity towards legume, composite and solanaceous weeds [32]. In fact, one of the primary registrations for this herbicide is for the control of tropical soda apple (*Solanum viarum*) in southeastern U.S. pastures [33]. In other areas of the U.S. the primary target species is composites such as thistles (*Cirsium spp.*) and species of knapweeds (*Centaurea spp.*) [34]. It is formulated as a salt and often

Aminocyclopyrachlor is the most recent herbicide to be registered for use in natural areas [35]. This herbicide possesses the typical growth regulator mode of action, but does not fit within the chemical classifications listed above. The uses of this compound are still being developed, but like aminopyralid and clopyralid, it has remarkable specificity at low use rates [36]. Aminocyclopyrachlor is primarily formulated as a salt, but ester formulations have been tested for basal bark applications in oil carriers. This herbicide is very active on a range of broadleaf species, but also possesses considerable activity on certain grasses, including many perennial

The mode of action of the synthetic auxin herbicides is not well understood, but appears to disrupt the normal cellular and tissue response to auxin. Auxin is present in plants at very small concentrations (nanomolar) and acts as a signaling molecular for a wide range of cellular functions and responses [38]. Auxin levels must be precisely controlled within the plant for

to be non-soil active [14].

208 Herbicides - Current Research and Case Studies in Use

and labeling restrictions.

composite type weeds [29,30,31].

grasses [37].

combined with other herbicides to increase weed spectrum.

Herbicides within this classification are considered auxin mimics, and are thought to act like auxin within plant tissues. Earlier research suggested that these herbicides acted to acidify the cell wall by activating a membrane bound ATPase proton pump and this acidification induced cell elongation [40]. Other work showed an increase in RNA polymerase, leading to increases in cell division and uncontrolled growth. Ethylene generation has also been reported, likely to counteract the stimulatory effect of auxin [41]. However, recent work has shown 2,4-D to be transported by influx carriers into the cell [42] and also through efflux carriers [43]. Due to limited metabolism, the auxin-effect of these herbicides presumably causes rapid cell division in some cells and a complete cessation of growth in other cells. This unregulated growth results in stem twisting, leaf strapping, puckering, and a plethora of other symptoms associated with growth regulator herbicides.

Synthetic auxin herbicides are chemically weak acids, and although some possess soil activity, these herbicides are applied to the foliage of plants. Once applied these herbicides are rapidly absorbed by leaf tissue and remobilized, similar to carbohydrate movement, to areas of meristematic growth via the phloem [14]. They possess the similar anion trapping mechanism as natural auxins, and this likely contributes to their effectiveness in herbicidal activity. Soil uptake of these herbicides occurs through the xylem where upward movement to shoots and leaves takes place. However, once diffusing from the xylem into leaf tissues, the herbicide is transported, in a similar manner to carbohydrates, to regions of meristematic growth.

The ability to metabolize is the primary selectivity mechanism for tolerant plant species. In most cases, grasses are moderately to highly tolerant to growth regulator herbicides through the ability to conjugate these herbicides with amino acids or sugars [25]. Most of these herbicides are slowly degraded regardless of plant species, but grasses appear to have the ability to shunt the herbicide conjugate to the vacuole, where it is either sequestered from sites of action, and/or slowly degraded. Many picolinic acid herbicides such as picloram, amino‐ pyralid and clopryralid are sequestered in the vacuole of tolerant plants, but the compound remains intact and thus herbicidally active [44]. This has lead to many issues with off-target damage due to removal of the herbicide sequestering plant tissue and subsequent release of the herbicide in the environment.

This phenomenon was first observed with picloram, and later with clopyralid and aminopyr‐ alid. In the case of picloram, animals grazing on treated forage grasses were observed to have the ability to transfer the herbicide through urination or defecation. Concentrating of the herbicide, coupled with soil persistence lead to problems with sensitive crops planted in fields after grazing. Dried hay, either degraded as plant biomass or via manure, transferred from treated fields to other areas has also been shown to cause problems [45]. Manure from animals fed on treated forage that is used for compost and fertilizer is another source of contamination. More recently, grass clippings from treated turf, primarily clopyralid, can also be a problem [46]. The sequestration rather than degradation, coupled with high sensitivity at very low rates (parts per billion) for many broadleaf crop species is the reason for this major problem. This issue has lead to the cancellation of this herbicide in many areas, due to contamination in municipal compost for use by the general public [47]. Product labels containing these herbi‐ cides explicitly restrict the movement of treated plant biomass, and manure from livestock fed with treated forage in an effort to minimize off-target injury.

of herbicides also contain labeling specific to natural areas. Metsulfuron has a special local needs (SLN) label for the control old world climbing fern (*Lygodium microphyllum*) in south

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Extremely low use rates and remarkable specificity set these herbicides apart from the traditional phenoxy herbicides [60]. It is difficult to make broad generalizations regarding the activity of the sulfonylureas because some species are controlled while other species, even within the same genus, are not. Therefore, uses for these products are regional or even local, depending on the species to be controlled and not controlled. These herbicides also have considerable soil activity, and this contributes to their long-lasting control in perennial systems [61]. However, this high level of activity can also cause problems with rotational crops, but this is not a common situation in areas where sulfometuron and metsulfuron are applied [60].

The imidazolinone herbicides used in natural areas include imazapyr, imazamox and ima‐ zapic. Imazapyr was first registered in 1985 for use in forestry and industrial sites such as railroads, rail yards, and powerline and highway rights-of-way [53]. At typical use rates, this herbicide has very broad spectrum activity that includes annual and perennial broadleaves, and several brush, vine and hardwood tree species. This herbicide also has tremendous activity on perennial grasses, both rhizomatous and bunch type grasses [62,63]. While initially developed for the industrial market, imazapyr is widely used in many natural areas for invasive species management. Imazapyr does have a registration for use in imidazolinone

Imazapic is registered for use in peanuts and certain forages, but is widely utilized for grass and broadleaf weed management in native perennial grass prairies [65]. Many perennial grasses such as eastern gamma grass, big bluestem grass (*Andropogon gerardii*), indiangrass (*Sorghastrum spp.*), switchgrass (*Panicum virgatum*) and buffalograss (*Bouteloua dactyloides*) have good tolerance to imazapic, although some injury is observed at seedling stages or during spring regrowth. Imazapic is also labeled for wildflower planting and for seedhead suppres‐ sion of bahiagrass in turf settings. Imazapic is also used for the control of several invasive species in natural areas. These include Dalmatian toadflax (*Linaria vulgaris*), yellow starthistle (*Centaurea solstitialis*), leafy spurge (*Euphorbia esula*), Russian knapweed (*Acroptilon repens*), and

Imazamox is the most recent registration from the imidazolinone herbicide group in natural areas for the control of submersed and emergent vegetation [70]. It is particularly effective on Chinese tallow tree (*Triadica sebifera*), which is a major invasive species throughout much of the southeastern United States. Imazamox is also effective for several emergent and ditchbank species, and preliminary research indicates good control of cattail (*Typha spp*.). This herbicide

The sulfonylurea herbicides chlorsulfuron, sulfometuron and metsulfuron are formulated as dry flowable granules that readily mix with water. Sulfonylureas are weak acid compounds with very high water solubility [14]. These herbicides are readily absorbed by roots from soil applications and transported via the xylem to shoots and leaves of plants. Once in the leaves, these herbicides are often remobilized in the phloem to growing regions - tracking a similar

has limited grass activity, and is most effective on broadleaf species.

Florida natural areas [59].

resistant crops, but its usage as such is limited [64].

tall fescue (*Schedonorus phoenix*) [66,67,68,69].

Recently genes for the metabolism of dicamba and 2,4-D have been inserted from bacteria into soybeans, cotton and corn, affording the ability to utilize these herbicides for weed control [48,49]. However, there are many concerns over the use of this technology, including the accelerated development of resistance by weeds as observed with the widespread use of glyphosate in glyphosate tolerant crops. Several weeds have developed resistance to growth regulator herbicides including kochia (*Kochia scoparia*) and lambsquarters (*Chenopodium album*) resistance to dicamba, yellow starthistle (*Centaurea solstitialis*) resistance to clopyralid and picloram and 2,4-D resistance in common chickweed (*Stellaria media*) and most recently common waterhemp (*Amaranthus tuberculatus*) [50,51]. The mechanism of resistance in most of these cases is not known.

#### **3.2. Acetolactate (ALS) inhibitors**

Herbicides within this classification are broadly represented by two major chemical families; the sulfonylureas and the imidazolinones. These herbicides are used in a wide range of cropping systems but many are also used in natural areas [14]. Both chemistries are highlighted by low use rates, low mammalian toxicity, and extreme specificity [52,53]. Interestingly, both classes of herbicide target the same plant enzyme, and were simultaneous discoveries by 2 separate agrochemical companies in the 1980's, DuPont for the sulfonylureas and American Cyanamid for the imidazolinones [54].

The first herbicide registered for use from this class was chlorsulfuron by DuPont in 1982 [52]. Chlorsulfuron is predominantly used in the western United States for broadleaf weed control in cereal grains and pasture/rangelands, but more recently for invasive species control by the Bureau of Land Management [55]. Other sulfonylurea herbicides developed by DuPont include sulfometuron and metsulfuron, which were initially labeled for use in forestry and industrial sites, but later labeling included uses for metsulfuron in pastures and natural areas and uses for sulfometuron for invasive species management [55,56].

Like the synthetic auxin herbicides, sulfonylurea herbicides have activity on a wide range of natural area broadleaf weeds but their activity also includes some grasses [57]. In general, and at rates labeled for use, chlorsulfuron is used for annual and short-lived perennial weed control in open rangeland and natural areas, while sulfometuron and metsulfuron have more control of woody brush and trees [58]. Both of these latter herbicides are used for hardwood control in commercial conifer forests and also for broad spectrum weed control in industrial sites such as railroads, rail yards, highway rights-of-way and electrical substations. However, all three of herbicides also contain labeling specific to natural areas. Metsulfuron has a special local needs (SLN) label for the control old world climbing fern (*Lygodium microphyllum*) in south Florida natural areas [59].

treated fields to other areas has also been shown to cause problems [45]. Manure from animals fed on treated forage that is used for compost and fertilizer is another source of contamination. More recently, grass clippings from treated turf, primarily clopyralid, can also be a problem [46]. The sequestration rather than degradation, coupled with high sensitivity at very low rates (parts per billion) for many broadleaf crop species is the reason for this major problem. This issue has lead to the cancellation of this herbicide in many areas, due to contamination in municipal compost for use by the general public [47]. Product labels containing these herbi‐ cides explicitly restrict the movement of treated plant biomass, and manure from livestock fed

Recently genes for the metabolism of dicamba and 2,4-D have been inserted from bacteria into soybeans, cotton and corn, affording the ability to utilize these herbicides for weed control [48,49]. However, there are many concerns over the use of this technology, including the accelerated development of resistance by weeds as observed with the widespread use of glyphosate in glyphosate tolerant crops. Several weeds have developed resistance to growth regulator herbicides including kochia (*Kochia scoparia*) and lambsquarters (*Chenopodium album*) resistance to dicamba, yellow starthistle (*Centaurea solstitialis*) resistance to clopyralid and picloram and 2,4-D resistance in common chickweed (*Stellaria media*) and most recently common waterhemp (*Amaranthus tuberculatus*) [50,51]. The mechanism of resistance in most

Herbicides within this classification are broadly represented by two major chemical families; the sulfonylureas and the imidazolinones. These herbicides are used in a wide range of cropping systems but many are also used in natural areas [14]. Both chemistries are highlighted by low use rates, low mammalian toxicity, and extreme specificity [52,53]. Interestingly, both classes of herbicide target the same plant enzyme, and were simultaneous discoveries by 2 separate agrochemical companies in the 1980's, DuPont for the sulfonylureas and American

The first herbicide registered for use from this class was chlorsulfuron by DuPont in 1982 [52]. Chlorsulfuron is predominantly used in the western United States for broadleaf weed control in cereal grains and pasture/rangelands, but more recently for invasive species control by the Bureau of Land Management [55]. Other sulfonylurea herbicides developed by DuPont include sulfometuron and metsulfuron, which were initially labeled for use in forestry and industrial sites, but later labeling included uses for metsulfuron in pastures and natural areas

Like the synthetic auxin herbicides, sulfonylurea herbicides have activity on a wide range of natural area broadleaf weeds but their activity also includes some grasses [57]. In general, and at rates labeled for use, chlorsulfuron is used for annual and short-lived perennial weed control in open rangeland and natural areas, while sulfometuron and metsulfuron have more control of woody brush and trees [58]. Both of these latter herbicides are used for hardwood control in commercial conifer forests and also for broad spectrum weed control in industrial sites such as railroads, rail yards, highway rights-of-way and electrical substations. However, all three

and uses for sulfometuron for invasive species management [55,56].

with treated forage in an effort to minimize off-target injury.

of these cases is not known.

**3.2. Acetolactate (ALS) inhibitors**

210 Herbicides - Current Research and Case Studies in Use

Cyanamid for the imidazolinones [54].

Extremely low use rates and remarkable specificity set these herbicides apart from the traditional phenoxy herbicides [60]. It is difficult to make broad generalizations regarding the activity of the sulfonylureas because some species are controlled while other species, even within the same genus, are not. Therefore, uses for these products are regional or even local, depending on the species to be controlled and not controlled. These herbicides also have considerable soil activity, and this contributes to their long-lasting control in perennial systems [61]. However, this high level of activity can also cause problems with rotational crops, but this is not a common situation in areas where sulfometuron and metsulfuron are applied [60].

The imidazolinone herbicides used in natural areas include imazapyr, imazamox and ima‐ zapic. Imazapyr was first registered in 1985 for use in forestry and industrial sites such as railroads, rail yards, and powerline and highway rights-of-way [53]. At typical use rates, this herbicide has very broad spectrum activity that includes annual and perennial broadleaves, and several brush, vine and hardwood tree species. This herbicide also has tremendous activity on perennial grasses, both rhizomatous and bunch type grasses [62,63]. While initially developed for the industrial market, imazapyr is widely used in many natural areas for invasive species management. Imazapyr does have a registration for use in imidazolinone resistant crops, but its usage as such is limited [64].

Imazapic is registered for use in peanuts and certain forages, but is widely utilized for grass and broadleaf weed management in native perennial grass prairies [65]. Many perennial grasses such as eastern gamma grass, big bluestem grass (*Andropogon gerardii*), indiangrass (*Sorghastrum spp.*), switchgrass (*Panicum virgatum*) and buffalograss (*Bouteloua dactyloides*) have good tolerance to imazapic, although some injury is observed at seedling stages or during spring regrowth. Imazapic is also labeled for wildflower planting and for seedhead suppres‐ sion of bahiagrass in turf settings. Imazapic is also used for the control of several invasive species in natural areas. These include Dalmatian toadflax (*Linaria vulgaris*), yellow starthistle (*Centaurea solstitialis*), leafy spurge (*Euphorbia esula*), Russian knapweed (*Acroptilon repens*), and tall fescue (*Schedonorus phoenix*) [66,67,68,69].

Imazamox is the most recent registration from the imidazolinone herbicide group in natural areas for the control of submersed and emergent vegetation [70]. It is particularly effective on Chinese tallow tree (*Triadica sebifera*), which is a major invasive species throughout much of the southeastern United States. Imazamox is also effective for several emergent and ditchbank species, and preliminary research indicates good control of cattail (*Typha spp*.). This herbicide has limited grass activity, and is most effective on broadleaf species.

The sulfonylurea herbicides chlorsulfuron, sulfometuron and metsulfuron are formulated as dry flowable granules that readily mix with water. Sulfonylureas are weak acid compounds with very high water solubility [14]. These herbicides are readily absorbed by roots from soil applications and transported via the xylem to shoots and leaves of plants. Once in the leaves, these herbicides are often remobilized in the phloem to growing regions - tracking a similar pattern of flow as carbohydrates. Sulfonylureas are also absorbed from applications to plant foliage, entering the leaves and stems, and translocated to areas of high meristematic activity in manner similar to root uptake [60].

will confer resistance, and several substitutions (single amino acid changes) will cause resistance in sulfonyl-ureas. Conversely, very few impart resistance in imidazolinones and only one confers resistance across both herbicide families. The substitutions that confer resistance also appear to have little to no effect on enzyme efficiency, and thus growth of

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Those herbicides that directly inhibit photosynthesis have been used for several years and were developed in the 1950's and 1960's [81]. While several chemical families are represented within this broad mode of action classification, the substituted ureas and triazines are those used most widely for natural area weed control. These products were originally developed for use in pasture/rangelands and forestry situations, but like several other herbicides, have been

The triazine herbicides used in natural areas include hexazinone, simazine, and prometon. Simazine was originally developed for broadleaf and grass weed control in corn and sorghum, but later uses included grass and broadleaf control in established fruit and nut crops, albeit much higher rates of application per acre [82,83]. It was also used in aquatic situations for algae control, sold under the trade name "Aquazine", but this was cancelled in the 1990's [83]. Its use in natural areas currently is limited, primarily because simazine lacks broadspectrum

Prometon has been used for many years in industrial settings for broad-spectrum annual and perennial grass and broadleaf weed control [85]. This herbicide has considerable activity on many hardwood tree species, and is often marketed as a soil sterilant. This tremendous activity limits its use in many situations that require selectivity, and that includes forestry and most natural areas. Therefore, labeling as such is confined to areas where little to no vegetation is desired such as powerline substations, under asphalt paving, sidewalks, railyards and similar

Hexazinone is an asymmetrical triazine that was originally developed for use in the conifer forest industry for hardwood control, and often used in a manner called pine release [87]. This situation occurs 2-4 years after pine seedling establishment, where hexazinone is broadcast applied to provide control of regenerating hardwood species, allowing the pines to be 'released' from the competing hardwood saplings. Hexazinone also has a label for use in bahiagrass (*Pasapalum notatum*) and bermudagrass (*Cynodon dactylon*) pastures for the control of broadleaf species, but most often targeting smutgrass (*Sporobulus indicus*) [88]. It can be used in many natural area settings where hardwood tree, brush/shrubs or possibly vines are the target, but many native forbs and some native grasses may also be injured. Hexazinone works wells in areas where pines are the primary species, possibly where undesirable species are dominant under pines, and understory selectivity is not paramount. Once these species have

Diuron and tebuthiuron comprise those herbicides in the substituted urea chemical family that are used in natural areas. Diuron is similar to simazine in that it was first developed for use in

industrial sites [86]. Consequently prometon use in natural areas is very limited.

herbicide resistant biotypes varies little from non-resistant biotypes [80].

control of perennial plants, particularly brush, vines and trees.

been removed, revegetation can then be accomplished.

**3.3. Photosynthetic inhibitors**

adopted for use in natural areas.

The imidazolinone herbicides are also highly water soluble but formulated as salts. They are generally marketed as aqueous solutions, but some older formulations were dry flowable granules. Imidiazolinone herbicides are variable in soil activity but if present can be readily absorbed by plant roots [71]. They are transported to leaves and stem tissues via the xylem and can be remobilized to meristematic tissues. This pattern of reallocation occurs in the phloem, similar to carbohydrate movement. Imidazolinones are also absorbed from applica‐ tions to plant foliage, entering the leaves and stems, and translocated to areas of high meris‐ tematic activity in manner similar to root uptake [72].

Mechanistically, the imidazolinones and sulfonyl-ureas act in the same manner by inhibiting the activity of the enzyme acetolactate synthase (ALS), which is also referred to as acetohy‐ droxy acid synthase (AHAS, EC 2.2.1.6) [73]. This enzyme catalyzes the conversion of 2 ketobutyrate to 2-acetohydroxybutyrate through the addition of a 2 carbon unit using hydroxymethyl thiamine pyrophosphate (TPP). This is the initial step in the formation of the amino acid isoleucine. The ALS enzyme also catalyzes the conversion of pyruvate to form 2 aectolactate, once again utilizing TPP to add a 2-carbon unit [74]. This reaction is the initial step in the formation of valine and leucine. Thus by inhibiting acetolactate synthase, the formation of three essential branched chain amino acids cannot occur and inhibition occurs through a binding of the herbicide across the channel leading to the active site [75]. Herbicides in both groups bind at entrance of this channel, effectively blocking entrance to substrates and co-factors needed for the reaction to occur.

The inability of the plant to produce these essential amino acids leads to a cessation of protein/ enzyme synthesis and plant growth. Since these compounds accumulate in areas of new growth, meristematic activity is stopped. The plant cannot continue to make new cells and eventually dies [60]. Symptoms from these herbicides are generally manifested as discoloration in the growing regions, especially newly emerging leaves and shoot tips. Internode length is markedly decreased, and leaves may be malformed or misshaped [76]. Generalized chlorosis is a common symptom, although imidazolinones may show purple discoloration, especially in effected grasses. In annual species, a characteristic symptom of sulfonylurea injury is a reddening of the abaxial leaf veins.

Selectivity of these herbicides in plants is primarily metabolism based, and is often mediated through mixed-function oxidases (MFO's) [77]. These compounds catalyze several reactions in plants, including the breakdown of harmful xenobiotics such as herbicides. Tolerant plants generally are able to metabolize suflonyl-ureas and/or imidazolinones through this mecha‐ nism, thus imparting selectivity [60]. In cropping systems, crop selectivity is compromised if certain insecticides, such organo-phosphates, are used that disrupt MFO activity, allowing the herbicide to affect the target enzyme [78].

Interestingly, resistance development by weedy species occurs through amino acid substitu‐ tions of the target enzyme at the binding site [79]. In most cases, only a single amino acid change will confer resistance, and several substitutions (single amino acid changes) will cause resistance in sulfonyl-ureas. Conversely, very few impart resistance in imidazolinones and only one confers resistance across both herbicide families. The substitutions that confer resistance also appear to have little to no effect on enzyme efficiency, and thus growth of herbicide resistant biotypes varies little from non-resistant biotypes [80].

#### **3.3. Photosynthetic inhibitors**

pattern of flow as carbohydrates. Sulfonylureas are also absorbed from applications to plant foliage, entering the leaves and stems, and translocated to areas of high meristematic activity

The imidazolinone herbicides are also highly water soluble but formulated as salts. They are generally marketed as aqueous solutions, but some older formulations were dry flowable granules. Imidiazolinone herbicides are variable in soil activity but if present can be readily absorbed by plant roots [71]. They are transported to leaves and stem tissues via the xylem and can be remobilized to meristematic tissues. This pattern of reallocation occurs in the phloem, similar to carbohydrate movement. Imidazolinones are also absorbed from applica‐ tions to plant foliage, entering the leaves and stems, and translocated to areas of high meris‐

Mechanistically, the imidazolinones and sulfonyl-ureas act in the same manner by inhibiting the activity of the enzyme acetolactate synthase (ALS), which is also referred to as acetohy‐ droxy acid synthase (AHAS, EC 2.2.1.6) [73]. This enzyme catalyzes the conversion of 2 ketobutyrate to 2-acetohydroxybutyrate through the addition of a 2 carbon unit using hydroxymethyl thiamine pyrophosphate (TPP). This is the initial step in the formation of the amino acid isoleucine. The ALS enzyme also catalyzes the conversion of pyruvate to form 2 aectolactate, once again utilizing TPP to add a 2-carbon unit [74]. This reaction is the initial step in the formation of valine and leucine. Thus by inhibiting acetolactate synthase, the formation of three essential branched chain amino acids cannot occur and inhibition occurs through a binding of the herbicide across the channel leading to the active site [75]. Herbicides in both groups bind at entrance of this channel, effectively blocking entrance to substrates and

The inability of the plant to produce these essential amino acids leads to a cessation of protein/ enzyme synthesis and plant growth. Since these compounds accumulate in areas of new growth, meristematic activity is stopped. The plant cannot continue to make new cells and eventually dies [60]. Symptoms from these herbicides are generally manifested as discoloration in the growing regions, especially newly emerging leaves and shoot tips. Internode length is markedly decreased, and leaves may be malformed or misshaped [76]. Generalized chlorosis is a common symptom, although imidazolinones may show purple discoloration, especially in effected grasses. In annual species, a characteristic symptom of sulfonylurea injury is a

Selectivity of these herbicides in plants is primarily metabolism based, and is often mediated through mixed-function oxidases (MFO's) [77]. These compounds catalyze several reactions in plants, including the breakdown of harmful xenobiotics such as herbicides. Tolerant plants generally are able to metabolize suflonyl-ureas and/or imidazolinones through this mecha‐ nism, thus imparting selectivity [60]. In cropping systems, crop selectivity is compromised if certain insecticides, such organo-phosphates, are used that disrupt MFO activity, allowing the

Interestingly, resistance development by weedy species occurs through amino acid substitu‐ tions of the target enzyme at the binding site [79]. In most cases, only a single amino acid change

in manner similar to root uptake [60].

212 Herbicides - Current Research and Case Studies in Use

tematic activity in manner similar to root uptake [72].

co-factors needed for the reaction to occur.

reddening of the abaxial leaf veins.

herbicide to affect the target enzyme [78].

Those herbicides that directly inhibit photosynthesis have been used for several years and were developed in the 1950's and 1960's [81]. While several chemical families are represented within this broad mode of action classification, the substituted ureas and triazines are those used most widely for natural area weed control. These products were originally developed for use in pasture/rangelands and forestry situations, but like several other herbicides, have been adopted for use in natural areas.

The triazine herbicides used in natural areas include hexazinone, simazine, and prometon. Simazine was originally developed for broadleaf and grass weed control in corn and sorghum, but later uses included grass and broadleaf control in established fruit and nut crops, albeit much higher rates of application per acre [82,83]. It was also used in aquatic situations for algae control, sold under the trade name "Aquazine", but this was cancelled in the 1990's [83]. Its use in natural areas currently is limited, primarily because simazine lacks broadspectrum control of perennial plants, particularly brush, vines and trees.

Prometon has been used for many years in industrial settings for broad-spectrum annual and perennial grass and broadleaf weed control [85]. This herbicide has considerable activity on many hardwood tree species, and is often marketed as a soil sterilant. This tremendous activity limits its use in many situations that require selectivity, and that includes forestry and most natural areas. Therefore, labeling as such is confined to areas where little to no vegetation is desired such as powerline substations, under asphalt paving, sidewalks, railyards and similar industrial sites [86]. Consequently prometon use in natural areas is very limited.

Hexazinone is an asymmetrical triazine that was originally developed for use in the conifer forest industry for hardwood control, and often used in a manner called pine release [87]. This situation occurs 2-4 years after pine seedling establishment, where hexazinone is broadcast applied to provide control of regenerating hardwood species, allowing the pines to be 'released' from the competing hardwood saplings. Hexazinone also has a label for use in bahiagrass (*Pasapalum notatum*) and bermudagrass (*Cynodon dactylon*) pastures for the control of broadleaf species, but most often targeting smutgrass (*Sporobulus indicus*) [88]. It can be used in many natural area settings where hardwood tree, brush/shrubs or possibly vines are the target, but many native forbs and some native grasses may also be injured. Hexazinone works wells in areas where pines are the primary species, possibly where undesirable species are dominant under pines, and understory selectivity is not paramount. Once these species have been removed, revegetation can then be accomplished.

Diuron and tebuthiuron comprise those herbicides in the substituted urea chemical family that are used in natural areas. Diuron is similar to simazine in that it was first developed for use in crops – corn and cotton, with later registrations including broadleaf and grassy weeds in established fruit and nut crops [89,90]. Diuron has good activity on a number of annual species, but lacks control of perennial plants. It is often a component in combination herbicides for broad-spectrum weed control in industrial sites such as railroads, railyards, powerline rights of way and substations [86]. The goal of these applications is to provide a vegetation free zone for extended periods of time. The use of diuron in natural areas is limited due to spectrum of activity; too much injury on desirable annual grasses and forbs and limited control of larger, more woody shrubs, vines and trees.

crops avoid herbicide injury primarily through limited uptake, since the roots of most trees are below the concentrated herbicide zone [100]. Conversely hexazinone and tebuthiuron are more water soluble and move deeper into the soil profile, which limits their utility for longterm vegetation management because annual weeds begin to infest the zone above the herbicide [101]. However, this places these herbicides into the root zone of many perennial forbs, vines, shrubs and trees where it is absorbed and translocated, causing injury and often mortality. Even large trees, especially oaks, can be killed if sufficient herbicide is placed in the root zone. Typically the leaves become chlorotic, necrotic and abscise. New leaves emerge, and follow the same chronological pattern, but generally do not expand to more than half normal size. After 2 to 3 cycles of leaf emergence and abscission, the trees succumb to death due to the lack of carbohydrate reserves needed for growth [102]. Depending on species, rate applied, and geographic location, death can take 1-2 years. Unfortunately, these herbicides are some‐ times used in malicious attacks to destroy trees or shrubs; and in some cases trees of historic value, such as the Toomer Oaks on the campus of Auburn University, Auburn, Alabama

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(tebuthiuron) in 2010 or the Treaty Oak of Austin, Texas (hexazinone) in 1989 [103].

stroyed, and the tissue degrades.

mechanism is not known.

Photosynthetic inhibitors, regardless of chemical family, work in the same manner to interrupt the light reactions of photosynthesis. These reactions serve to capture the light energy from sunlight through excitation of chlorophyll molecules and the subsequent removal of an electron from a molecule of water; producing free oxygen and hydrogen [104]. Electrochemical energy is passed through a series of reactions (mainly photosystem II, cytochrome B, plasto‐ cyanin and photosystem I) to form NADPH+H. During this transfer, a proton gradient is formed across the chloroplast membrane, sufficient to generate ATP. These herbicides bind to a protein (specifically the D1 protein) within the photosystem II complex that does not allow electron transfer to occur [81]. This blockage of electron flow inhibits the formation of NADPH +H, and indirectly inhibits ATP formation as well. Energy continues to be absorbed by the chlorophyll molecules and transferred to the reaction centers associated with photosystem II, but cannot be dissipated [105]. This excess, or non-transferable, energy is then passed on to free oxygen, creating radical oxygen. Oxygen is a highly toxic radical that quickly reacts within the chloroplast to form hydroxyl radicals, peroxide, and/or lipoxides. Ultimately chloroplast and other cellular membranes become damaged and leaky, chlorophyll molecules are de‐

While many photosynthetic inhibitors can be considered total vegetation control herbicides, certain species have the ability to tolerate these herbicides through metabolism. Metabolism is achieved primarily by glutathione and/or carbohydrate conjugation, whereby the herbicide molecules are bound with these compounds and shuttled to the vacuole for further breakdown [106]. However, in natural area systems - especially at rates typically used, placement and differential uptake is the primary mechanism of selectivity. Many conifers, pines (*Pinus spp.*) in particular, have the ability to tolerate hexazinone presumably through metabolism, but the

Tebuthiuron however, has tremendous activity on a wide range of woody species, particularly hardwood trees such as as oaks (*Quercus spp.*), maple (*Acer spp.*), poplar (*Populus spp.*), and sweet gum (*Liquidambar styraciflua*). [91]. This species is also very effective on shrubs, vines and herbaceous perennials [92]. It is often used in non-crop land and industrial settings for broadleaf vegetation control, including vines and hardwoods. Tebuthiuron is utilized in powerline corridors and around utility poles to promote healthy grass stands to maintain cover for grazing for livestock and wildlife and also erosion control [93]. This herbicide also is labeled for use in certain forestry situations, primarily for non-desirable vegetation control in conifers [94].

Bromacil is another photosynthetic inhibitor that belongs to the uracil chemical family that has limited uses in natural areas. It has similar use patterns as diuron and simazine, including vegetation management in industrial sites such as powerline substations, railroads, railyards, and rights-of-way [86]. Bromacil can also be used in certain fruit crops such as citrus for broadleaf and grass weed control [14,95]. While this herbicide has tremendous activity on annual species, it has less than adequate control of perennial vines, trees and shrubs compared to other herbicides; therefore wide spread utility in natural areas is limited [96].

As a group, photosynthetic inhibitors have low water solubility and limited foliar uptake [97]. Most are formulated dry as wettable powders or pellets, or liquid as clay-suspended flowables. Hexazinone is the only exception with a liquid formulation. These herbicides are soil applied; even applications over the top of existing foliage are active only when reaching the soil [14]. Photosynthetic inhibitors are readily taken up by plant roots and translocated to leaves and shoots through the water stream facilitated by xylem tissue [98]. Once reaching leaves, these herbicides partition into individual cells. As the plant continues to transpire, more herbicide is moved to the leaves, with older leaves and leaf tips transpiring the most water. These areas tend to demonstrate chlorosis first and most strongly simply because these tissues have transpired more water, and thus taken up more herbicide, compared to newer tissues. This causes the characteristic pattern of chlorosis often observed with these herbicides. Subtle differences in water solubility between herbicides and subsequent partitioning into leaf tissue of various species produce variations in chlorotic patterns, such as veinal chlorosis and/or interveinal chlorosis [99].

Differences in water solubility and to a lesser extent degradation, dictate the uses and selec‐ tivity of these products. Diuron, simazine, prometon, and bromacil are very non-water soluble and tend to remain in the upper soil profile [14]. This maintains the herbicides in the zone of germinating annual weeds, thus providing extended weed control. Perennial fruit and nut crops avoid herbicide injury primarily through limited uptake, since the roots of most trees are below the concentrated herbicide zone [100]. Conversely hexazinone and tebuthiuron are more water soluble and move deeper into the soil profile, which limits their utility for longterm vegetation management because annual weeds begin to infest the zone above the herbicide [101]. However, this places these herbicides into the root zone of many perennial forbs, vines, shrubs and trees where it is absorbed and translocated, causing injury and often mortality. Even large trees, especially oaks, can be killed if sufficient herbicide is placed in the root zone. Typically the leaves become chlorotic, necrotic and abscise. New leaves emerge, and follow the same chronological pattern, but generally do not expand to more than half normal size. After 2 to 3 cycles of leaf emergence and abscission, the trees succumb to death due to the lack of carbohydrate reserves needed for growth [102]. Depending on species, rate applied, and geographic location, death can take 1-2 years. Unfortunately, these herbicides are some‐ times used in malicious attacks to destroy trees or shrubs; and in some cases trees of historic value, such as the Toomer Oaks on the campus of Auburn University, Auburn, Alabama (tebuthiuron) in 2010 or the Treaty Oak of Austin, Texas (hexazinone) in 1989 [103].

crops – corn and cotton, with later registrations including broadleaf and grassy weeds in established fruit and nut crops [89,90]. Diuron has good activity on a number of annual species, but lacks control of perennial plants. It is often a component in combination herbicides for broad-spectrum weed control in industrial sites such as railroads, railyards, powerline rights of way and substations [86]. The goal of these applications is to provide a vegetation free zone for extended periods of time. The use of diuron in natural areas is limited due to spectrum of activity; too much injury on desirable annual grasses and forbs and limited control of larger,

Tebuthiuron however, has tremendous activity on a wide range of woody species, particularly hardwood trees such as as oaks (*Quercus spp.*), maple (*Acer spp.*), poplar (*Populus spp.*), and sweet gum (*Liquidambar styraciflua*). [91]. This species is also very effective on shrubs, vines and herbaceous perennials [92]. It is often used in non-crop land and industrial settings for broadleaf vegetation control, including vines and hardwoods. Tebuthiuron is utilized in powerline corridors and around utility poles to promote healthy grass stands to maintain cover for grazing for livestock and wildlife and also erosion control [93]. This herbicide also is labeled for use in certain forestry situations, primarily

Bromacil is another photosynthetic inhibitor that belongs to the uracil chemical family that has limited uses in natural areas. It has similar use patterns as diuron and simazine, including vegetation management in industrial sites such as powerline substations, railroads, railyards, and rights-of-way [86]. Bromacil can also be used in certain fruit crops such as citrus for broadleaf and grass weed control [14,95]. While this herbicide has tremendous activity on annual species, it has less than adequate control of perennial vines, trees and shrubs compared

As a group, photosynthetic inhibitors have low water solubility and limited foliar uptake [97]. Most are formulated dry as wettable powders or pellets, or liquid as clay-suspended flowables. Hexazinone is the only exception with a liquid formulation. These herbicides are soil applied; even applications over the top of existing foliage are active only when reaching the soil [14]. Photosynthetic inhibitors are readily taken up by plant roots and translocated to leaves and shoots through the water stream facilitated by xylem tissue [98]. Once reaching leaves, these herbicides partition into individual cells. As the plant continues to transpire, more herbicide is moved to the leaves, with older leaves and leaf tips transpiring the most water. These areas tend to demonstrate chlorosis first and most strongly simply because these tissues have transpired more water, and thus taken up more herbicide, compared to newer tissues. This causes the characteristic pattern of chlorosis often observed with these herbicides. Subtle differences in water solubility between herbicides and subsequent partitioning into leaf tissue of various species produce variations in chlorotic patterns, such as veinal chlorosis and/or

Differences in water solubility and to a lesser extent degradation, dictate the uses and selec‐ tivity of these products. Diuron, simazine, prometon, and bromacil are very non-water soluble and tend to remain in the upper soil profile [14]. This maintains the herbicides in the zone of germinating annual weeds, thus providing extended weed control. Perennial fruit and nut

to other herbicides; therefore wide spread utility in natural areas is limited [96].

more woody shrubs, vines and trees.

214 Herbicides - Current Research and Case Studies in Use

interveinal chlorosis [99].

for non-desirable vegetation control in conifers [94].

Photosynthetic inhibitors, regardless of chemical family, work in the same manner to interrupt the light reactions of photosynthesis. These reactions serve to capture the light energy from sunlight through excitation of chlorophyll molecules and the subsequent removal of an electron from a molecule of water; producing free oxygen and hydrogen [104]. Electrochemical energy is passed through a series of reactions (mainly photosystem II, cytochrome B, plasto‐ cyanin and photosystem I) to form NADPH+H. During this transfer, a proton gradient is formed across the chloroplast membrane, sufficient to generate ATP. These herbicides bind to a protein (specifically the D1 protein) within the photosystem II complex that does not allow electron transfer to occur [81]. This blockage of electron flow inhibits the formation of NADPH +H, and indirectly inhibits ATP formation as well. Energy continues to be absorbed by the chlorophyll molecules and transferred to the reaction centers associated with photosystem II, but cannot be dissipated [105]. This excess, or non-transferable, energy is then passed on to free oxygen, creating radical oxygen. Oxygen is a highly toxic radical that quickly reacts within the chloroplast to form hydroxyl radicals, peroxide, and/or lipoxides. Ultimately chloroplast and other cellular membranes become damaged and leaky, chlorophyll molecules are de‐ stroyed, and the tissue degrades.

While many photosynthetic inhibitors can be considered total vegetation control herbicides, certain species have the ability to tolerate these herbicides through metabolism. Metabolism is achieved primarily by glutathione and/or carbohydrate conjugation, whereby the herbicide molecules are bound with these compounds and shuttled to the vacuole for further breakdown [106]. However, in natural area systems - especially at rates typically used, placement and differential uptake is the primary mechanism of selectivity. Many conifers, pines (*Pinus spp.*) in particular, have the ability to tolerate hexazinone presumably through metabolism, but the mechanism is not known.

#### **3.4. Glyphosate**

Glyphosate is one of the most widely used herbicides in the world, and has been extensively used in natural areas for nearly 4 decades [107]. It is non-selective and provides control of a wide range of species, including annual and perennial grasses, annual forbs, short lived perennials, vines and many tree species [108,109,110]. It has limited activity on conifers, but time of year dictates use during periods of no or slow growth. This is generally the fall months prior to winter, termed hardening-off [111]. While active on many species of larger perennials, it is often mixed with other herbicides for greater control.

**3.5. Fosamine**

tree to remove unwanted limbs and foliage [86].

multiple treatments are usually required.

**3.6. Inhibitors of Acetyl CoA Carboxylase (ACCase inhibitors)**

through binding to soil particles and rapid microbial degradation.

symptom is the water soaked browning of stems when pulled from the whorl.

Fosamine has been used in industrial right of way situations for many years and more recently used for invasive species control in natural areas such as natural savannahs and prairies. Brush control is the target for this herbicide, but it can be used for the control of herbaceous weeds such as leafy spurge (*Euphorbia esula*). Fosamine is tolerated by certain species of conifers, but hardwoods and other deciduous trees are often damaged. Fosamine is applied to the foliage of target plants where it is slowly absorbed by leaf tissues [118]. This herbicide has little to no soil activity and is rapidly degraded by soil microbes, limiting its environmental persistence [119]. This herbicide is recommended for late summer/autumn applications – typically one to two months prior to leaf drop. Fosamine appears to have limited translocation out of treated foliage and does not exhibit symptoms on treated tissue [120,121]. The effect of fosamine is not apparent until the following spring where leaves often fail to emerge or if emerged will be small and spindly in appearance. The mechanism of fosamine is not clear, but some evidence suggests an inhibition of mitosis or the inability of new developing cells to effectively transport calcium [14]. The limited translocation within plant tissues allows the use of this herbicide as a 'side-trim' treatment, where a portion of tree can be controlled without affecting the entire tree. This type of application is used in powerline and railroad situations to chemically trim a

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Herbicides within this group fall into two broad chemical families – the cyclohexanediones or the aryl-oxy-phenoxy propionates [14]. There are several herbicides within these families labeled for use in non-crop/natural areas, but the most widely utilized include sethoxydim, clethodim and fluazifop-butyl [86]. These herbicides are characterized by their selectivity towards annual and perennial grasses, with minimal to no activity on other monocots or dicot species [122]. They are primarily applied to the foliage due to a lack of appreciable soil activity

ACCase inhibiting herbicides are applied to the foliage of grasses, where they are readily absorbed. Similar to other weak acid herbicides, they are translocated to areas of meristematic growth following the pattern of carbohydrate flow [112]. Cyclohexanediones and aryl-oxyphenoxy propionate herbicides inhibit the activity of acetyl CoA carboxylase [123]. This enzyme is the initial step in the formation of fatty acids, which are the primary building blocks of cell membranes and other cellular components necessary for normal growth. New growth is stopped and grasses often become chlorotic or purple in color. Another characteristic

The utility of these herbicides is limited to annual and perennial grass control. Clethodim and fluazifop have superior activity on perennial grasses, and are often used for the control/ suppression of reed canarygrass (*Phalaris arundinacea*), cogongrass (*Imperata cylindrica*), Japanese stiltgrass (*Microstegium vimineum*) to name a few [124,125,126]. However, complete control of well established grass stands is often not achieved with a single application and

Glyphosate is chemically a weak acid, and is readily translocated in phloem tissues to areas of new growth. It is absorbed through foliar tissues such as leaves, shoot tips and green stems, but uptake is limited by woody tissues. Root uptake is possible, but rarely occurs due to irreversible binding of glyphosate to soil particles once the herbicide comes in contact with the soil. As in the case of other weak acid herbicides, glyphosate accumulates in meristematic regions, following a similar movement to that of carbohydrates [112]. Glyphosate affects the ability of plants to produce essential aromatic amino acids by blocking an initial step in the shikimic acid pathway. More specifically, this herbicide inhibits the activity of 5-enolpyruvyl‐ shikimate-3phosphate synthase (EPSP synthase) which catalyzes the conversion of EPSP from shikimate-3-phosphate and phosphoenolpyruvate [14]. This enzyme is a key enzyme in the shikimate acid pathway, which produces the aromatic amino acids tryptophan, phenylalanine, and tyrosine, along with a multitude of other secondary compounds including phenolics, flavonoids and coumarins [113]. Glyphosate also greatly influences carbon allocation and flow within the cell, as uncontrolled shikimate accumulation occurs as a result of this inhibition.

The typical symptoms of glyphosate injury include an initial cessation of growth followed by chlorosis in the meristematic regions of growth [14]. Chlorosis is often lighter in color com‐ pared to the photosynthetic inhibitors, and in some species may almost appear white or cream colored. Necrosis occurs several days after initial symptoms and complete plant death results in 21 to 35 days depending on species and maturity/size of treated plants. Glyphosate is extremely difficult to metabolize by plants and is readily translocated to areas of new growth [114]. This stability within plant tissues is the reason it has excellent activity on many perennial plants, allowing glyphosate to be 'stored' in overwintering tissues such rhizomes and root‐ stocks [115]. When plants begin to reallocate carbohydrates for spring regrowth, glyphosate is remobilized to these areas. Another unique symptom of glyphosate, particularly in regrow‐ ing perennial species, is the phenomenon of bud fasciation [116]. Bud fasciation is where several buds/shoot tips arise from a single meristematic region, forming a cluster of tightly packed shoots and leaves. The exact mechanism is not well understood, but appears to be related to a loss of apical dominance and deregulation of auxin activity.

Resistance to glyphosate has increased in annual cropping systems (Roundup-Ready technol‐ ogy) but resistance has not been documented in natural areas systems [117]. Several plants have the ability to tolerate and outgrow applications of glyphosate, especially trees, shrubs and woody vines. In these cases, limited uptake and/or dilution within non-metabolically active tissues is the likely reason for poor activity.

#### **3.5. Fosamine**

**3.4. Glyphosate**

216 Herbicides - Current Research and Case Studies in Use

Glyphosate is one of the most widely used herbicides in the world, and has been extensively used in natural areas for nearly 4 decades [107]. It is non-selective and provides control of a wide range of species, including annual and perennial grasses, annual forbs, short lived perennials, vines and many tree species [108,109,110]. It has limited activity on conifers, but time of year dictates use during periods of no or slow growth. This is generally the fall months prior to winter, termed hardening-off [111]. While active on many species of larger perennials,

Glyphosate is chemically a weak acid, and is readily translocated in phloem tissues to areas of new growth. It is absorbed through foliar tissues such as leaves, shoot tips and green stems, but uptake is limited by woody tissues. Root uptake is possible, but rarely occurs due to irreversible binding of glyphosate to soil particles once the herbicide comes in contact with the soil. As in the case of other weak acid herbicides, glyphosate accumulates in meristematic regions, following a similar movement to that of carbohydrates [112]. Glyphosate affects the ability of plants to produce essential aromatic amino acids by blocking an initial step in the shikimic acid pathway. More specifically, this herbicide inhibits the activity of 5-enolpyruvyl‐ shikimate-3phosphate synthase (EPSP synthase) which catalyzes the conversion of EPSP from shikimate-3-phosphate and phosphoenolpyruvate [14]. This enzyme is a key enzyme in the shikimate acid pathway, which produces the aromatic amino acids tryptophan, phenylalanine, and tyrosine, along with a multitude of other secondary compounds including phenolics, flavonoids and coumarins [113]. Glyphosate also greatly influences carbon allocation and flow within the cell, as uncontrolled shikimate accumulation occurs as a result of this inhibition.

The typical symptoms of glyphosate injury include an initial cessation of growth followed by chlorosis in the meristematic regions of growth [14]. Chlorosis is often lighter in color com‐ pared to the photosynthetic inhibitors, and in some species may almost appear white or cream colored. Necrosis occurs several days after initial symptoms and complete plant death results in 21 to 35 days depending on species and maturity/size of treated plants. Glyphosate is extremely difficult to metabolize by plants and is readily translocated to areas of new growth [114]. This stability within plant tissues is the reason it has excellent activity on many perennial plants, allowing glyphosate to be 'stored' in overwintering tissues such rhizomes and root‐ stocks [115]. When plants begin to reallocate carbohydrates for spring regrowth, glyphosate is remobilized to these areas. Another unique symptom of glyphosate, particularly in regrow‐ ing perennial species, is the phenomenon of bud fasciation [116]. Bud fasciation is where several buds/shoot tips arise from a single meristematic region, forming a cluster of tightly packed shoots and leaves. The exact mechanism is not well understood, but appears to be

Resistance to glyphosate has increased in annual cropping systems (Roundup-Ready technol‐ ogy) but resistance has not been documented in natural areas systems [117]. Several plants have the ability to tolerate and outgrow applications of glyphosate, especially trees, shrubs and woody vines. In these cases, limited uptake and/or dilution within non-metabolically

related to a loss of apical dominance and deregulation of auxin activity.

active tissues is the likely reason for poor activity.

it is often mixed with other herbicides for greater control.

Fosamine has been used in industrial right of way situations for many years and more recently used for invasive species control in natural areas such as natural savannahs and prairies. Brush control is the target for this herbicide, but it can be used for the control of herbaceous weeds such as leafy spurge (*Euphorbia esula*). Fosamine is tolerated by certain species of conifers, but hardwoods and other deciduous trees are often damaged. Fosamine is applied to the foliage of target plants where it is slowly absorbed by leaf tissues [118]. This herbicide has little to no soil activity and is rapidly degraded by soil microbes, limiting its environmental persistence [119]. This herbicide is recommended for late summer/autumn applications – typically one to two months prior to leaf drop. Fosamine appears to have limited translocation out of treated foliage and does not exhibit symptoms on treated tissue [120,121]. The effect of fosamine is not apparent until the following spring where leaves often fail to emerge or if emerged will be small and spindly in appearance. The mechanism of fosamine is not clear, but some evidence suggests an inhibition of mitosis or the inability of new developing cells to effectively transport calcium [14]. The limited translocation within plant tissues allows the use of this herbicide as a 'side-trim' treatment, where a portion of tree can be controlled without affecting the entire tree. This type of application is used in powerline and railroad situations to chemically trim a tree to remove unwanted limbs and foliage [86].

#### **3.6. Inhibitors of Acetyl CoA Carboxylase (ACCase inhibitors)**

Herbicides within this group fall into two broad chemical families – the cyclohexanediones or the aryl-oxy-phenoxy propionates [14]. There are several herbicides within these families labeled for use in non-crop/natural areas, but the most widely utilized include sethoxydim, clethodim and fluazifop-butyl [86]. These herbicides are characterized by their selectivity towards annual and perennial grasses, with minimal to no activity on other monocots or dicot species [122]. They are primarily applied to the foliage due to a lack of appreciable soil activity through binding to soil particles and rapid microbial degradation.

ACCase inhibiting herbicides are applied to the foliage of grasses, where they are readily absorbed. Similar to other weak acid herbicides, they are translocated to areas of meristematic growth following the pattern of carbohydrate flow [112]. Cyclohexanediones and aryl-oxyphenoxy propionate herbicides inhibit the activity of acetyl CoA carboxylase [123]. This enzyme is the initial step in the formation of fatty acids, which are the primary building blocks of cell membranes and other cellular components necessary for normal growth. New growth is stopped and grasses often become chlorotic or purple in color. Another characteristic symptom is the water soaked browning of stems when pulled from the whorl.

The utility of these herbicides is limited to annual and perennial grass control. Clethodim and fluazifop have superior activity on perennial grasses, and are often used for the control/ suppression of reed canarygrass (*Phalaris arundinacea*), cogongrass (*Imperata cylindrica*), Japanese stiltgrass (*Microstegium vimineum*) to name a few [124,125,126]. However, complete control of well established grass stands is often not achieved with a single application and multiple treatments are usually required.

#### **3.7. Glufosinate and paraquat**

Glufosinate and paraquat are contact type herbicides that can be used in a wide range of noncropland, industrial, rights-of-way areas and natural areas [86]. Both of these herbicides are contact in activity, requiring complete coverage of the target foliage to attain good control [14]. In addition, both paraquat and glufosinate do not possess soil activity due to immediate and irreversible binding to soil particles [127]. These herbicides are very effective on annual broadleaf and grassy weeds, but only marginally effective on well established perennial plants. Since these herbicides do not translocate out of treated foliage, perennial plants can usually regrow following treatment [112].

Secondly, these herbicides are non-selective - causing damage to any plant that is contacted, desirable and undesirable vegetation [14]. Thirdly, these herbicides lack soil activity so long

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There are several herbicides and herbicide families that encompass this mode of action category [14]. Protox inhibitors are primarily used in annual cropping systems for broadleaf, grasses and nutsedge (*Cyperus spp*.) control [134,135,136]. These herbicides possess good soil activity with moderate to long soil persistence and many also have

Protox inhibitors are readily taken up by plant roots and translocated to leaves and shoots through the water stream facilitated by xylem tissue [138]. Once reaching leaves, these herbicides partition into individual cells. As plant continues to transpire, more herbicide is moved to the leaves, with older leaves and leaf tips transpiring the most water. These areas tend to demonstrate damage initially and most strongly simply because these tissues have transpired more water, and thus taken up more herbicide, compared to newer tissues. Damage appears as bronzing or necrotic lesions in leaf tissue. These lesions generally lack pattern, but eventually coalesce into more wide-spread damage and eventual leaf drop. Stem tissues may also exhibit similar necrotic injury. In grasses and sedges, a browning of leaf tissue along the

Foliar activity shows a similar pattern, with necrotic lesions developing in random areas on leaf tissues, with complete necrosis occurring in 3-5 days [139]. Even tolerant plants will show some damage from foliar applications on treated tissue, but to a much lesser extent and quickly outgrow the injury. There is no translocation from foliar applications of protox inhibiting herbicides, only those areas contacted will be damaged [139]. However, subsequent damage may occur from root uptake, if an appreciable amount of herbicide reaches the soil and remains active. This is highly dependent on whether the herbicide has soil activity, application rate,

Protox inhibiting herbicides have a very unique mode of action that was not clearly understood for many years [140]. Mechanistically, these herbicides inhibit the enzyme protoporphyrino‐ gen oxidase which catalyzes the conversion of protoporphyrinogen IX to protoprophyrin IX in the chloroplast [141]. This step is an intermediate process in the production of chlorophyll molecules. Excess protoporphyrinogen IX leaks out of the chloroplast envelope into the cytoplasm where is it converted by a cytoplasmic (insensitive) version of protoporphyrinogen oxidase to protoprophyrin IX [142]. This molecule has the ability to absorb light energy, but can only dissipate this energy to oxygen. This forms singlet oxygen, a highly reactive form of oxygen that quickly interacts to form other highly toxic radicals that destroy cell membranes.

Utility of the protox inhibiting herbicides in natural areas is limited. Foliar activity is contact only, therefore perennial plants quickly regrow. In addition, at the rates needed to garner control, selectivity is lost or severely compromised. Appreciable control can be achieved from

Cells become leaky, rupture, die and eventual tissue degradation follows.

term control cannot be realized.

tremendous foliar activity [137].

midvein is often observed.

and foliar coverage at the time of application.

**3.8. Protox inhibitors**

Glufosinate is rapidly absorbed by leaf tissue and is active in the chloroplast of cells. Specifi‐ cally, glufosinate inhibits the enzyme glutamine synthase, which catalyzes the incorporation of free ammonia into the amino acid glutamate to form glutamine [128]. This reaction is the primary mechanism by which plants incorporate nitrogen for use in cellular products such as amino acids, nucleotides, enzymes and storage proteins. The lack of nitrogen incorporation, however, is not the primary means by which the plant dies. Free ammonia levels increase in the chloroplast where this molecule begins to uncouple membranes. Uncoupling is the action where membranes can no longer maintain a gradient that drives energy formation in photo‐ synthesis [129]. Damage becomes visible generally after 4 to 5 days and appears as chlorotic lesions followed by rapid necrosis of treated leaves.

Paraquat herbicide was developed in the early 1960's for broad spectrum weed control in non-crop land and other vegetation free sites. Paraquat is rapidly absorbed by leaf tissues and is active primarily in the chloroplast, although it may also impede mitochondrial function [112]. Paraquat affects the light reactions of photosynthesis in the photosystem I complex, more specifically at the site of electron transfer from ferrodoxin to NADPH+H reductase [130,131]. Paraquat does not bind or disrupt enzyme activity, but rather steals/ diverts the electron to become a reduced paraquat molecule. Paraquat in this reduced form quickly passes the electron energy to oxygen, creating oxidized paraquat and radical oxygen (O2-). Paraquat becomes reduced again by another electron, oxidized through transfer to oxygen and the cycle continues. Subsequently, the ability of the plant to make NADPH+H is compromised, but more importantly radical oxygen reacts with water and lipids to produce hydrogen peroxide, hydroxyl radicals and lipoxides. These radicals interact with the lipid fraction of membranes, destroying the chloroplast and eventually the plasma membrane [132]. Symptoms from paraquat can be evident within 12 to 24 hours after application. Leaves first appear water soaked, followed quickly by necrotic lesions that coalesce to encompass the entire leaf. High light levels promote faster necrosis, and complete damage is generally achieved within 2-4 days.

Glufosinate and paraquat require good coverage and therefore must be applied in higher carrier volumes compared to systemic herbicides. There appears to be some movement with glufosinate out of treated tissues, but translocation to perennial structures such as rhizomes or tubers does not occur to an appreciable extent [133]. The utility of paraquat and glufosinate for natural area plant management is limited for several reasons. First most weeds in natural areas are perennials, so applications of these herbicides will only provide temporary control. Secondly, these herbicides are non-selective - causing damage to any plant that is contacted, desirable and undesirable vegetation [14]. Thirdly, these herbicides lack soil activity so long term control cannot be realized.

### **3.8. Protox inhibitors**

**3.7. Glufosinate and paraquat**

218 Herbicides - Current Research and Case Studies in Use

regrow following treatment [112].

lesions followed by rapid necrosis of treated leaves.

complete damage is generally achieved within 2-4 days.

Glufosinate and paraquat are contact type herbicides that can be used in a wide range of noncropland, industrial, rights-of-way areas and natural areas [86]. Both of these herbicides are contact in activity, requiring complete coverage of the target foliage to attain good control [14]. In addition, both paraquat and glufosinate do not possess soil activity due to immediate and irreversible binding to soil particles [127]. These herbicides are very effective on annual broadleaf and grassy weeds, but only marginally effective on well established perennial plants. Since these herbicides do not translocate out of treated foliage, perennial plants can usually

Glufosinate is rapidly absorbed by leaf tissue and is active in the chloroplast of cells. Specifi‐ cally, glufosinate inhibits the enzyme glutamine synthase, which catalyzes the incorporation of free ammonia into the amino acid glutamate to form glutamine [128]. This reaction is the primary mechanism by which plants incorporate nitrogen for use in cellular products such as amino acids, nucleotides, enzymes and storage proteins. The lack of nitrogen incorporation, however, is not the primary means by which the plant dies. Free ammonia levels increase in the chloroplast where this molecule begins to uncouple membranes. Uncoupling is the action where membranes can no longer maintain a gradient that drives energy formation in photo‐ synthesis [129]. Damage becomes visible generally after 4 to 5 days and appears as chlorotic

Paraquat herbicide was developed in the early 1960's for broad spectrum weed control in non-crop land and other vegetation free sites. Paraquat is rapidly absorbed by leaf tissues and is active primarily in the chloroplast, although it may also impede mitochondrial function [112]. Paraquat affects the light reactions of photosynthesis in the photosystem I complex, more specifically at the site of electron transfer from ferrodoxin to NADPH+H reductase [130,131]. Paraquat does not bind or disrupt enzyme activity, but rather steals/ diverts the electron to become a reduced paraquat molecule. Paraquat in this reduced form quickly passes the electron energy to oxygen, creating oxidized paraquat and radical oxygen (O2-). Paraquat becomes reduced again by another electron, oxidized through transfer to oxygen and the cycle continues. Subsequently, the ability of the plant to make NADPH+H is compromised, but more importantly radical oxygen reacts with water and lipids to produce hydrogen peroxide, hydroxyl radicals and lipoxides. These radicals interact with the lipid fraction of membranes, destroying the chloroplast and eventually the plasma membrane [132]. Symptoms from paraquat can be evident within 12 to 24 hours after application. Leaves first appear water soaked, followed quickly by necrotic lesions that coalesce to encompass the entire leaf. High light levels promote faster necrosis, and

Glufosinate and paraquat require good coverage and therefore must be applied in higher carrier volumes compared to systemic herbicides. There appears to be some movement with glufosinate out of treated tissues, but translocation to perennial structures such as rhizomes or tubers does not occur to an appreciable extent [133]. The utility of paraquat and glufosinate for natural area plant management is limited for several reasons. First most weeds in natural areas are perennials, so applications of these herbicides will only provide temporary control. There are several herbicides and herbicide families that encompass this mode of action category [14]. Protox inhibitors are primarily used in annual cropping systems for broadleaf, grasses and nutsedge (*Cyperus spp*.) control [134,135,136]. These herbicides possess good soil activity with moderate to long soil persistence and many also have tremendous foliar activity [137].

Protox inhibitors are readily taken up by plant roots and translocated to leaves and shoots through the water stream facilitated by xylem tissue [138]. Once reaching leaves, these herbicides partition into individual cells. As plant continues to transpire, more herbicide is moved to the leaves, with older leaves and leaf tips transpiring the most water. These areas tend to demonstrate damage initially and most strongly simply because these tissues have transpired more water, and thus taken up more herbicide, compared to newer tissues. Damage appears as bronzing or necrotic lesions in leaf tissue. These lesions generally lack pattern, but eventually coalesce into more wide-spread damage and eventual leaf drop. Stem tissues may also exhibit similar necrotic injury. In grasses and sedges, a browning of leaf tissue along the midvein is often observed.

Foliar activity shows a similar pattern, with necrotic lesions developing in random areas on leaf tissues, with complete necrosis occurring in 3-5 days [139]. Even tolerant plants will show some damage from foliar applications on treated tissue, but to a much lesser extent and quickly outgrow the injury. There is no translocation from foliar applications of protox inhibiting herbicides, only those areas contacted will be damaged [139]. However, subsequent damage may occur from root uptake, if an appreciable amount of herbicide reaches the soil and remains active. This is highly dependent on whether the herbicide has soil activity, application rate, and foliar coverage at the time of application.

Protox inhibiting herbicides have a very unique mode of action that was not clearly understood for many years [140]. Mechanistically, these herbicides inhibit the enzyme protoporphyrino‐ gen oxidase which catalyzes the conversion of protoporphyrinogen IX to protoprophyrin IX in the chloroplast [141]. This step is an intermediate process in the production of chlorophyll molecules. Excess protoporphyrinogen IX leaks out of the chloroplast envelope into the cytoplasm where is it converted by a cytoplasmic (insensitive) version of protoporphyrinogen oxidase to protoprophyrin IX [142]. This molecule has the ability to absorb light energy, but can only dissipate this energy to oxygen. This forms singlet oxygen, a highly reactive form of oxygen that quickly interacts to form other highly toxic radicals that destroy cell membranes. Cells become leaky, rupture, die and eventual tissue degradation follows.

Utility of the protox inhibiting herbicides in natural areas is limited. Foliar activity is contact only, therefore perennial plants quickly regrow. In addition, at the rates needed to garner control, selectivity is lost or severely compromised. Appreciable control can be achieved from soil uptake and activity, but those rates of herbicide application necessary may also reduce selectivity, and in some cases may not be within label guidelines. Flumioxazin and oxyfluorfen are protox inhibiting herbicides that may be used in non-crop areas, but applicability to natural areas has not been widely studied. These herbicides may have some use in restoration situations, providing control of undesirable vegetation prior to or immediately after an augmented restoration planting. However, there has been limited research to determine which herbicide product is most effective as a function of selectivity and desirable persistence.

#### **3.9. Growth inhibitors**

Herbicides that are categorized as growth inhibitors fall into three major mechanisms of action, but produce the common effect of inhibiting seedling emergence. The three mechanisms include: 1) interruption of mitosis through a blockage of spindle fiber formation, 2) interruption of cell wall formation through an inhibition of cellulose biosynthesis, and 3) interruption of cell membrane formation through a blockage of very long chain fatty acid synthesis. In nearly all situations, these herbicides are applied to the soil where they are absorbed by germinating seedlings, preventing seedling growth [14].

These compounds are characterized by extremely low water solubility, maintaining the herbicides in the upper soil profile [127]. As seeds germinate, the roots and emerging shoots come in contact with the herbicide, where it is rapidly absorbed, inhibiting growth and killing seedlings before they emerge from the soil. These herbicides do not translocate within plant tissues, so the growing regions of the plant must come in contact to be effective. Foliar applications are ineffective because the herbicides remain in the cuticle or epidermal cells, and cannot come in contact with meristematic tissues which are generally shielded within the bud structure. Selectivity is achieved through placement, whereby the shoots of tolerant germi‐ nating seedlings can emerge with minimal herbicide uptake in meristematic regions *and* the roots can grow below the treated layer. In cropping systems, this is most often achieved with broadleaf crops possessing hypogeal germination patterns. Perennial crops also exhibit good tolerance because the roots are well below the treated soil layer and foliar uptake is minimal. Examples of growth inhibiting herbicides used in non-crop areas include diclobenil, pendi‐ methalin and metolachlor, but applicability to natural areas has not been widely studied.

area weed management, only one species is the target and damage to other species is not

**Figure 1.** Postemergence application of herbicide to woody brush. Photo Courtesy James Miller, U.S. Department of

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This is the most common method of application, whereby the herbicide in diluted solution is applied as a spray over the top of targeted species (Figure 1). For larger areas, treatments are made to both target and non-target species utilizing an aerial (propeller plane or helicopter), tractor or all-terrain vehicle (ATV) mounted broadcast spray boom. Herbicides are applied as the amount of active ingredient per unit land area, and calibrated to deliver this amount based on carrier volume output. Smaller, more isolated or higher selectivity required sites will utilize a backpack sprayer with a hand-held spray wand or boom. Backpack applications cannot be calibrated in the same manner; herbicides are applied as a percentage of undiluted herbicide

Aerial applications are highly restricted and only certain herbicides can be applied aerially, and in some cases only during certain times of the year to minimize off-target injury. For

desirable – especially injury to rare or endangered plants.

**4.1. Post-emergence foliar applications**

Agriculture, Forest Service, http://www.forestryimages.org

in a variable carrier output [18,143].

### **4. Herbicide application methods in natural area weed management**

This section will detail the various methods used for applying herbicides for management of weedy species in natural areas. The complexity of natural areas dictates a unique and often non-conventional approach to herbicide application to 1) maintain selectivity, 2) provide control of large specimens, and 3) minimize off-target damage to the natural environment. Selectivity is much more difficult to achieve and maintain in natural areas. Herbicides are generally developed for weed control in cropping systems, and then secondarily labeled for use in non-cropland areas. In crops only selectivity towards the crop plant is desired, and damage to all other plants is beneficial, advantageous or inconsequential. However, in natural

**Figure 1.** Postemergence application of herbicide to woody brush. Photo Courtesy James Miller, U.S. Department of Agriculture, Forest Service, http://www.forestryimages.org

area weed management, only one species is the target and damage to other species is not desirable – especially injury to rare or endangered plants.

#### **4.1. Post-emergence foliar applications**

soil uptake and activity, but those rates of herbicide application necessary may also reduce selectivity, and in some cases may not be within label guidelines. Flumioxazin and oxyfluorfen are protox inhibiting herbicides that may be used in non-crop areas, but applicability to natural areas has not been widely studied. These herbicides may have some use in restoration situations, providing control of undesirable vegetation prior to or immediately after an augmented restoration planting. However, there has been limited research to determine which herbicide product is most effective as a function of selectivity and desirable persistence.

Herbicides that are categorized as growth inhibitors fall into three major mechanisms of action, but produce the common effect of inhibiting seedling emergence. The three mechanisms include: 1) interruption of mitosis through a blockage of spindle fiber formation, 2) interruption of cell wall formation through an inhibition of cellulose biosynthesis, and 3) interruption of cell membrane formation through a blockage of very long chain fatty acid synthesis. In nearly all situations, these herbicides are applied to the soil where they are absorbed by germinating

These compounds are characterized by extremely low water solubility, maintaining the herbicides in the upper soil profile [127]. As seeds germinate, the roots and emerging shoots come in contact with the herbicide, where it is rapidly absorbed, inhibiting growth and killing seedlings before they emerge from the soil. These herbicides do not translocate within plant tissues, so the growing regions of the plant must come in contact to be effective. Foliar applications are ineffective because the herbicides remain in the cuticle or epidermal cells, and cannot come in contact with meristematic tissues which are generally shielded within the bud structure. Selectivity is achieved through placement, whereby the shoots of tolerant germi‐ nating seedlings can emerge with minimal herbicide uptake in meristematic regions *and* the roots can grow below the treated layer. In cropping systems, this is most often achieved with broadleaf crops possessing hypogeal germination patterns. Perennial crops also exhibit good tolerance because the roots are well below the treated soil layer and foliar uptake is minimal. Examples of growth inhibiting herbicides used in non-crop areas include diclobenil, pendi‐ methalin and metolachlor, but applicability to natural areas has not been widely studied.

**4. Herbicide application methods in natural area weed management**

This section will detail the various methods used for applying herbicides for management of weedy species in natural areas. The complexity of natural areas dictates a unique and often non-conventional approach to herbicide application to 1) maintain selectivity, 2) provide control of large specimens, and 3) minimize off-target damage to the natural environment. Selectivity is much more difficult to achieve and maintain in natural areas. Herbicides are generally developed for weed control in cropping systems, and then secondarily labeled for use in non-cropland areas. In crops only selectivity towards the crop plant is desired, and damage to all other plants is beneficial, advantageous or inconsequential. However, in natural

**3.9. Growth inhibitors**

220 Herbicides - Current Research and Case Studies in Use

seedlings, preventing seedling growth [14].

This is the most common method of application, whereby the herbicide in diluted solution is applied as a spray over the top of targeted species (Figure 1). For larger areas, treatments are made to both target and non-target species utilizing an aerial (propeller plane or helicopter), tractor or all-terrain vehicle (ATV) mounted broadcast spray boom. Herbicides are applied as the amount of active ingredient per unit land area, and calibrated to deliver this amount based on carrier volume output. Smaller, more isolated or higher selectivity required sites will utilize a backpack sprayer with a hand-held spray wand or boom. Backpack applications cannot be calibrated in the same manner; herbicides are applied as a percentage of undiluted herbicide in a variable carrier output [18,143].

Aerial applications are highly restricted and only certain herbicides can be applied aerially, and in some cases only during certain times of the year to minimize off-target injury. For example, the state of Florida restricts the use of organo-auxin from aerial applications from January 1 until May 1 of each year [144]. Aerial treatments often utilize very low gallon spray volumes (3-10 gallons per acre) to maximize efficiency with weight and spray volume [145]. This restricts aerial applications to systemic herbicides that are not dependent on high carrier volume for effectiveness.

by placing a known amount of liquid in the sprayer and sprays a defined area. Once the area has been completely sprayed, the amount of liquid used by the applicator is calculated to

Soil basal applications are used for 2 primary purposes - 1) provide control of an existing plant or group of plants, or 2) provide preventative control of potential plant problems around stationary objects such as power poles. In either scenario, the herbicide is placed in often high concentrations around the base of the treated plant or object. The herbicide may be applied in liquid or granular form, and in a variety of placement patterns to achieve maximum root uptake of the intended target(s). Some herbicides, especially soil active photosynthetic herbicides, are formulated as pellets, which are essentially larger, more concentrated granules. Dry formulated granules or pellets are often easier and more accurate to apply as basal soil treatments. In these situations a certain number of pellets or dry volume of granule is placed as a function of targeted plant circumference. The pattern of placement varies considerably among applicators and may include circular, piles of pellets, or even gridlines in the case of larger infestations [146]. Soil basal herbicides include many of the photosynthetic inhibitors and several of the ALS and growth regulating herbicides. While the growth inhibiting and protox herbicides possess good soil activity, their effectiveness on established and larger plants is limited due to lack of root uptake and translocation or short-term control. Uses are generally

restricted to those situations where preventative control is the primary objective.

Basal-bark applications are utilized to provide control of larger specimens, where over-thetop foliar applications are not feasible for logistical or selectivity reasons. As the name suggests, basal-bark treatments are made near the ground to the trunks of small trees or shrubs [143]. Treatments are applied using a hand-held spray bottle or backpack sprayer to provide a tight stream of liquid onto the bark (Figure 2). Techniques for basal-bark applications vary widely among practitioners and weed specialists, but most agree that complete coverage around the trunk base is necessary for control. The width of the spray band around the tree varies as a function of species, size and herbicide being used, but most common is a 12 inch (30 cm) width band. Applications are generally made to the point of visual dripping or running of the liquid

Basal-bark treatments utilize an oil carrier (often referred to as basal oil) in which the herbicide is diluted at a high concentration or undiluted [147]. Diesel fuel or kerosene was used as carriers for many years, but environmental and economic restrictions limit current usages in many areas. In some cases, depending on herbicide formulation, the herbicide may be applied in undiluted form. Regardless of carrier, the herbicide must be in an oil soluble/lipophilic form to allow for penetration into the bark tissues. The objective is to maximize herbicide penetration through the outer epidermal layers (periderm) and reach the secondary phloem and cambium [143]. Once reaching these layers, the herbicide may be remobilized in the phloem, penetrate and affect the dividing cambium cells, or possibly enter the water stream via the xylem

).

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determine individual spray output per area (in most cases ft2

**4.2. Soil basal applications**

**4.3. Basal-bark applications**

down the bark surface.

Tractor or ATV boom-mounted sprayer applications can utilize a range of carrier volumes and thus not restricted to systemic herbicides only. These types of application equipment generally utilize a rear mounted boom with flat fan nozzles. The size or width of the boom varies, but ATV mounted booms are generally less than 15 feet while tractor booms may reach 30 feet or greater. Regardless, boom width is restricted compared to traditional agricultural applications due to unevenness of terrain to be covered, obstacles such as trees, shrubs, etc. and limitations on pump and tank capacity on smaller tractors and ATVs. Boom applications, especially those utilizing boom widths greater than 15-20 feet, require relatively flat ground, uniform height and high density of target species. As such, many land managers cannot utilize this type of equipment in many natural area systems.

Boom-less nozzles are often used in industrial applications and have some merit for use in natural area weed control. These nozzles are specifically designed to produce a multi-stream pattern across a 12-15 foot-wide spray swath. When mounted on an ATV or truck, these nozzles can produce a sizable sprayed area, without the issues associated with a fix boom to avoid obstacles and uneven terrain. However, coverage with these types of nozzles is not uniform and generally high volume output is required to maintain proper spray pattern. In addition, the actual nozzle is very expensive compared to a standard fan flan system. Due to difficulties with application uniformity and issues with achieving selectivity, most natural area weed managers will rely heavily on small backpack sprayers. This type of sprayer consists of a 5 gallon/20 liter (on average) tank, a hand-held, single nozzle spray wand, and a small dia‐ phragm pump with an attached lever. The operator uses the pump to pressurize the tank, forcing the liquid spray mixture through the spray wand. Pressurization is under the control of the operator, and is generally maintained to provide a proper pattern from the adjustable orifice on the spray wand. As the name suggests, the apparatus is worn on the back of the applicator using shoulder straps and often a waist strap to stabilize weight distribution. In most cases, the user operates the wand with one hand and pressurizes the tank with the other.

Backpack applications utilize diluted herbicide solution and mixed as a percent solution; in most cases between 0.5 and 3% solution. Applications are made to target species on a visual 'spray to wetness' observation. To achieve some degree of uniformity among applicators, the basis for adequate spray delivery is when spray droplets begin to drip from the leaf surfaces. This 'spray to runoff' technique is common regardless of target species or herbicide. While it is difficult to accurately measure volume output on a per acre basis, most researchers estimate these types of applications to range from 30 to 50 gallons per acre. In many cases, postemergence foliar applications contain herbicides with soil residual activity, either from an herbicide that possesses both foliar and soil activity or soil active herbicides that are tank-mixed to provide extended control. Regardless, the application technique is the same for most boommounted sprayers. For soil applications using a backpack sprayer, the applicator self-calibrates by placing a known amount of liquid in the sprayer and sprays a defined area. Once the area has been completely sprayed, the amount of liquid used by the applicator is calculated to determine individual spray output per area (in most cases ft2 ).

### **4.2. Soil basal applications**

example, the state of Florida restricts the use of organo-auxin from aerial applications from January 1 until May 1 of each year [144]. Aerial treatments often utilize very low gallon spray volumes (3-10 gallons per acre) to maximize efficiency with weight and spray volume [145]. This restricts aerial applications to systemic herbicides that are not dependent on high carrier

Tractor or ATV boom-mounted sprayer applications can utilize a range of carrier volumes and thus not restricted to systemic herbicides only. These types of application equipment generally utilize a rear mounted boom with flat fan nozzles. The size or width of the boom varies, but ATV mounted booms are generally less than 15 feet while tractor booms may reach 30 feet or greater. Regardless, boom width is restricted compared to traditional agricultural applications due to unevenness of terrain to be covered, obstacles such as trees, shrubs, etc. and limitations on pump and tank capacity on smaller tractors and ATVs. Boom applications, especially those utilizing boom widths greater than 15-20 feet, require relatively flat ground, uniform height and high density of target species. As such, many land managers cannot utilize this type of

Boom-less nozzles are often used in industrial applications and have some merit for use in natural area weed control. These nozzles are specifically designed to produce a multi-stream pattern across a 12-15 foot-wide spray swath. When mounted on an ATV or truck, these nozzles can produce a sizable sprayed area, without the issues associated with a fix boom to avoid obstacles and uneven terrain. However, coverage with these types of nozzles is not uniform and generally high volume output is required to maintain proper spray pattern. In addition, the actual nozzle is very expensive compared to a standard fan flan system. Due to difficulties with application uniformity and issues with achieving selectivity, most natural area weed managers will rely heavily on small backpack sprayers. This type of sprayer consists of a 5 gallon/20 liter (on average) tank, a hand-held, single nozzle spray wand, and a small dia‐ phragm pump with an attached lever. The operator uses the pump to pressurize the tank, forcing the liquid spray mixture through the spray wand. Pressurization is under the control of the operator, and is generally maintained to provide a proper pattern from the adjustable orifice on the spray wand. As the name suggests, the apparatus is worn on the back of the applicator using shoulder straps and often a waist strap to stabilize weight distribution. In most cases, the user operates the wand with one hand and pressurizes the tank with the other.

Backpack applications utilize diluted herbicide solution and mixed as a percent solution; in most cases between 0.5 and 3% solution. Applications are made to target species on a visual 'spray to wetness' observation. To achieve some degree of uniformity among applicators, the basis for adequate spray delivery is when spray droplets begin to drip from the leaf surfaces. This 'spray to runoff' technique is common regardless of target species or herbicide. While it is difficult to accurately measure volume output on a per acre basis, most researchers estimate these types of applications to range from 30 to 50 gallons per acre. In many cases, postemergence foliar applications contain herbicides with soil residual activity, either from an herbicide that possesses both foliar and soil activity or soil active herbicides that are tank-mixed to provide extended control. Regardless, the application technique is the same for most boommounted sprayers. For soil applications using a backpack sprayer, the applicator self-calibrates

volume for effectiveness.

222 Herbicides - Current Research and Case Studies in Use

equipment in many natural area systems.

Soil basal applications are used for 2 primary purposes - 1) provide control of an existing plant or group of plants, or 2) provide preventative control of potential plant problems around stationary objects such as power poles. In either scenario, the herbicide is placed in often high concentrations around the base of the treated plant or object. The herbicide may be applied in liquid or granular form, and in a variety of placement patterns to achieve maximum root uptake of the intended target(s). Some herbicides, especially soil active photosynthetic herbicides, are formulated as pellets, which are essentially larger, more concentrated granules. Dry formulated granules or pellets are often easier and more accurate to apply as basal soil treatments. In these situations a certain number of pellets or dry volume of granule is placed as a function of targeted plant circumference. The pattern of placement varies considerably among applicators and may include circular, piles of pellets, or even gridlines in the case of larger infestations [146]. Soil basal herbicides include many of the photosynthetic inhibitors and several of the ALS and growth regulating herbicides. While the growth inhibiting and protox herbicides possess good soil activity, their effectiveness on established and larger plants is limited due to lack of root uptake and translocation or short-term control. Uses are generally restricted to those situations where preventative control is the primary objective.

#### **4.3. Basal-bark applications**

Basal-bark applications are utilized to provide control of larger specimens, where over-thetop foliar applications are not feasible for logistical or selectivity reasons. As the name suggests, basal-bark treatments are made near the ground to the trunks of small trees or shrubs [143]. Treatments are applied using a hand-held spray bottle or backpack sprayer to provide a tight stream of liquid onto the bark (Figure 2). Techniques for basal-bark applications vary widely among practitioners and weed specialists, but most agree that complete coverage around the trunk base is necessary for control. The width of the spray band around the tree varies as a function of species, size and herbicide being used, but most common is a 12 inch (30 cm) width band. Applications are generally made to the point of visual dripping or running of the liquid down the bark surface.

Basal-bark treatments utilize an oil carrier (often referred to as basal oil) in which the herbicide is diluted at a high concentration or undiluted [147]. Diesel fuel or kerosene was used as carriers for many years, but environmental and economic restrictions limit current usages in many areas. In some cases, depending on herbicide formulation, the herbicide may be applied in undiluted form. Regardless of carrier, the herbicide must be in an oil soluble/lipophilic form to allow for penetration into the bark tissues. The objective is to maximize herbicide penetration through the outer epidermal layers (periderm) and reach the secondary phloem and cambium [143]. Once reaching these layers, the herbicide may be remobilized in the phloem, penetrate and affect the dividing cambium cells, or possibly enter the water stream via the xylem

**4.4. Stem injection applications**

application site.

ture, Forest Service, http://www.forestryimages.org

Stem injection applications are generally made to trees or shrubs with larger than 4 inch (20 cm) diameter trunk bases, which is the upper limit for effective basal treatments. In this type of application – also called hack and squirt, the herbicide is placed into a cut or frill made into the bark of the specimen (Figure 3). A hatchet, axe, machete, or other hand-held cutting device is used to make a downward cut/incision that penetrates the bark to the cambium layer, creating a cavity to contain a small amount of herbicide solution [147]. Although highly dependent on herbicide and species, incisions are made evenly around the trunk, or in the case of larger trees a complete girdle might be necessary. One rule of thumb is one incision per inch of trunk diameter [149]; another is no incisions more than 3 inches (10 cm) apart [150]. Herbicide activity on a given species is generally what dictates the number of cuts that is required. Additionally, it is useful to place these cuts near the base of the stem. Making the application higher on the stem will often increase the likelihood of stem-sprouting below the

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**Figure 3.** Hack and squirt application to larger diameter tree. Photo courtesy James Miller, U.S. Department of Agricul‐

Unlike basal bark applications, this type of application can utilize water and oil soluble formulations, providing greater flexibility in herbicide options. In addition to those herbicides mentioned for basal bark, glyphosate, triclopyr amine salt, and hexazinone can be effectively used.Typical concentrations forinjections range from33 to50%solutioninwater.Insome cases, undiluted herbicide is used. Only a small amount of liquid is placed per cut (< 5 ml) and applied using a single nozzle backpack sprayer, or a hand-held spray bottle. A marker dye is often used to help applicators visualize and keep track of treatment applications. There have been several pieces of equipment developed to 'inject' herbicide into woody plant tissues, combining the mechanicalcuttingoperationwithliquiddispensingoperation[151].The'hypo-hatchet'delivers a pre-measured amount of liquid through a pore in the hatchet blade when inserted into the trunk tissue [152]. Injector bars (Figure 4) contain the herbicide mixture within the bar which is

**Figure 2.** Basal bark application to small tree. Photo Courtesy BASF.

sapwood. There is little research as to the actual mechanism of mortality but is surmised that the herbicide is translocated slowly throughout the plant, accumulating in regions of active growth and killing meristematic tissues. The resiliency of many large woody trees and shrubs requires that the herbicide remain available within the plant, and presumably in translocatable form, for a period of time that allows the specimen to exhaust food reserves and/or meristems to provide complete control.

Basal bark herbicides are limited to ester formulations of triclopyr, picloram, 2,4-D, 2,4-DP. Dicamba and oil-soluble formulation of imazapyr have also been used, often in combination with other herbicides [148]. To be effective as a basal treatment, the herbicide must be able to solubilize in oil, which is needed to penetrate the bark layers. The herbicide must also be systemic to allow translocation once reaching the vascular tissues. For these reasons, basal bark treatments are exclusively weak acid herbicides, but only those chemistries that can be formulated to be oil soluble such as esters. Several weak acid herbicides, including the sulfonylureas, are not effective as basal treatments because of low oil solubility.

#### **4.4. Stem injection applications**

sapwood. There is little research as to the actual mechanism of mortality but is surmised that the herbicide is translocated slowly throughout the plant, accumulating in regions of active growth and killing meristematic tissues. The resiliency of many large woody trees and shrubs requires that the herbicide remain available within the plant, and presumably in translocatable form, for a period of time that allows the specimen to exhaust food reserves and/or meristems

Basal bark herbicides are limited to ester formulations of triclopyr, picloram, 2,4-D, 2,4-DP. Dicamba and oil-soluble formulation of imazapyr have also been used, often in combination with other herbicides [148]. To be effective as a basal treatment, the herbicide must be able to solubilize in oil, which is needed to penetrate the bark layers. The herbicide must also be systemic to allow translocation once reaching the vascular tissues. For these reasons, basal bark treatments are exclusively weak acid herbicides, but only those chemistries that can be formulated to be oil soluble such as esters. Several weak acid herbicides, including the

sulfonylureas, are not effective as basal treatments because of low oil solubility.

to provide complete control.

**Figure 2.** Basal bark application to small tree. Photo Courtesy BASF.

224 Herbicides - Current Research and Case Studies in Use

Stem injection applications are generally made to trees or shrubs with larger than 4 inch (20 cm) diameter trunk bases, which is the upper limit for effective basal treatments. In this type of application – also called hack and squirt, the herbicide is placed into a cut or frill made into the bark of the specimen (Figure 3). A hatchet, axe, machete, or other hand-held cutting device is used to make a downward cut/incision that penetrates the bark to the cambium layer, creating a cavity to contain a small amount of herbicide solution [147]. Although highly dependent on herbicide and species, incisions are made evenly around the trunk, or in the case of larger trees a complete girdle might be necessary. One rule of thumb is one incision per inch of trunk diameter [149]; another is no incisions more than 3 inches (10 cm) apart [150]. Herbicide activity on a given species is generally what dictates the number of cuts that is required. Additionally, it is useful to place these cuts near the base of the stem. Making the application higher on the stem will often increase the likelihood of stem-sprouting below the application site.

**Figure 3.** Hack and squirt application to larger diameter tree. Photo courtesy James Miller, U.S. Department of Agricul‐ ture, Forest Service, http://www.forestryimages.org

Unlike basal bark applications, this type of application can utilize water and oil soluble formulations, providing greater flexibility in herbicide options. In addition to those herbicides mentioned for basal bark, glyphosate, triclopyr amine salt, and hexazinone can be effectively used.Typical concentrations forinjections range from33 to50%solutioninwater.Insome cases, undiluted herbicide is used. Only a small amount of liquid is placed per cut (< 5 ml) and applied using a single nozzle backpack sprayer, or a hand-held spray bottle. A marker dye is often used to help applicators visualize and keep track of treatment applications. There have been several pieces of equipment developed to 'inject' herbicide into woody plant tissues, combining the mechanicalcuttingoperationwithliquiddispensingoperation[151].The'hypo-hatchet'delivers a pre-measured amount of liquid through a pore in the hatchet blade when inserted into the trunk tissue [152]. Injector bars (Figure 4) contain the herbicide mixture within the bar which is

**Figure 4.** Stem injection of herbicide into trunk of target tree. Phot credit James Miller, U.S. Department of Agricul‐ ture, Forest Service, http://www.forestryimages.org

off the stump or root crown. Regardless, applicators are encouraged to use only the amount

**Figure 5.** Herbicide application with marker dye made to cut stump, targeting only outer cambium region. Photo

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This unique approach to applying herbicides has been developed by Dr. James Leary with the University of Hawaii [153]. In this system, herbicides are encapsulated in paint ball pellets and distributed to the target species via a commercially available paint ball gun. Each 'ball' contains a known amount of herbicide and rate is calculated by the number of balls fired at each specimen. The applicator targets the apical regions of the plant, or the larger stems to increase

Dr. Leary has performed nearly all initial testing with imazapyr and triclopyr, which readily translocates within plant tissues. Imazapyr also possesses good soil residual activity, which aids in effectiveness. This technology is still in the evaluation phase, but holds good promise for treating invasive species in remote and inaccessible areas. Most of the treatment evaluations have been performed on the slopes of tropical mountains in Hawaii, where the only means of treatment has previously been a single nozzle suspended from a helicopter. The nozzle is embedded in a heavy ball that helps reduce swaying and the pilot attempts to direct the nozzle over the crown of the targeted specimen. This approach is time consuming, precarious and expensive. With the ballistic approach, the applicator fires a number of balls into the crown to deliver the herbicide. This allows for more specimens to be treated per helicopter flying time, eliminates the need for unwieldy spray equipment and provides for more precise herbicide

the 'splatter' effect that helps distribute the herbicide within the plant architecture.

necessary to provide control of resprouting, and limit excessive herbicide use.

credit James Miller, U.S. Department of Agriculture, Forest Service, http://www.forestryimages.org

**4.6. Ballistic herbicide application**

jabbedintoatree,andaleverispulledallowingapre-measuredamountofliquidtoflowthrough the end of the bar [147]. Some bar type devices will insert a granular pellet during each injec‐ tion. Other injection tools include a hand-held gun, with a large diameter needle that can be inserted into softer perennial tissues, once again with a premeasured amount that is injected.

#### **4.5. Cut-stump applications**

Cut-stump applications occur, as the name implies, to the cut portion of a felled tree or shrub. The purpose of the application is to prevent regrowth of the plant from shoots arising from the cambium layer of the cut stump. Herbicide is applied to the cut surface, making sure to cover the entire outer cambium layer [86,147]. Placement of the herbicide across the entire stump is not necessary, since the majority of the inner tissues consist of non-living heartwood (Figure 5). Applications should occur within 30 minutes of cutting to avoid the layer becoming scabbed over, reducing herbicide uptake and penetration.

Triclopyr amine or ester, picloram, 2,4-D, dicamba, imazapyr or glyphosate can be used for cut stump applications. Ester formulations can be applied as 25% solution in basal oil, while amine/ salt formulations are applied as 50% solution in water. Sometimes undiluted herbicide can be used,butcaremustbetakentoavoid'flashback'.Flashbackisaphenomenonwheretheherbicide is absorbed by the trunk and roots of the felled specimen, translocated through the root system, and passed through root grafting to the roots of neighboring plants [149]. Neighboring plant rootscanalsoabsorbtheherbicidefromsoilaroundthetreatedstump,whereherbicideiswashed

**Figure 5.** Herbicide application with marker dye made to cut stump, targeting only outer cambium region. Photo credit James Miller, U.S. Department of Agriculture, Forest Service, http://www.forestryimages.org

off the stump or root crown. Regardless, applicators are encouraged to use only the amount necessary to provide control of resprouting, and limit excessive herbicide use.

#### **4.6. Ballistic herbicide application**

jabbedintoatree,andaleverispulledallowingapre-measuredamountofliquidtoflowthrough the end of the bar [147]. Some bar type devices will insert a granular pellet during each injec‐ tion. Other injection tools include a hand-held gun, with a large diameter needle that can be inserted into softer perennial tissues, once again with a premeasured amount that is injected.

**Figure 4.** Stem injection of herbicide into trunk of target tree. Phot credit James Miller, U.S. Department of Agricul‐

Cut-stump applications occur, as the name implies, to the cut portion of a felled tree or shrub. The purpose of the application is to prevent regrowth of the plant from shoots arising from the cambium layer of the cut stump. Herbicide is applied to the cut surface, making sure to cover the entire outer cambium layer [86,147]. Placement of the herbicide across the entire stump is not necessary, since the majority of the inner tissues consist of non-living heartwood (Figure 5). Applications should occur within 30 minutes of cutting to avoid the layer becoming

Triclopyr amine or ester, picloram, 2,4-D, dicamba, imazapyr or glyphosate can be used for cut stump applications. Ester formulations can be applied as 25% solution in basal oil, while amine/ salt formulations are applied as 50% solution in water. Sometimes undiluted herbicide can be used,butcaremustbetakentoavoid'flashback'.Flashbackisaphenomenonwheretheherbicide is absorbed by the trunk and roots of the felled specimen, translocated through the root system, and passed through root grafting to the roots of neighboring plants [149]. Neighboring plant rootscanalsoabsorbtheherbicidefromsoilaroundthetreatedstump,whereherbicideiswashed

**4.5. Cut-stump applications**

ture, Forest Service, http://www.forestryimages.org

226 Herbicides - Current Research and Case Studies in Use

scabbed over, reducing herbicide uptake and penetration.

This unique approach to applying herbicides has been developed by Dr. James Leary with the University of Hawaii [153]. In this system, herbicides are encapsulated in paint ball pellets and distributed to the target species via a commercially available paint ball gun. Each 'ball' contains a known amount of herbicide and rate is calculated by the number of balls fired at each specimen. The applicator targets the apical regions of the plant, or the larger stems to increase the 'splatter' effect that helps distribute the herbicide within the plant architecture.

Dr. Leary has performed nearly all initial testing with imazapyr and triclopyr, which readily translocates within plant tissues. Imazapyr also possesses good soil residual activity, which aids in effectiveness. This technology is still in the evaluation phase, but holds good promise for treating invasive species in remote and inaccessible areas. Most of the treatment evaluations have been performed on the slopes of tropical mountains in Hawaii, where the only means of treatment has previously been a single nozzle suspended from a helicopter. The nozzle is embedded in a heavy ball that helps reduce swaying and the pilot attempts to direct the nozzle over the crown of the targeted specimen. This approach is time consuming, precarious and expensive. With the ballistic approach, the applicator fires a number of balls into the crown to deliver the herbicide. This allows for more specimens to be treated per helicopter flying time, eliminates the need for unwieldy spray equipment and provides for more precise herbicide application [154]. As mentioned, this technology is still under intense evaluation, and com‐ mercialization of the process has not been undertaken.

the Center for Aquatic and Invasive Plants at the University of Florida for support in publi‐

, Jason A. Ferrell1

1 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐

2 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐

3 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Range Cattle Research and Education Center, 3401 Experiment Station Rd., Ona, FL,

[1] Shrader-Frechette, K.S. and E.D. McCoy. 1995. Natural landscapes, natural communi‐

[2] Maser, C. 1990. On the "naturalness" of natural areas: A perspective for the future. Nat‐

[3] Daily, G.C., S. Alexander, P.R. Ehrlich, L. Goulder, J. Lubchenco, P.A. Matson, H.A. Mooney, S. Postel, S.H. Schneider, D. Tilman, and G.M. Woodwell. 1997. Ecosystem services: benefits supplied to human societies by natural ecosystems. Issues in Ecology

[4] Walls, M. 2009. Parks and recreation in the United States: The National Park System.

[5] U.S. National Vegetation Classification. 2012. http://usnvc.org/explore-classification/

[6] Anonymous. 2006. Management Policies 2006. National Park Service. http://

[7] Anonymous. 2012. Conserving the Future: Wildlife Refuges and the Next Generation.

Resources for the Future (RFF) Backgrounder, January 2009, 14p.

www.nps.gov/policy/mp2006.pdf. 180p. (accessed 9 February 2013).

ties, and natural ecosystems. Forest and Conservation History 39:138-142.

my, Fort Lauderdale Research and Education Center, Davie, FL, USA

and Brent A. Sellers3

Herbicides for Natural Area Weed Management

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229

cation of this document.

my, Gainesville, FL, USA

USA

**References**

Gregory E. MacDonald1\*, Lyn A. Gettys2

\*Address all correspondence to: pineacre@ufl.edu

ural Areas Journal 10:129-133.

Number 2, Spring 1997 18p.

(accessed 9 February 2013).

U.S. Fish and Wildlife Service. 49 p.

**Author details**

### **5. Integrated approaches to natural area weed management**

Regardless of herbicide or application method used, chemical weed control must be used in an integrated approach for controlling weeds. Other methods of weed control such as prevention, biological, cultural and mechanical techniques are often utilized to complement chemical control programs. For example, mechanical felling of large trees, followed by chemical treatment of regrowth, is a common operational strategy for forestry. Chemical control to provide initial kill of vegetation, followed by the introduction of a biocontrol agent is very effective for management of *Melaleuca quinquenervia* in south Florida. A critical aspect of management in many systems is the use of fire to reduce ground litter, promote seed germination and flowering, and provide control of undesirable species. Fire can also be used to reduce biomass and promote regrowth, which often results in more efficacious herbicide treatments. Conversely, intense fires by excessive fuel generated from invasive species can cause severe damage, especially to desirable over-story trees.

Restoration is another very important component of natural area management. This aspect involves: 1) promoting the existing desirable vegetation through regrowth or regeneration from a seed bank, or 2) intentional planting of desirable species through physical transplanting or sowing of seed. Previous control methods can have a profound effect on restoration. Mechanical tillage can disrupt the seedbank through exposure seed on the surface or bury beyond the point of emergence. Residual activity from herbicides used to control invasive plants can also be deleterious to recolonizing desirable species. Studies to determine herbicide longevity and sensitivity of species is important when developing both control strategies and subsequent restoration plans as components of an overall management plan.

### **6. Conclusions**

Herbicides are a critical component to managing undesirable species in natural areas. How‐ ever, several considerations must be addressed for effective and environmentally safe usage. Proper herbicide selection, timing of application, type of application methodology and application rate must be adhered according to the product label. Actual site of usage must also fall within product label guidelines. Herbicides should never be used as a stand-alone approach but rather as a component of an integrated long-term management strategy for invasive species control and natural area restoration.

### **Acknowledgements**

This publication is a contribution of the University of Florida Institute for Food and Agricul‐ tural Sciences and the Florida Agricultural Experiment Station. The authors also wish to thank the Center for Aquatic and Invasive Plants at the University of Florida for support in publi‐ cation of this document.

### **Author details**

application [154]. As mentioned, this technology is still under intense evaluation, and com‐

Regardless of herbicide or application method used, chemical weed control must be used in an integrated approach for controlling weeds. Other methods of weed control such as prevention, biological, cultural and mechanical techniques are often utilized to complement chemical control programs. For example, mechanical felling of large trees, followed by chemical treatment of regrowth, is a common operational strategy for forestry. Chemical control to provide initial kill of vegetation, followed by the introduction of a biocontrol agent is very effective for management of *Melaleuca quinquenervia* in south Florida. A critical aspect of management in many systems is the use of fire to reduce ground litter, promote seed germination and flowering, and provide control of undesirable species. Fire can also be used to reduce biomass and promote regrowth, which often results in more efficacious herbicide treatments. Conversely, intense fires by excessive fuel generated from invasive species can

Restoration is another very important component of natural area management. This aspect involves: 1) promoting the existing desirable vegetation through regrowth or regeneration from a seed bank, or 2) intentional planting of desirable species through physical transplanting or sowing of seed. Previous control methods can have a profound effect on restoration. Mechanical tillage can disrupt the seedbank through exposure seed on the surface or bury beyond the point of emergence. Residual activity from herbicides used to control invasive plants can also be deleterious to recolonizing desirable species. Studies to determine herbicide longevity and sensitivity of species is important when developing both control strategies and

Herbicides are a critical component to managing undesirable species in natural areas. How‐ ever, several considerations must be addressed for effective and environmentally safe usage. Proper herbicide selection, timing of application, type of application methodology and application rate must be adhered according to the product label. Actual site of usage must also fall within product label guidelines. Herbicides should never be used as a stand-alone approach but rather as a component of an integrated long-term management strategy for

This publication is a contribution of the University of Florida Institute for Food and Agricul‐ tural Sciences and the Florida Agricultural Experiment Station. The authors also wish to thank

subsequent restoration plans as components of an overall management plan.

mercialization of the process has not been undertaken.

228 Herbicides - Current Research and Case Studies in Use

cause severe damage, especially to desirable over-story trees.

invasive species control and natural area restoration.

**6. Conclusions**

**Acknowledgements**

**5. Integrated approaches to natural area weed management**

Gregory E. MacDonald1\*, Lyn A. Gettys2 , Jason A. Ferrell1 and Brent A. Sellers3

\*Address all correspondence to: pineacre@ufl.edu

1 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Gainesville, FL, USA

2 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Fort Lauderdale Research and Education Center, Davie, FL, USA

3 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Range Cattle Research and Education Center, 3401 Experiment Station Rd., Ona, FL, USA

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[143] Ferrell, J.A., K.A. Langeland, and B.A. Sellers. 2012. Herbicide Application Techniques for Woody Plant Control. Agronomy Department, Florida Cooperative Extension Serv‐ ice, Institute of Food and Agricultural Sciences, University of Florida. SS-AGR-260.

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**Chapter 10**

**Integrated Weed Management Practices for**

The earth is undergoing a number of irreversible changes as a result of the activities of man, many of which are adversely affecting the environment. Inappropriate methods of agricultural production, especially those stimulated by efforts in pursuit of short-term gains, have been

In earlier times, traditional farming in the tropics involved the use of natural resources in adequate quantity for the sustenance of its population, without diminishing the natural resource base. Key elements of that system included multiple cropping and mixed farming, minimum tillage and water conservation techniques, the use of simple hand tools and other

These sustainable farming methods have been described in pejorative terms as drudgery, laborious, and inefficient. Many have been rejected and new technologies and other high energy based inputs have been embraced. These technologies are costly and heavily foreignexchange dependent. They also disturb the delicate ecological balance resulting in increased occurrence of pests and diseases, shift in noxious weed populations, soil erosion and pollution

The situation in the tropical world is exacerbated as many tropical countries are characterised by conditions that are ideal for the prolific growth and development of a range of plant species. Many of these species are generally non-harmful. However, when inappropriate methods of weed control and/or poor crop management strategies are employed, weeds assume noxious potentials. Ready examples are corn grass (*Rottboellia cochinchinensis*), white-top (*Parthenium*

> © 2013 Isaac et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Isaac et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Adoption in the Tropics**

Wayne G. Ganpat

**1. Introduction**

low input technologies.

of the air and water resources.

*hysterophorus*) and nutgrass (*Cyperus rotundus*) [33].

http://dx.doi.org/10.5772/55950

Wendy-Ann P. Isaac, Puran Bridgemohan and

Additional information is available at the end of the chapter

identified as prime contributors to this environmental degradation.

## **Integrated Weed Management Practices for Adoption in the Tropics**

Wendy-Ann P. Isaac, Puran Bridgemohan and Wayne G. Ganpat

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55950

### **1. Introduction**

The earth is undergoing a number of irreversible changes as a result of the activities of man, many of which are adversely affecting the environment. Inappropriate methods of agricultural production, especially those stimulated by efforts in pursuit of short-term gains, have been identified as prime contributors to this environmental degradation.

In earlier times, traditional farming in the tropics involved the use of natural resources in adequate quantity for the sustenance of its population, without diminishing the natural resource base. Key elements of that system included multiple cropping and mixed farming, minimum tillage and water conservation techniques, the use of simple hand tools and other low input technologies.

These sustainable farming methods have been described in pejorative terms as drudgery, laborious, and inefficient. Many have been rejected and new technologies and other high energy based inputs have been embraced. These technologies are costly and heavily foreignexchange dependent. They also disturb the delicate ecological balance resulting in increased occurrence of pests and diseases, shift in noxious weed populations, soil erosion and pollution of the air and water resources.

The situation in the tropical world is exacerbated as many tropical countries are characterised by conditions that are ideal for the prolific growth and development of a range of plant species. Many of these species are generally non-harmful. However, when inappropriate methods of weed control and/or poor crop management strategies are employed, weeds assume noxious potentials. Ready examples are corn grass (*Rottboellia cochinchinensis*), white-top (*Parthenium hysterophorus*) and nutgrass (*Cyperus rotundus*) [33].

© 2013 Isaac et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Isaac et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Present farming involves substantial reliance on a range of manufactured inputs. The high dependence on herbicides for weed control in the cultivation of rice, maize, bananas, citrus, sugarcane, onions, white potatoes and vegetable crops is not unnoticed. The competition among suppliers of herbicides has resulted in lower costs of these products which has fuelled their use and abuse in the region.

**•** destroying vegetative propagules of perennial weeds;

**•** whenever possible, depleting the soil weed seed bank;

**•** preventing weeds from going to seed in crop fields;

*1.1.2. Competitive crops and/or smother crops*

intercrop, cover crop and green manure selection [41].

rapid canopy closure, thus suppressing emerging weeds.

*rotundus*) and small broadleaved weeds.

**•** cleaning farm machinery before movement into fields;

**•** propagating seeds and seedling transplants in media free of weed propagules;

**•** minimizing the presence of weed seed in livestock feed, manures and composts;

ition of machinery, seed, water and fertilizers, to crop harvest and processing.

For preventive strategies to be fully adopted in an IWM approach, there must be an attitudinal change by farmers and agricultural educators in the tropics. Prevention, although complex, is a very efficient technique for any property size at all crop production stages, from the acquis‐

Integrated Weed Management Practices for Adoption in the Tropics

http://dx.doi.org/10.5772/55950

243

Crops differ in their competitiveness with weeds based on their emergence, leaf-area expan‐ sion, light interception, canopy architecture, leaf-angle, shape and competitiveness. Within a crop species, cultivars may vary in their competitiveness. While the improved varieties may be high yielding, the traditional varieties exhibit multiple adaptations, competitive ability against weeds and require less agricultural input. The use of competitive crops to discourage weeds is an important IWM strategy. To maximise crop production by minimising the impact of weeds, replacement series and addition series designs have been recommended for

Plant height and leaf area index correlate with competitive ability in row crops. These characters allow the crop to outgrow and cover the weeds. Indeterminate varieties of bean, cowpea, squash and cucumber appear to be better competitive than determinate varieties [38, 39]. The indeterminate varieties of these crops have a vining or spreading habit which allows

Some plants are able to exude chemical substances which suppress the growth of other neighbouring plants. Research in plants with allelopathic potential is ongoing and has revealed

Smother crops are quickly established and usurp the resources that weeds would otherwise use. The suppression of weeds may be through both competition (resources) and allelopathy [11]. Smother crops include cowpea (*Vigna unguiculata)*, forage soya beans (*Glycine max* L.Merril), Sudan grass (*Sorghum bicolor* subsp. *drummondii*), kudzu (*Pueraria phaseoloides*) and pumpkins (*Cucurbita maxima*), which are very effective in suppressing nutgrass (*Cyperus*

a clearer understanding into the genetics of allelopathic activity in certain crops [29].

**•** preventing weed seed introduction into rivers and irrigation canals.

Low-input, sustainable agriculture addresses multiple objectives from increasing profits to maintaining the environment, and builds on multiple systems as integrated pest management (IPM), integrated weed management (IWM), and crop rotation. Integrated weed management involves the combination of a number of weed control practices that reduces the dependence on any one type of control method and also lowers the input of herbicides. This approach is important for the control of perennial weeds that are inadequately controlled by any single method [8]. The application of IWM also includes the knowledge of past annual and perennial weed populations in fields and weed seed bank [7], competitive crop cultivars, improved crop and soil management practices, and appropriate selection of herbicides [52]. In the context of sustainable agriculture, the concept of IWM seems enlightening and applicable.

The objective of this paper is to discuss the various weed management practices for the control of noxious weeds in major cereal, root and vegetable crops in tropical sustainable agriculture and the strategies used over time to promote their adoption by small farmers.

#### **1.1. Common Integrated Weed Management (IWM) strategies in the tropics**

Integrated weed management systems are based on an agro-ecosystem approach for the management and control of weeds at economic threshold levels [12]. Many farmers in the tropics today practise the same weed control measures as was practised before the introduction of herbicides [35]. The IWM systems approach includes any or a combination of the following practices that give a crop a comparative advantage in competing with weeds.

#### *1.1.1. Prevention strategies*

Prevention strategies include field sanitation and harvesting methods that do not spread weed seeds and vegetative propagules at every step of production (such as seed selection, field preparation, planting, fertilization, irrigation, weed control, harvest and transport) [19]. Such strategies can significantly reduce the infestation of noxious weeds such as nutgrass and whitetop [7]. The use of clean crop seed, especially those direct seeded, e.g., maize and legumes, is critical in the prevention of weed problems in new and existing fields. Prevention should be a daily activity, incorporated into the routine of all workers involved in agricultural produc‐ tion, at farm, state and national levels [19]. It is recommended that managers make simple, cost effective modifications to their farm practices to mitigate the risk of introducing new weed seeds to the field. Some of the key considerations as outlined in [19] include:


**•** destroying vegetative propagules of perennial weeds;

Present farming involves substantial reliance on a range of manufactured inputs. The high dependence on herbicides for weed control in the cultivation of rice, maize, bananas, citrus, sugarcane, onions, white potatoes and vegetable crops is not unnoticed. The competition among suppliers of herbicides has resulted in lower costs of these products which has fuelled

Low-input, sustainable agriculture addresses multiple objectives from increasing profits to maintaining the environment, and builds on multiple systems as integrated pest management (IPM), integrated weed management (IWM), and crop rotation. Integrated weed management involves the combination of a number of weed control practices that reduces the dependence on any one type of control method and also lowers the input of herbicides. This approach is important for the control of perennial weeds that are inadequately controlled by any single method [8]. The application of IWM also includes the knowledge of past annual and perennial weed populations in fields and weed seed bank [7], competitive crop cultivars, improved crop and soil management practices, and appropriate selection of herbicides [52]. In the context of

The objective of this paper is to discuss the various weed management practices for the control of noxious weeds in major cereal, root and vegetable crops in tropical sustainable agriculture

Integrated weed management systems are based on an agro-ecosystem approach for the management and control of weeds at economic threshold levels [12]. Many farmers in the tropics today practise the same weed control measures as was practised before the introduction of herbicides [35]. The IWM systems approach includes any or a combination of the following

Prevention strategies include field sanitation and harvesting methods that do not spread weed seeds and vegetative propagules at every step of production (such as seed selection, field preparation, planting, fertilization, irrigation, weed control, harvest and transport) [19]. Such strategies can significantly reduce the infestation of noxious weeds such as nutgrass and whitetop [7]. The use of clean crop seed, especially those direct seeded, e.g., maize and legumes, is critical in the prevention of weed problems in new and existing fields. Prevention should be a daily activity, incorporated into the routine of all workers involved in agricultural produc‐ tion, at farm, state and national levels [19]. It is recommended that managers make simple, cost effective modifications to their farm practices to mitigate the risk of introducing new weed

sustainable agriculture, the concept of IWM seems enlightening and applicable.

and the strategies used over time to promote their adoption by small farmers.

**1.1. Common Integrated Weed Management (IWM) strategies in the tropics**

practices that give a crop a comparative advantage in competing with weeds.

seeds to the field. Some of the key considerations as outlined in [19] include:

plant materials or soil from one location to another;

**•** diligent monitoring for sources of new weed introductions to the agro-ecosystem;

**•** proactive government laws and regulations controlling the introduction and movement of

their use and abuse in the region.

242 Herbicides - Current Research and Case Studies in Use

*1.1.1. Prevention strategies*


For preventive strategies to be fully adopted in an IWM approach, there must be an attitudinal change by farmers and agricultural educators in the tropics. Prevention, although complex, is a very efficient technique for any property size at all crop production stages, from the acquis‐ ition of machinery, seed, water and fertilizers, to crop harvest and processing.

#### *1.1.2. Competitive crops and/or smother crops*

Crops differ in their competitiveness with weeds based on their emergence, leaf-area expan‐ sion, light interception, canopy architecture, leaf-angle, shape and competitiveness. Within a crop species, cultivars may vary in their competitiveness. While the improved varieties may be high yielding, the traditional varieties exhibit multiple adaptations, competitive ability against weeds and require less agricultural input. The use of competitive crops to discourage weeds is an important IWM strategy. To maximise crop production by minimising the impact of weeds, replacement series and addition series designs have been recommended for intercrop, cover crop and green manure selection [41].

Plant height and leaf area index correlate with competitive ability in row crops. These characters allow the crop to outgrow and cover the weeds. Indeterminate varieties of bean, cowpea, squash and cucumber appear to be better competitive than determinate varieties [38, 39]. The indeterminate varieties of these crops have a vining or spreading habit which allows rapid canopy closure, thus suppressing emerging weeds.

Some plants are able to exude chemical substances which suppress the growth of other neighbouring plants. Research in plants with allelopathic potential is ongoing and has revealed a clearer understanding into the genetics of allelopathic activity in certain crops [29].

Smother crops are quickly established and usurp the resources that weeds would otherwise use. The suppression of weeds may be through both competition (resources) and allelopathy [11]. Smother crops include cowpea (*Vigna unguiculata)*, forage soya beans (*Glycine max* L.Merril), Sudan grass (*Sorghum bicolor* subsp. *drummondii*), kudzu (*Pueraria phaseoloides*) and pumpkins (*Cucurbita maxima*), which are very effective in suppressing nutgrass (*Cyperus rotundus*) and small broadleaved weeds.

#### *1.1.3. Optimum plant population*

Row spacing and seeding rate may influence the ability of the crop to compete with weeds for resources and, therefore, may affect weed management [24, 45]. The rapid closure of the crop canopy can be obtained with a reduction in row spacing [1], an increase in seeding rate [42], and selection of varieties with traits that favour rapid canopy development [13]. It has been reported that rows of 38 cm or less could increase yields and reduce tillage and herbicide requirements because of faster canopy closure [1]. Cereal and vegetable crops can compete with weed growth if they are established at the optimum plant population that allows them to more effectively usurp resources. If crops can reduce incident light by 50 % or more, weeds will seldom become a problem [6, 15]. This approach requires closer intra-and interrow spacings and higher crop densities than normally used.

because they improve soil organic matter and nutrient status, prevent erosion and suppress weeds [30]. The use of legume covers is, however, expensive because of the cost of seeds and labour for their establishment [53, 56]. It is important to use legume and other crop covers which will not compete with the crop for resources. Moreover, any crop cover used must

Integrated Weed Management Practices for Adoption in the Tropics

http://dx.doi.org/10.5772/55950

245

Some of the weed species that are easily smothered by live legume covers include: *Ageratum conyzoides* L., *Alternenthera sessilis* L. R. Br. ex Roth, *Mimosa invisa* Mart, *Digitaria orizontalis* Willd, and *Panicum maximum* Jacq. However, some sedges and grasses like *Cyperus rotundus* L (purple nutsedge), *Rottboellia cochinchinensis* (Lour) Clayton (Raoul grass), *Sorghum hale‐ pense* L. Pers (Johnson grass) and *Ipomoea* sp. (morning glory) are noxious weeds and difficult

Both cover crops and mulches offer great agro-ecological potential. They serve as a physical barrier against weed emergence, both conserve the soil and improve the ecological balance of the soil, enhance crop yield and provide several environmental services. These new technol‐ ogies, however, are not easily accepted by small farmers in the tropics. Notwithstanding, they offer a complex combination of interrelated practices which include: (i) necessary practices so as to ensure the production and retention of sufficient mulch and (ii) complementary practices in order to be able to grow a crop and/or maintain yield levels. This typically implies several adaptations to the entire farm production system. Whether mulching actually is a viable component for smallholder conservation farming in developing countries depends on a number of factors, including bio-physical, technological, farm level and institutional factors. The combination of these factors determines the feasibility of and the economic returns to

The development and dissemination of cover crops and mulches for small farmers in tropical developing countries highlights a number of promising experiences, particularly among banana growers i in St. Vincent, in the Caribbean [25]. The technology offers significant savings through reduced tillage and alleviation of some major crop production constraints such as

The basic principles of IWM which include: suppression of weed growth, prevention or suppression of weed seed production, reduction in weed seed bank and prevention or reduction in weed spread, are key elements of all improved husbandry practices. All crop husbandry practices, particularly precision placement and timing of fertiliser application, enhance maximum stimulation of the crop and minimum stimulation of the weed population. Additionally, the use of clean certified seeds, clean farm implements, effective seedbed preparation and seeding methods that improve crop growth, all reduce weed competition [7, 36]. Other management practices including: cultural weed control (intercropping, early planting, optimum plant crop density, and tillage), chemical (minimum herbicide) weed control, mechanical weed control and hoe weeding, have been shown to reduce the competitive

effects of weeds on vegetable and cereal crops growth, development and yield [36].

water conservation, timeliness of land preparation and crop establishment.

directly benefit the farmer if adoption of the practice is to be sustained.

to control in root and cereal crops [20, 46].

mulching practices—and thereby farmer acceptance.

*1.1.5. Improved husbandry*

#### *1.1.4. Cover crops and mulches*

Cover crops have long been used extensively in the tropics for soil and water conservation, to maintain soil structure and enhance soil fertility, especially on steep or difficult terrain. They are often referred to as living mulches. The use of leguminous cover plants to suppress weeds in plantation crops in the tropical world dates back many decades, but the integration of the legumes into arable cropping systems has not been developed to a level acceptable to farmers. Cover crops also contribute to pest management and help to suppress unwanted weeds. Its use has been mainly in plantation crops. The introduction of inexpensive nitrogen fertilizers and herbicides encouraged many farmers to discontinue this practice. Cover crops can be intercropped or interplanted with a crop of economic significance. They work by excluding light and limiting weed emergence. Examples of cover crops in the tropics include: *Mucuna pruriens* (L.) DC. (velvet beans), *Desmodium heterocarpon* var *ovalifolium* and *Arachis pintoi* Crap. & Greg. (wild or perennial peanut).

Mulches, on the other hand, may be in the natural form of plant or crop residues or in synthetic form as plastic films or woven synthetic fibres. Other non-living mulches can be either natural materials (plant leaves, stalks, straw, compost and dry soil) or synthetic materials, such as polyethylene, which are used widely in pineapple production. The major disadvantages of plastic films are material costs and difficulty in removal after cropping season. Organic mulches or living mulches are considered cover crops, e.g., mungbean (*Vigna radiata* (L.) Wilczek cv. Local) and have been shown to be an economical alternative to synthetic mulches [36]. Watermelon and tomato farmers in Dominica, West Indies use Guinea grass (*Panicum fasiculatum*) as a mulch and cover crop. The grass is killed using a weed killer such as paraquat, and when it re-grows, it is brush-cut before crop emergence or otherwise left as a residue. The crop is planted directly into the cover crop residue which enhances soil and water conservation and protection from wind.

In root crops, for example cassava, live green legumes e.g. *Desmodium heterophyllum* (Willd) DC [20] with bean *(Phaseolus* sp.) have been used successfully. Both legumes gave better weed control and crop yields than the herbicide and mulch treatments and *Desmodium heterocar‐ pon* var *ovalifolium* in banana [25]. *Stylosanthes guianensis* (Aubl.) SW, too, has been used as a cover crop to suppress weeds in cassava [43, 44]. Legume and dry mulch covers are beneficial because they improve soil organic matter and nutrient status, prevent erosion and suppress weeds [30]. The use of legume covers is, however, expensive because of the cost of seeds and labour for their establishment [53, 56]. It is important to use legume and other crop covers which will not compete with the crop for resources. Moreover, any crop cover used must directly benefit the farmer if adoption of the practice is to be sustained.

Some of the weed species that are easily smothered by live legume covers include: *Ageratum conyzoides* L., *Alternenthera sessilis* L. R. Br. ex Roth, *Mimosa invisa* Mart, *Digitaria orizontalis* Willd, and *Panicum maximum* Jacq. However, some sedges and grasses like *Cyperus rotundus* L (purple nutsedge), *Rottboellia cochinchinensis* (Lour) Clayton (Raoul grass), *Sorghum hale‐ pense* L. Pers (Johnson grass) and *Ipomoea* sp. (morning glory) are noxious weeds and difficult to control in root and cereal crops [20, 46].

Both cover crops and mulches offer great agro-ecological potential. They serve as a physical barrier against weed emergence, both conserve the soil and improve the ecological balance of the soil, enhance crop yield and provide several environmental services. These new technol‐ ogies, however, are not easily accepted by small farmers in the tropics. Notwithstanding, they offer a complex combination of interrelated practices which include: (i) necessary practices so as to ensure the production and retention of sufficient mulch and (ii) complementary practices in order to be able to grow a crop and/or maintain yield levels. This typically implies several adaptations to the entire farm production system. Whether mulching actually is a viable component for smallholder conservation farming in developing countries depends on a number of factors, including bio-physical, technological, farm level and institutional factors. The combination of these factors determines the feasibility of and the economic returns to mulching practices—and thereby farmer acceptance.

The development and dissemination of cover crops and mulches for small farmers in tropical developing countries highlights a number of promising experiences, particularly among banana growers i in St. Vincent, in the Caribbean [25]. The technology offers significant savings through reduced tillage and alleviation of some major crop production constraints such as water conservation, timeliness of land preparation and crop establishment.

#### *1.1.5. Improved husbandry*

*1.1.3. Optimum plant population*

244 Herbicides - Current Research and Case Studies in Use

*1.1.4. Cover crops and mulches*

& Greg. (wild or perennial peanut).

and protection from wind.

spacings and higher crop densities than normally used.

Row spacing and seeding rate may influence the ability of the crop to compete with weeds for resources and, therefore, may affect weed management [24, 45]. The rapid closure of the crop canopy can be obtained with a reduction in row spacing [1], an increase in seeding rate [42], and selection of varieties with traits that favour rapid canopy development [13]. It has been reported that rows of 38 cm or less could increase yields and reduce tillage and herbicide requirements because of faster canopy closure [1]. Cereal and vegetable crops can compete with weed growth if they are established at the optimum plant population that allows them to more effectively usurp resources. If crops can reduce incident light by 50 % or more, weeds will seldom become a problem [6, 15]. This approach requires closer intra-and interrow

Cover crops have long been used extensively in the tropics for soil and water conservation, to maintain soil structure and enhance soil fertility, especially on steep or difficult terrain. They are often referred to as living mulches. The use of leguminous cover plants to suppress weeds in plantation crops in the tropical world dates back many decades, but the integration of the legumes into arable cropping systems has not been developed to a level acceptable to farmers. Cover crops also contribute to pest management and help to suppress unwanted weeds. Its use has been mainly in plantation crops. The introduction of inexpensive nitrogen fertilizers and herbicides encouraged many farmers to discontinue this practice. Cover crops can be intercropped or interplanted with a crop of economic significance. They work by excluding light and limiting weed emergence. Examples of cover crops in the tropics include: *Mucuna pruriens* (L.) DC. (velvet beans), *Desmodium heterocarpon* var *ovalifolium* and *Arachis pintoi* Crap.

Mulches, on the other hand, may be in the natural form of plant or crop residues or in synthetic form as plastic films or woven synthetic fibres. Other non-living mulches can be either natural materials (plant leaves, stalks, straw, compost and dry soil) or synthetic materials, such as polyethylene, which are used widely in pineapple production. The major disadvantages of plastic films are material costs and difficulty in removal after cropping season. Organic mulches or living mulches are considered cover crops, e.g., mungbean (*Vigna radiata* (L.) Wilczek cv. Local) and have been shown to be an economical alternative to synthetic mulches [36]. Watermelon and tomato farmers in Dominica, West Indies use Guinea grass (*Panicum fasiculatum*) as a mulch and cover crop. The grass is killed using a weed killer such as paraquat, and when it re-grows, it is brush-cut before crop emergence or otherwise left as a residue. The crop is planted directly into the cover crop residue which enhances soil and water conservation

In root crops, for example cassava, live green legumes e.g. *Desmodium heterophyllum* (Willd) DC [20] with bean *(Phaseolus* sp.) have been used successfully. Both legumes gave better weed control and crop yields than the herbicide and mulch treatments and *Desmodium heterocar‐ pon* var *ovalifolium* in banana [25]. *Stylosanthes guianensis* (Aubl.) SW, too, has been used as a cover crop to suppress weeds in cassava [43, 44]. Legume and dry mulch covers are beneficial

The basic principles of IWM which include: suppression of weed growth, prevention or suppression of weed seed production, reduction in weed seed bank and prevention or reduction in weed spread, are key elements of all improved husbandry practices. All crop husbandry practices, particularly precision placement and timing of fertiliser application, enhance maximum stimulation of the crop and minimum stimulation of the weed population. Additionally, the use of clean certified seeds, clean farm implements, effective seedbed preparation and seeding methods that improve crop growth, all reduce weed competition [7, 36]. Other management practices including: cultural weed control (intercropping, early planting, optimum plant crop density, and tillage), chemical (minimum herbicide) weed control, mechanical weed control and hoe weeding, have been shown to reduce the competitive effects of weeds on vegetable and cereal crops growth, development and yield [36].

Weeds have a life cycle synchronised to that of the crop such that more weeds emerge with the crop with the onset of rains [33]. Intermittent wetting and drying of weed seeds brought about by early rains preceded by dry spells break seed dormancy [8]. Tillage operations bring buried weed seeds to the surface where they germinate. However, early planting of the crop gives the crop a competitive advantage over the weeds [33, 35].

ment and enhance the efficiency of minimum herbicide input through soil incorporation of

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Successive inter-row cultivation has been effective in reducing weed growth and density. Many weed species exhibit morphological plasticity in response to environmental variation and density. Weeds can compensate for density changes so that total biomass per unit area is held relatively constant. Inter-row cultivation improved crop yield by 33% and 78% [20]. However, herbicide application resulted in yield increases of 57 to 300% [27]. Benefits from inter-row cultivation can be limited by in-row weed growth. Most uncontrolled weed growth occurs in the uncultivated area adjacent to and within the crop row. Therefore, the integration of other mechanical or cultural methods often improves with inter-row cultivation. Inter-row cultivation also has potential as a means of controlling late flushes of weeds, but it should not be considered a stand-alone weed management technique since significant in-row weed

Tillage operations have a major impact on distribution of weeds in the soil, weed survival and persistency [8, 10], weed species diversity in a given cropping system [15] and the selection pressure on the weed population. Although not much research has gone into the effect that tillage has on tropical weeds, studies have shown that grass weeds, *Setaria* spp and *Corchorus tridens* were higher under the ripper and basins compared to conventional tillage. Also, broadleaf weeds were less in minimum tillage compared to conventional tillage. Rotation with conventional tillage systems controls the grasses and perennials but other weeds or weed groups may assume numerical dominance. To balance the pressure of tillage, there may be

Tillage affects vertical weed seed distribution in a soil profile and this seed distribution affects weed seed germination by influencing the soil environment surrounding the seeds [12, 13]. There is less soil disturbance with minimum or zero-till systems and, as such, most of the weed seeds are on or near the soil surface after crop planting. In systems with high soil disturbance using conventional tillage, mixing weed seeds uniformly in the tilled-soil depth has been found to be beneficial. It was also found that on direct-seeded rice, 77% of the weed seeds were retained in the top 2 cm soil layer under a zero-till system, whereas soil disturbance under a conventional tillage system resulted in 62% of the seeds being buried to a depth of 2-5 cm. The

The conditions for seed germination are conducive near the soil surface and therefore there is high germination of the weed seeds that are close to the soil surface under zero-till systems, for example, *Ageratum conyzoides*, *Eclipta prostrata, Echinochloa colona, Digitaria ciliaris* and *Portulaca oleracea*. The weed seed populations on the top that are not dormant are easily destroyed by the stale seedbed practice. In this practice, weed seeds are allowed to germinate after a light irrigation or shower and are then killed by using a non-selective herbicide or shallow tillage. This practice helps to reduce the size of the weed seed bank in the soil [8]. Conservation agriculture, or zero tillage farming, is an effective solution to stopping agricul‐ tural land degradation, for rehabilitation, and sustainable crop production intensification in

seeds were not present in the 5-10 cm soil layer in the zero-till system [22, 23].

pre-plant herbicides.

growth may limit benefits [7].

the tropics [21, 22].

need to consider rotational tillage where appropriate [35].

#### *1.1.6. Irrigation practices*

Judicious irrigation practices such as the use of clean water, channels and canals, can reduce the spread of weed seeds to uninfested fields [3, 38]. Flooding is an important component of weed management in rice in the tropical world. In irrigated and flooded systems, the envi‐ ronment in which weed seeds have to germinate is characterized by the existence of low oxygen concentrations. Differential responses between rice and weeds to flooding could be an important component of weed management for the direct-seeded rice crop, since rice is tolerant to flooding, but many weeds, e.g., *Cyperus iria, Fimbristylis miliacea, Leptochloa chinensis, Ludwigia hyssopifolia* and similar weed species are not. However, the timing, duration, and depth of flooding and intensity and frequency of irrigation are critical if germination and growth of a number of weed species are to be effectively suppressed.

Irrigated and upland rice and cereal crops are typically grown with few agricultural inputs. A wide range of weeds infest upland rice, many of which are pan-tropical, including the grass weeds: *Digitaria* spp., *Echinochloa colona*, *Eleusine indica*, *Paspalum* spp., and *Rottboellia cochin‐ chinensis*, and the broadleaf weeds: *Commelina* spp., *Ageratum conyzoides*, *Portulaca oleracea*, *Amaranthus* spp. and *Euphorbia* spp. The variability of weed species composition in upland rice tends to be greater than in the other production systems, and is dependent upon the ecology the cropping system and the management practices used.

Once weed seedlings have emerged and passed the seedling stage, their growth will not be reduced by flooding. In an irrigated environment, there was no emergence of *Leptochloa chinensis* when rice was flooded 5 days after seeding, but its emergence increased to more than 70 plants m-2 when flooding was delayed until 20 days after seeding. In such situations where water is not readily available, early flooding would make the best use of water to control weeds. Introducing flooding after herbicide application or weeding or hoeing could help reduce future weed growth and the need for additional interventions [17, 27].

#### *1.1.7. Inter-row cultivation and minimum tillage*

Inter-row cultivation is practical in widely spaced row crops, such as maize, vegetables, sugarcane and banana [8, 19], which have interrow distances of 60 cm or more. Interrow cultivations are done by tractor drawn implements or hand operated rotary tillers. The efficiency of this method is higher than manual methods. Minimum tillage, on the other hand, involves the use of the minimum amount of tillage required for crop production for meeting the tillage requirement under existing soil and climatic conditions [56]. It refers to eliminating excess tillage, e.g., reducing four secondary tillage steps to two [8]. Both operations comple‐ ment and enhance the efficiency of minimum herbicide input through soil incorporation of pre-plant herbicides.

Weeds have a life cycle synchronised to that of the crop such that more weeds emerge with the crop with the onset of rains [33]. Intermittent wetting and drying of weed seeds brought about by early rains preceded by dry spells break seed dormancy [8]. Tillage operations bring buried weed seeds to the surface where they germinate. However, early planting of the crop

Judicious irrigation practices such as the use of clean water, channels and canals, can reduce the spread of weed seeds to uninfested fields [3, 38]. Flooding is an important component of weed management in rice in the tropical world. In irrigated and flooded systems, the envi‐ ronment in which weed seeds have to germinate is characterized by the existence of low oxygen concentrations. Differential responses between rice and weeds to flooding could be an important component of weed management for the direct-seeded rice crop, since rice is tolerant to flooding, but many weeds, e.g., *Cyperus iria, Fimbristylis miliacea, Leptochloa chinensis, Ludwigia hyssopifolia* and similar weed species are not. However, the timing, duration, and depth of flooding and intensity and frequency of irrigation are critical if germination and

Irrigated and upland rice and cereal crops are typically grown with few agricultural inputs. A wide range of weeds infest upland rice, many of which are pan-tropical, including the grass weeds: *Digitaria* spp., *Echinochloa colona*, *Eleusine indica*, *Paspalum* spp., and *Rottboellia cochin‐ chinensis*, and the broadleaf weeds: *Commelina* spp., *Ageratum conyzoides*, *Portulaca oleracea*, *Amaranthus* spp. and *Euphorbia* spp. The variability of weed species composition in upland rice tends to be greater than in the other production systems, and is dependent upon the ecology

Once weed seedlings have emerged and passed the seedling stage, their growth will not be reduced by flooding. In an irrigated environment, there was no emergence of *Leptochloa chinensis* when rice was flooded 5 days after seeding, but its emergence increased to more than 70 plants m-2 when flooding was delayed until 20 days after seeding. In such situations where water is not readily available, early flooding would make the best use of water to control weeds. Introducing flooding after herbicide application or weeding or hoeing could help reduce future

Inter-row cultivation is practical in widely spaced row crops, such as maize, vegetables, sugarcane and banana [8, 19], which have interrow distances of 60 cm or more. Interrow cultivations are done by tractor drawn implements or hand operated rotary tillers. The efficiency of this method is higher than manual methods. Minimum tillage, on the other hand, involves the use of the minimum amount of tillage required for crop production for meeting the tillage requirement under existing soil and climatic conditions [56]. It refers to eliminating excess tillage, e.g., reducing four secondary tillage steps to two [8]. Both operations comple‐

gives the crop a competitive advantage over the weeds [33, 35].

growth of a number of weed species are to be effectively suppressed.

the cropping system and the management practices used.

weed growth and the need for additional interventions [17, 27].

*1.1.7. Inter-row cultivation and minimum tillage*

*1.1.6. Irrigation practices*

246 Herbicides - Current Research and Case Studies in Use

Successive inter-row cultivation has been effective in reducing weed growth and density. Many weed species exhibit morphological plasticity in response to environmental variation and density. Weeds can compensate for density changes so that total biomass per unit area is held relatively constant. Inter-row cultivation improved crop yield by 33% and 78% [20]. However, herbicide application resulted in yield increases of 57 to 300% [27]. Benefits from inter-row cultivation can be limited by in-row weed growth. Most uncontrolled weed growth occurs in the uncultivated area adjacent to and within the crop row. Therefore, the integration of other mechanical or cultural methods often improves with inter-row cultivation. Inter-row cultivation also has potential as a means of controlling late flushes of weeds, but it should not be considered a stand-alone weed management technique since significant in-row weed growth may limit benefits [7].

Tillage operations have a major impact on distribution of weeds in the soil, weed survival and persistency [8, 10], weed species diversity in a given cropping system [15] and the selection pressure on the weed population. Although not much research has gone into the effect that tillage has on tropical weeds, studies have shown that grass weeds, *Setaria* spp and *Corchorus tridens* were higher under the ripper and basins compared to conventional tillage. Also, broadleaf weeds were less in minimum tillage compared to conventional tillage. Rotation with conventional tillage systems controls the grasses and perennials but other weeds or weed groups may assume numerical dominance. To balance the pressure of tillage, there may be need to consider rotational tillage where appropriate [35].

Tillage affects vertical weed seed distribution in a soil profile and this seed distribution affects weed seed germination by influencing the soil environment surrounding the seeds [12, 13]. There is less soil disturbance with minimum or zero-till systems and, as such, most of the weed seeds are on or near the soil surface after crop planting. In systems with high soil disturbance using conventional tillage, mixing weed seeds uniformly in the tilled-soil depth has been found to be beneficial. It was also found that on direct-seeded rice, 77% of the weed seeds were retained in the top 2 cm soil layer under a zero-till system, whereas soil disturbance under a conventional tillage system resulted in 62% of the seeds being buried to a depth of 2-5 cm. The seeds were not present in the 5-10 cm soil layer in the zero-till system [22, 23].

The conditions for seed germination are conducive near the soil surface and therefore there is high germination of the weed seeds that are close to the soil surface under zero-till systems, for example, *Ageratum conyzoides*, *Eclipta prostrata, Echinochloa colona, Digitaria ciliaris* and *Portulaca oleracea*. The weed seed populations on the top that are not dormant are easily destroyed by the stale seedbed practice. In this practice, weed seeds are allowed to germinate after a light irrigation or shower and are then killed by using a non-selective herbicide or shallow tillage. This practice helps to reduce the size of the weed seed bank in the soil [8]. Conservation agriculture, or zero tillage farming, is an effective solution to stopping agricul‐ tural land degradation, for rehabilitation, and sustainable crop production intensification in the tropics [21, 22].

#### *1.1.8. Minimum herbicides*

Herbicide use continues to be one of the most important tools in weed management. However, an IWM approach creates an opportunity to reduce herbicide rates and in some instances, just forgo the use of herbicides altogether.

may fit into the economics of the small farmer and hence has the potential to be a 'small hammer' in the IWM programme [51]. However, as mentioned before because of the risk of

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Crop and herbicide rotation reduce selection pressure on weeds and this allows for the development of resistant ecotypes and biotypes [35]. Crop rotation should include crops with either different cultural practices or morphology that will upset the life cycle of weeds as in white-top (*Partheniuim hysterophorous*) and corn grass (*R. cochininensis*) [7, 33]. Crop rotations and crop diversification are useful tools for weed management, as they encourage operational diversity that in turn can facilitate improved weed management [31]. Manipulating different planting and harvesting dates among crops provides more opportunities for producers to prevent either plant establishment or seed production by weeds. If sufficient differences exist in the germination requirements of crops and weeds, then seed date can be manipulated to the benefit of the crop for example. Weeds then germinate after canopy closure and they become

However, in the small farm production systems, crop diversification in rotation and even crop succession are limited. The effectiveness of crop rotation in weed suppression may be en‐ hanced by crop sequences that create varying patterns of resource competition, allelopathy, soil disturbance and mechanical damage to certain species. Diversified crop rotations are likely to provide best opportunities for exploiting diverse sets of tactics and ecological processes to

There are only eight modes of action in available herbicides, and as a consequence rotating herbicides is as important as alternating crops, as overuse will increase the risk of single-, cross-, and multiple resistance [29].There is also the potential for a "species shift," as new weed species take over when the population of another diminishes, as a result of an effective herbicide or other control practice. Resistance, however, poses a more serious problem, as it depends on the weed species, the efficacy of the herbicide, and the frequency of herbicide use. Continuous use of a particular herbicide will contribute to resistance, and farmers should rotate two or three herbicides [49]. Additionally, using herbicides with the same mode of action will create an environment for resistance development. To reduce the risk of resistance the

**•** Rotate herbicides, including mode of action of herbicides with the same site of action. Example, Maverick is a sulfonylurea herbicide and Pursuit is an imidazolinone herbicide,

**•** Rotate crops which differ in their competitiveness against weeds based on life cycle, growth habit, maturity length, etc., so rotating to different crops can help prevent some weed species

**•** Tank mix different modes of action to apply different types of materials.

herbicide resistance, this decision must be taken carefully.

*1.1.9. Crop and herbicide rotation*

non competitive [35].

suppress weeds [57]

following guidelines should be considered:

but both are group 2 herbicides.

**•** Alternate non-chemical with chemical control methods.

Given the high cost of herbicides in the tropics, smallholders sometimes either reduce the herbicide rate or mix with other herbicides with differing modes of action. These practices are not without risk. Oftentimes, smallholders realise that these practices are inconsequential and there is no recourse with pesticide retail outlets regarding poor herbicide performance if label rates have not been followed. Yet, farmers often cut rates as a cost saving strategy.

The effectiveness of a reduced rate usually depends on the type of herbicide, weed species present, weed pressure, environmental conditions and, of course, the competitiveness of the crop stand. If the weed pressure is high or the weeds are under stress, it is probably advisable to use an integrated approach. However, reduced rates of herbicide may lead to some level of herbicide resistance and thus the approach to be taken must be carefully considered.

The extent of herbicide use in the tropics is closely related to the cost and availability of labour. Large scale rice and banana production in the tropics receive more than two herbicide applications. However, in the smaller farms, only about 50% of the rice area is treated, particularly where rural labour is available. Herbicides replace hand weeding and enable direct seeding which is less labour demanding, compared to transplanting. Herbicides are also used in the transplanted systems, though to a much lesser extent, and in systems particularly where crop rotation is practised.

There is a need to reduce herbicide input in crop production which can complement cultural practices. With proper timing and selected application methods, good control may be achieved with one-fourth to one-half rates of application [7]. Herbicides are becoming more expensive, and by reducing the pesticide load into the environment, the risk of pollution is reduced. This can be achieved by:


Using lower herbicide dosages would reduce expenditure on herbicides to a fraction of the cost of full label herbicide rates while maintaining efficacy and other benefits derived from herbicide use. Research has revealed that half the recommended dosages of atrazine and nicosulfuron resulted in the lowest weed biomass. Mixing a third of the recommended herbicides of Atrazine and Nicosulfuron resulted in equivalent weed control to the atrazine label recommended dosages. Weed seed production was reduced. Reduced herbicide dosages may fit into the economics of the small farmer and hence has the potential to be a 'small hammer' in the IWM programme [51]. However, as mentioned before because of the risk of herbicide resistance, this decision must be taken carefully.

#### *1.1.9. Crop and herbicide rotation*

*1.1.8. Minimum herbicides*

forgo the use of herbicides altogether.

248 Herbicides - Current Research and Case Studies in Use

where crop rotation is practised.

**i.** banded application of herbicides

**iii.** proper timing of post emergence herbicides **iv.** the use of herbicide combinations at low rates

**vi.** monitoring fields to achieve spray decisions

**ii.** the use of low volumes to improve glyphosate performance

**v.** the use of newer, more active and more rapidly degradable herbicides, and

Using lower herbicide dosages would reduce expenditure on herbicides to a fraction of the cost of full label herbicide rates while maintaining efficacy and other benefits derived from herbicide use. Research has revealed that half the recommended dosages of atrazine and nicosulfuron resulted in the lowest weed biomass. Mixing a third of the recommended herbicides of Atrazine and Nicosulfuron resulted in equivalent weed control to the atrazine label recommended dosages. Weed seed production was reduced. Reduced herbicide dosages

can be achieved by:

Herbicide use continues to be one of the most important tools in weed management. However, an IWM approach creates an opportunity to reduce herbicide rates and in some instances, just

Given the high cost of herbicides in the tropics, smallholders sometimes either reduce the herbicide rate or mix with other herbicides with differing modes of action. These practices are not without risk. Oftentimes, smallholders realise that these practices are inconsequential and there is no recourse with pesticide retail outlets regarding poor herbicide performance if label

The effectiveness of a reduced rate usually depends on the type of herbicide, weed species present, weed pressure, environmental conditions and, of course, the competitiveness of the crop stand. If the weed pressure is high or the weeds are under stress, it is probably advisable to use an integrated approach. However, reduced rates of herbicide may lead to some level of

The extent of herbicide use in the tropics is closely related to the cost and availability of labour. Large scale rice and banana production in the tropics receive more than two herbicide applications. However, in the smaller farms, only about 50% of the rice area is treated, particularly where rural labour is available. Herbicides replace hand weeding and enable direct seeding which is less labour demanding, compared to transplanting. Herbicides are also used in the transplanted systems, though to a much lesser extent, and in systems particularly

There is a need to reduce herbicide input in crop production which can complement cultural practices. With proper timing and selected application methods, good control may be achieved with one-fourth to one-half rates of application [7]. Herbicides are becoming more expensive, and by reducing the pesticide load into the environment, the risk of pollution is reduced. This

rates have not been followed. Yet, farmers often cut rates as a cost saving strategy.

herbicide resistance and thus the approach to be taken must be carefully considered.

Crop and herbicide rotation reduce selection pressure on weeds and this allows for the development of resistant ecotypes and biotypes [35]. Crop rotation should include crops with either different cultural practices or morphology that will upset the life cycle of weeds as in white-top (*Partheniuim hysterophorous*) and corn grass (*R. cochininensis*) [7, 33]. Crop rotations and crop diversification are useful tools for weed management, as they encourage operational diversity that in turn can facilitate improved weed management [31]. Manipulating different planting and harvesting dates among crops provides more opportunities for producers to prevent either plant establishment or seed production by weeds. If sufficient differences exist in the germination requirements of crops and weeds, then seed date can be manipulated to the benefit of the crop for example. Weeds then germinate after canopy closure and they become non competitive [35].

However, in the small farm production systems, crop diversification in rotation and even crop succession are limited. The effectiveness of crop rotation in weed suppression may be en‐ hanced by crop sequences that create varying patterns of resource competition, allelopathy, soil disturbance and mechanical damage to certain species. Diversified crop rotations are likely to provide best opportunities for exploiting diverse sets of tactics and ecological processes to suppress weeds [57]

There are only eight modes of action in available herbicides, and as a consequence rotating herbicides is as important as alternating crops, as overuse will increase the risk of single-, cross-, and multiple resistance [29].There is also the potential for a "species shift," as new weed species take over when the population of another diminishes, as a result of an effective herbicide or other control practice. Resistance, however, poses a more serious problem, as it depends on the weed species, the efficacy of the herbicide, and the frequency of herbicide use. Continuous use of a particular herbicide will contribute to resistance, and farmers should rotate two or three herbicides [49]. Additionally, using herbicides with the same mode of action will create an environment for resistance development. To reduce the risk of resistance the following guidelines should be considered:


from becoming dominant in a given field and control "suspect" herbicide-resistant weeds as if they were an invasive weed species.

Classical biological control is the best among the viable options available for sustainable management of invasive weeds, especially where other technologies such as chemical and mechanical control are unacceptable due to cost and adverse impact on the environment [40]. Some of the techniques described for biological control of weeds in developed countries can be safely and efficiently transferred to developing countries with minimal expense for the initial institutional and human-capacity building. It is essential to know the organism to be used as well as the methods for rearing and release and its host range in order to avoid problems with crops. *The Code of Conduct for the Import and Release of Exotic Biological Control Agents* (FAO, 1996), gives good guidance on how to proceed in order to introduce new exotic

**Weeds Biological control agents**

Weevils: *Neochetina eichhorniae*, *N. Bruchi*

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Fungus: *Puccinia abrupta* var. Partheniicola

Moth: *Parauchaetes pseudoinsulata*

Lacebug: *Teleonemia scrupulosa*

Fungus: *Puccina canaliculata*

Fungus: *Sporisorium ophiuri*

Dactylaria higginsii Moth: Bactra spp.

The traditional top-down approaches, participatory approaches and discovery based teaching

The top-down method has been by far the most predominant method and widely used in training on weeds and their control. The focus of these sessions was to train farmers how to apply, mostly synthetic pesticides, and emphasised the need for continuous application.

*Amaranthus* spp. Fungus: *Phomopsis amaranthicola*

methods have all been used to promote integrated weed management.

**Table 1.** Some organisms used for the biological control of selected weeds

Moth: *Sameodes albigutalis*

Zygogramma bicolorata Epiblema strenuana

organisms for biological control.

*Eichhornia crassipes* (Mart.) Solms

*Chromolaena odorata* (L.) King & Robins.

*Rottboellia cochinchinensis* (Lour.)

**2. Adoption strategies**

*Parthenium hysterophorus* L.

Water hyacinth

Christmas bush

*Lantana camara* L. Black sage, Lantana

*Cyperus rotundus* L.

Nutgrass

Corn grass

Adapted from: [29]

White-top

Multiple management practices can be used in an integrated plan to prevent or delay the development of herbicide-resistant weed populations. In addition, avoid using herbicides with the same site of action in both fallow years and in the succession crops. Herbicide diversifica‐ tion is the key to preventing resistance, since using one system will create resistant weeds. Herbicide rotation is critical to maintaining grade and delaying resistance. Rotating herbicides with multiple modes of action is critical to delaying the spread of resistance and preventing weeds and volunteers [27].

Currently, there is an increase in the number of resistant weed biotypes, including those resistant to glyphosate, PPO, ALS, dicamba and triazine chemistries. The rapid growth of Respect the RotationTM is a testament to the urgency with which thousands of growers treat the issue of weed resistance [36]. Glyphosate-resistant weeds are spreading at alarming rates from rampant infestations; 358 biotypes have developed resistance to one or more herbicide groups, including glyphosate, PPO, ALS, dicamba and triazine chemistries.

#### *1.1.10. Intercropping or relay cropping*

Intercropping or relay cropping systems are based on the principle that space should be occupied by crops and not weeds [57]. Relay cropping can be practised by market gardeners who harvest their crops by hand. These crops should be planted in such a way that the intercrop provides an effective canopy to shade weeds, or that previous crop residue can be used as a mulch to prevent weed growth in successional crops, e.g., pigeon pea (*Cajanus cajan*) inter‐ planted with maize (*Zea mays*). Occasionally, the second crop in some intercropping systems is for the purpose of weed management. Crops such as velvetbean (*Mucuna pruriens*), lablab (*Lablab purpureus*), *Desmodium heterocarpon* and tropical kudzu (*Pueraria phaseoloides*) have been used successfully as intercrops in banana (*Musa* sp.), cassava (*Manihot esculenta*) and maize for the management of weeds such as watergrass (*Commelina* sp.) and cogongrass (*Imperata cylindrica*) [18, 25, 26] across tropical environments. It was found that intercrops may inhibit weeds by limiting resource capture by weeds or through allelopathic interactions [31], and that weed biomass was reduced in 90 % of the cases when a main crop was intercropped with a "smother" crop. It has also been reported that self-regenerating intercrops reduce establish‐ ment costs and can provide weed suppression over years [37].

#### *1.1.11. Biological agents*

The use of biological agents such as mycoherbicides, insects and pathogens to control weeds in the tropics is not common. However, the potential for its application to control noxious weeds using monophagous/oligophagous natural enemies must not be overlooked [29]. Table 1.0 shows some of the most successful achievements using this method of control which include: water hyacinth (*Eichhornia crassipes* (Mart.) Solms) using specific insects, white-top (*Parthenium hysterophorus* L.) using a fungus, Christmas bush (*Chromolaena odorata* (L.) King & Robins) using an insect and nutgrass (*Cyperus* spp.) using a fungus.

Classical biological control is the best among the viable options available for sustainable management of invasive weeds, especially where other technologies such as chemical and mechanical control are unacceptable due to cost and adverse impact on the environment [40].

Some of the techniques described for biological control of weeds in developed countries can be safely and efficiently transferred to developing countries with minimal expense for the initial institutional and human-capacity building. It is essential to know the organism to be used as well as the methods for rearing and release and its host range in order to avoid problems with crops. *The Code of Conduct for the Import and Release of Exotic Biological Control Agents* (FAO, 1996), gives good guidance on how to proceed in order to introduce new exotic organisms for biological control.


**Table 1.** Some organisms used for the biological control of selected weeds

### **2. Adoption strategies**

from becoming dominant in a given field and control "suspect" herbicide-resistant weeds

Multiple management practices can be used in an integrated plan to prevent or delay the development of herbicide-resistant weed populations. In addition, avoid using herbicides with the same site of action in both fallow years and in the succession crops. Herbicide diversifica‐ tion is the key to preventing resistance, since using one system will create resistant weeds. Herbicide rotation is critical to maintaining grade and delaying resistance. Rotating herbicides with multiple modes of action is critical to delaying the spread of resistance and preventing

Currently, there is an increase in the number of resistant weed biotypes, including those resistant to glyphosate, PPO, ALS, dicamba and triazine chemistries. The rapid growth of Respect the RotationTM is a testament to the urgency with which thousands of growers treat the issue of weed resistance [36]. Glyphosate-resistant weeds are spreading at alarming rates from rampant infestations; 358 biotypes have developed resistance to one or more herbicide

Intercropping or relay cropping systems are based on the principle that space should be occupied by crops and not weeds [57]. Relay cropping can be practised by market gardeners who harvest their crops by hand. These crops should be planted in such a way that the intercrop provides an effective canopy to shade weeds, or that previous crop residue can be used as a mulch to prevent weed growth in successional crops, e.g., pigeon pea (*Cajanus cajan*) inter‐ planted with maize (*Zea mays*). Occasionally, the second crop in some intercropping systems is for the purpose of weed management. Crops such as velvetbean (*Mucuna pruriens*), lablab (*Lablab purpureus*), *Desmodium heterocarpon* and tropical kudzu (*Pueraria phaseoloides*) have been used successfully as intercrops in banana (*Musa* sp.), cassava (*Manihot esculenta*) and maize for the management of weeds such as watergrass (*Commelina* sp.) and cogongrass (*Imperata cylindrica*) [18, 25, 26] across tropical environments. It was found that intercrops may inhibit weeds by limiting resource capture by weeds or through allelopathic interactions [31], and that weed biomass was reduced in 90 % of the cases when a main crop was intercropped with a "smother" crop. It has also been reported that self-regenerating intercrops reduce establish‐

The use of biological agents such as mycoherbicides, insects and pathogens to control weeds in the tropics is not common. However, the potential for its application to control noxious weeds using monophagous/oligophagous natural enemies must not be overlooked [29]. Table 1.0 shows some of the most successful achievements using this method of control which include: water hyacinth (*Eichhornia crassipes* (Mart.) Solms) using specific insects, white-top (*Parthenium hysterophorus* L.) using a fungus, Christmas bush (*Chromolaena odorata* (L.) King &

groups, including glyphosate, PPO, ALS, dicamba and triazine chemistries.

ment costs and can provide weed suppression over years [37].

Robins) using an insect and nutgrass (*Cyperus* spp.) using a fungus.

as if they were an invasive weed species.

250 Herbicides - Current Research and Case Studies in Use

weeds and volunteers [27].

*1.1.10. Intercropping or relay cropping*

*1.1.11. Biological agents*

The traditional top-down approaches, participatory approaches and discovery based teaching methods have all been used to promote integrated weed management.

The top-down method has been by far the most predominant method and widely used in training on weeds and their control. The focus of these sessions was to train farmers how to apply, mostly synthetic pesticides, and emphasised the need for continuous application. Farmers responded well to these instructional approaches given the severe losses they sustain because of the extent and vigour of weed growth in the tropics and the quick, highly visible effect of synthetic herbicide applications. These class and field sessions have been historically conducted either as stand-alone modules in training courses or as part of the general agronomic practices for field crops. Over the years, extension agents conducted these courses in com‐ munities or at centralised farmer training centres. The concept of integrated weed management was not part of the landscape at this time.

farming experiences and farmer circumstances in the tropical world, scientists, educators and farmers will have to dedicate increased energies towards finding an approach that is econom‐

Integrated Weed Management Practices for Adoption in the Tropics

http://dx.doi.org/10.5772/55950

253

**3. Research needs for integrated weed management systems for the tropics**

There is a need to encompass weed management into improved/integrated crop management systems and to develop research and development programmes that will facilitate a more comprehensive understanding of ecology, physiology, biochemistry, competitiveness/

The key to a successful weed management programme is the effective insertion into crop management programmes of those control techniques that will minimise the impacts of weeds not controlled by the competing crop. The dependence on overly generalized and increasingly expensive chemical input packages, developed elsewhere under a different set of conditions, and aggressively promoted by Researchers, Extension agents and Agro-chemical companies,

The IWM systems approach fits into the work habit of many farmers and gives more effective control than when only chemical methods are used. In addition, yield improvements in the order of 40 to 100 % are realized. While IWM systems are considered technologically sound, the social and environmental advantages, as well as the economic costs associated with the practice, need to be ascertained. If farmers are not convinced of the economic viability of the

1 Department of Food Production, Faculty of Science and Agriculture, The University of the

2 Department of Agricultural Economics and Extension, Faculty of Science and Agriculture,

and Wayne G. Ganpat2

system, then the technology no matter how sound will not be adopted.

, Puran Bridgemohan3

The University of the West Indies, St. Augustine, Trinidad

3 The University of Trinidad and Tobago, Trinidad

ical, culturally acceptable and environment friendly.

allelopathic potential and threshold of weeds.

**4. Conclusion**

must be broken.

**Author details**

Wendy-Ann P. Isaac1

West Indies, St. Augustine, Trinidad

In the 1990s, the emergence of farmer participatory approaches to educating farmers gained momentum. Although the focus was on Integrated Pest Management (IPM), weed manage‐ ment was incorporated into learning activities. Farmers, for the first time, were presented with the option of applying a mix of weed management strategies instead of a single chemical option. The aim of farmer participatory approaches is to strengthen farmers' decision-making skills through an understanding of the agro-ecology of their fields. The approach is widely recognised as an integral part of more sustainable and environmentally friendly crop produc‐ tion practices. The flagship method, Farmer Field Schools (FFS), continues to be used as the preferred approach to integrated management mostly of pests and diseases but increasingly included is the management of noxious weeds.

The Farmer Field School approach involved farmers in activities mostly in the field to under‐ stand weed dynamics and to involve farmers in decisions to manage weeds using more sustainable approaches. These activities, done on farmers' fields, have been conducted across the Caribbean as part of the FFS approach to integrated pest management. Farmers have been exposed to different weed management strategies which stressed integrated approaches. FFS have been conducted in St Lucia, Suriname, Trinidad and Dominica [4].

The FFS model is flexible, and, in recent times, one component has been singled out for increased use because of the enhanced learning it provides. Discovery-based learning is based on the principles of experiential learning; farmers are guided by a trained facilitator who draws out their knowledge and helps them construct meanings based on their rich field experiences. This has been done in several countries of the Caribbean. Discovery-based learning activities have been used in St Vincent in a Farmer Participatory Research (FPR) process to manage weeds in bananas [25, 26]. Farmers were encouraged to plant several cover crops on their farms to evaluate the efficacy of these crops on weed control in bananas. As farmers carried out these activities, they took the weekly measurements and did simple statistical analysis. They were able to discover for themselves the benefits of alternative approaches to the pesticide approach both for their health and that of consumers in foreign countries who purchase their bananas.

Farmers in Trinidad have also conducted community experiments using paper, used cartons, grass much, plastic, precision irrigation all in an attempt to evaluate alternative weed man‐ agement strategies. Farmers have discovered for themselves the effects of the various treat‐ ments and some of these have been adopted by farmers who are tending to move to the low pesticide/ organic farming methods.

A mix of adoption strategies has been used over the years in an effort to get the right approach to IWM. No silver bullet has been found. It is a work in progress. Given the diverse weed flora, farming experiences and farmer circumstances in the tropical world, scientists, educators and farmers will have to dedicate increased energies towards finding an approach that is econom‐ ical, culturally acceptable and environment friendly.

### **3. Research needs for integrated weed management systems for the tropics**

There is a need to encompass weed management into improved/integrated crop management systems and to develop research and development programmes that will facilitate a more comprehensive understanding of ecology, physiology, biochemistry, competitiveness/ allelopathic potential and threshold of weeds.

### **4. Conclusion**

Farmers responded well to these instructional approaches given the severe losses they sustain because of the extent and vigour of weed growth in the tropics and the quick, highly visible effect of synthetic herbicide applications. These class and field sessions have been historically conducted either as stand-alone modules in training courses or as part of the general agronomic practices for field crops. Over the years, extension agents conducted these courses in com‐ munities or at centralised farmer training centres. The concept of integrated weed management

In the 1990s, the emergence of farmer participatory approaches to educating farmers gained momentum. Although the focus was on Integrated Pest Management (IPM), weed manage‐ ment was incorporated into learning activities. Farmers, for the first time, were presented with the option of applying a mix of weed management strategies instead of a single chemical option. The aim of farmer participatory approaches is to strengthen farmers' decision-making skills through an understanding of the agro-ecology of their fields. The approach is widely recognised as an integral part of more sustainable and environmentally friendly crop produc‐ tion practices. The flagship method, Farmer Field Schools (FFS), continues to be used as the preferred approach to integrated management mostly of pests and diseases but increasingly

The Farmer Field School approach involved farmers in activities mostly in the field to under‐ stand weed dynamics and to involve farmers in decisions to manage weeds using more sustainable approaches. These activities, done on farmers' fields, have been conducted across the Caribbean as part of the FFS approach to integrated pest management. Farmers have been exposed to different weed management strategies which stressed integrated approaches. FFS

The FFS model is flexible, and, in recent times, one component has been singled out for increased use because of the enhanced learning it provides. Discovery-based learning is based on the principles of experiential learning; farmers are guided by a trained facilitator who draws out their knowledge and helps them construct meanings based on their rich field experiences. This has been done in several countries of the Caribbean. Discovery-based learning activities have been used in St Vincent in a Farmer Participatory Research (FPR) process to manage weeds in bananas [25, 26]. Farmers were encouraged to plant several cover crops on their farms to evaluate the efficacy of these crops on weed control in bananas. As farmers carried out these activities, they took the weekly measurements and did simple statistical analysis. They were able to discover for themselves the benefits of alternative approaches to the pesticide approach both for their health and that of consumers in foreign countries who purchase their bananas.

Farmers in Trinidad have also conducted community experiments using paper, used cartons, grass much, plastic, precision irrigation all in an attempt to evaluate alternative weed man‐ agement strategies. Farmers have discovered for themselves the effects of the various treat‐ ments and some of these have been adopted by farmers who are tending to move to the low

A mix of adoption strategies has been used over the years in an effort to get the right approach to IWM. No silver bullet has been found. It is a work in progress. Given the diverse weed flora,

have been conducted in St Lucia, Suriname, Trinidad and Dominica [4].

was not part of the landscape at this time.

252 Herbicides - Current Research and Case Studies in Use

included is the management of noxious weeds.

pesticide/ organic farming methods.

The key to a successful weed management programme is the effective insertion into crop management programmes of those control techniques that will minimise the impacts of weeds not controlled by the competing crop. The dependence on overly generalized and increasingly expensive chemical input packages, developed elsewhere under a different set of conditions, and aggressively promoted by Researchers, Extension agents and Agro-chemical companies, must be broken.

The IWM systems approach fits into the work habit of many farmers and gives more effective control than when only chemical methods are used. In addition, yield improvements in the order of 40 to 100 % are realized. While IWM systems are considered technologically sound, the social and environmental advantages, as well as the economic costs associated with the practice, need to be ascertained. If farmers are not convinced of the economic viability of the system, then the technology no matter how sound will not be adopted.

### **Author details**

Wendy-Ann P. Isaac1 , Puran Bridgemohan3 and Wayne G. Ganpat2

1 Department of Food Production, Faculty of Science and Agriculture, The University of the West Indies, St. Augustine, Trinidad

2 Department of Agricultural Economics and Extension, Faculty of Science and Agriculture, The University of the West Indies, St. Augustine, Trinidad

3 The University of Trinidad and Tobago, Trinidad

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**Chapter 11**

**Integrated Plant Invasion and Bush Encroachment**

Rangeland could be defined as the land on which indigenous vegetation (climax or natural potential) is predominantly grass, grass-like plants, forbs, or shrubs that are grazed or have the potential to be grazed, and which is managed as a natural ecosystem for grazing livestock and wildlife habitat [1]. Rangeland productivity is threatened by land degradation mostly characterised by soil erosion and invasion by alien plant species. Plant invasion is considered a threat to rangelands because of the suppression of productivity of herbaceous plant species due to the increase of bush cover [2]. In an endeavour to understand the concepts of plant invasion in rangelands, it is important to acknowledge that the terms invasion and encroach‐ ment are normally used loosely and commonly interchangeably. However, it is crucial to understand their distinction so that the approaches in addressing their different characteristics and effects on rangelands are informed by clear comprehension. Bush encroachment refers to the spread of plant species into an area where previously it did not occur [18]. Invasion on the other hand, refers to the introduction and spread of an exotic plant species into an area where previously did not occur. Thus, bush encroachment could occur even with indigenous species and it is more defined by plant density than species themselves. Whilst invasion on the other hand, although it includes plant density, focuses on the exoticism of species in question and it is, therefore, more species specific. Furthermore, while encroachment focuses on the woodiness of the species, invasion is not limited to woody species but includes the alien herbaceous species; thus, there are grasses that are classified as invaders. However, in this chapter bush encroachment and invasion are used interchangeably and treated as synonyms.

> © 2013 Lesoli et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Lesoli et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

**Management on Southern African Rangelands**

M. S. Lesoli, M. Gxasheka, T. B. Solomon and

Additional information is available at the end of the chapter

B. Moyo

http://dx.doi.org/10.5772/56182

**1. Introduction**

**1.1. Background**


## **Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands**

M. S. Lesoli, M. Gxasheka, T. B. Solomon and B. Moyo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56182

### **1. Introduction**

[55] Swanton, C. J, Maloney, K. J, Chandler, K, & Gulden, R. (2008). Integrated weed management: Knowledge based weed management systems, Weed Science 2008; , 56,

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168-172.

258 Herbicides - Current Research and Case Studies in Use

#### **1.1. Background**

Rangeland could be defined as the land on which indigenous vegetation (climax or natural potential) is predominantly grass, grass-like plants, forbs, or shrubs that are grazed or have the potential to be grazed, and which is managed as a natural ecosystem for grazing livestock and wildlife habitat [1]. Rangeland productivity is threatened by land degradation mostly characterised by soil erosion and invasion by alien plant species. Plant invasion is considered a threat to rangelands because of the suppression of productivity of herbaceous plant species due to the increase of bush cover [2]. In an endeavour to understand the concepts of plant invasion in rangelands, it is important to acknowledge that the terms invasion and encroach‐ ment are normally used loosely and commonly interchangeably. However, it is crucial to understand their distinction so that the approaches in addressing their different characteristics and effects on rangelands are informed by clear comprehension. Bush encroachment refers to the spread of plant species into an area where previously it did not occur [18]. Invasion on the other hand, refers to the introduction and spread of an exotic plant species into an area where previously did not occur. Thus, bush encroachment could occur even with indigenous species and it is more defined by plant density than species themselves. Whilst invasion on the other hand, although it includes plant density, focuses on the exoticism of species in question and it is, therefore, more species specific. Furthermore, while encroachment focuses on the woodiness of the species, invasion is not limited to woody species but includes the alien herbaceous species; thus, there are grasses that are classified as invaders. However, in this chapter bush encroachment and invasion are used interchangeably and treated as synonyms.

© 2013 Lesoli et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lesoli et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Other than the suppression of herbaceous by encroaching species, the higher bush density in rangelands reduces land accessibility by livestock, and that subsequently negatively affects the utilisation of rangelands. Furthermore, due to competition for light, water, and nutrients between native and invading species, the grazing capacity of rangelands declines [2, 4] and plant biodiversity becomes compromised [3]. Therefore, invasions are considered one of the largest threats to the ecosystems of the earth [5- 6], and the services that they provide to humanity [5]. These species are characterised by rapid spread and they displace native vegetation and disrupt important ecosystem processes, and that leads to serious environmen‐ tal impacts [5- 7]. There are a number of sources for invading species, however, in natural ecosystems such as rangelands some alien tree species used in commercial forestry and agroforestry cause major problems as invaders [8]. The effects of bush encroachment, such as an increase in woody vegetation density and cover, and reduction of biomass production in rangelands [9], have been widely reported in Southern Africa [10 – 11]. Invader species can be found in different ecosystems, however, in South Africa, they are a significant environmental problem in terrestrial and freshwater ecosystems [12]. Bush encroachment and invasion on rangelands, therefore, have negative effects on rangeland biological and economic value. Thus, bush encroachment and invasion results in rangeland degradation, which leads to declination of rangeland functional capacity and subsequently on the increased food insecurity and poverty. Hence, introduction of woody plant cover in grasslands and their increase in savanna ecosystems is an indication of rangeland degradation [13]. The foregoing assertion is aligned with the definition of rangeland degradation, which states the reduction or loss of biological and economic productivity arising from inappropriate land use practices [13]. Therefore, if bush encroachment in rangeland is left unchecked, it progresses within grassland ecosystems until a closed canopy woodland thicket occurs [15], which influences vegetation species composition and in turn threatens the sustainability of livestock production as well as wildlife habitat [16] and grassland birds [17]. Thus, the increase in vegetation cover of encroaching species can significantly reduce grass productivity through competition, shading and allelo‐ pathic effects.

increasing costs of management and production of livestock, and eventually reducing land value. In the wildlife ecosystems, these species affect the wildlife habitat and forage produc‐ tion, deplete soil and water resources, and reduce plant and animal diversity [25]. In general, woody and succulent species invasion in rangelands result in a decline in biodiversity [26], reduction in ecosystem resilience [27] and a greater likelihood of irreversible changes in plant

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261

Grazing is one of the economic ways of utilising rangelands especially in communal and/or pastoral areas. The provisions of biodiversity conservation and ecosystem stability within rangelands maintain the ecological value of these ecosystems. Therefore, maintaining or restoring rangeland ecosystem health and resilience is a critical social imperative to ensure the future supply of the ecosystem services, which are vital for the future well-being of human societies [29]. Such services include provision of stable soils, reliable and clean supply of water, and the natural occurrence of plants, animals and other organisms to meet the aesthetic and cultural values, and to enhance the livelihoods of people living around rangelands [30]. This review chapter explores the phenomenon of plant invasion and bush encroachment in the southern African region; however, reference is made to invasion and encroachment reported beyond the southern African boundaries. Furthermore, although this chapter emphasises bush encroachment and invasion in rangelands or natural ecosystems, the reference is further made from other ecosystems such as cultivated, riparian, and marine areas. This chapter explores plant invasion and encroachment phenomenon in terms of its identified causes, its ecological and economic impact. Furthermore, bush encroachment control practices in rangeland ecosystems and their significance in restoring invaded ecosystems were evaluated. Finally, different methods and approaches used in management of invasion in rangeland are synthes‐

Bush encroachment could be defined as an increase in woody plant abundance in grassland and savanna regions accompanied by changes in the herbaceous cover and composition of the natural vegetation [31 - 33]. This section addresses the question of whether bush encroachment and/or invasion are the problem in rangelands and if the phenomenon poses a challenge to natural ecosystems and human livelihoods. South Africa's natural ecosystems such as rangelands are under threat from invasive alien plants [12, 34], the scale of the problem facing mangers of invasive alien plants in South Africa is huge, and thus, about 10 million ha has been invaded [35]. There is some sort of cosmopolitan concern about the effects of bush encroachment and invasion on rangeland ecosystem productivity and sustainability. Thus, human communities and natural ecosystems worldwide are under siege from a growing number of destructive invasive alien species [36]. These species erode natural capital, com‐ promise ecosystem stability, and threaten economic productivity of rangeland ecosystems. Besides the effects of invasion in agriculture, forestry, and human health, biological invasions

species composition [28].

ised into an integrated rangeland management approach.

**2.1. The concept of bush encroachment and invasion**

**2. Bush encroachment and invasion in rangelands**

Invasion phenomenon is becoming an increasing concern to land managers who are seeking cost-effective ways of combating the spread of invasive species [6]. It is important to acknowl‐ edge that factors causing invasion are complex [10, 19]. This is because of a large number of predisposing factors and that species behave differently at various environments. Therefore, any ecological and/or economic intervention in managing bush encroachment in rangelands should be anteceded by the comprehensive understanding of the drivers for this phenomenon. Nevertheless, bush encroachment is often associated with overgrazing [20]. This is because of a positive relationship between grazing pressure and woody vegetation cover [13]. There are other reported drivers of bush encroachment such as increased rainfall [21], fire suppression [22], and soil characteristics [23]. It is acknowledged, therefore, that bush encroachment threatens livestock production particularly, grazers [24] and in turn livelihoods of pastoral communities hence researchers, policy makers and practitioners need to understand bush encroachment dynamics and characteristics in order to adapt to live with or control it. Invasive plants in rangelands in the long-term affect livestock industry by lowering forage yield and quality, interfering with grazing accessibility and poisoning animals and subsequently increasing costs of management and production of livestock, and eventually reducing land value. In the wildlife ecosystems, these species affect the wildlife habitat and forage produc‐ tion, deplete soil and water resources, and reduce plant and animal diversity [25]. In general, woody and succulent species invasion in rangelands result in a decline in biodiversity [26], reduction in ecosystem resilience [27] and a greater likelihood of irreversible changes in plant species composition [28].

Grazing is one of the economic ways of utilising rangelands especially in communal and/or pastoral areas. The provisions of biodiversity conservation and ecosystem stability within rangelands maintain the ecological value of these ecosystems. Therefore, maintaining or restoring rangeland ecosystem health and resilience is a critical social imperative to ensure the future supply of the ecosystem services, which are vital for the future well-being of human societies [29]. Such services include provision of stable soils, reliable and clean supply of water, and the natural occurrence of plants, animals and other organisms to meet the aesthetic and cultural values, and to enhance the livelihoods of people living around rangelands [30]. This review chapter explores the phenomenon of plant invasion and bush encroachment in the southern African region; however, reference is made to invasion and encroachment reported beyond the southern African boundaries. Furthermore, although this chapter emphasises bush encroachment and invasion in rangelands or natural ecosystems, the reference is further made from other ecosystems such as cultivated, riparian, and marine areas. This chapter explores plant invasion and encroachment phenomenon in terms of its identified causes, its ecological and economic impact. Furthermore, bush encroachment control practices in rangeland ecosystems and their significance in restoring invaded ecosystems were evaluated. Finally, different methods and approaches used in management of invasion in rangeland are synthes‐ ised into an integrated rangeland management approach.

### **2. Bush encroachment and invasion in rangelands**

#### **2.1. The concept of bush encroachment and invasion**

Other than the suppression of herbaceous by encroaching species, the higher bush density in rangelands reduces land accessibility by livestock, and that subsequently negatively affects the utilisation of rangelands. Furthermore, due to competition for light, water, and nutrients between native and invading species, the grazing capacity of rangelands declines [2, 4] and plant biodiversity becomes compromised [3]. Therefore, invasions are considered one of the largest threats to the ecosystems of the earth [5- 6], and the services that they provide to humanity [5]. These species are characterised by rapid spread and they displace native vegetation and disrupt important ecosystem processes, and that leads to serious environmen‐ tal impacts [5- 7]. There are a number of sources for invading species, however, in natural ecosystems such as rangelands some alien tree species used in commercial forestry and agroforestry cause major problems as invaders [8]. The effects of bush encroachment, such as an increase in woody vegetation density and cover, and reduction of biomass production in rangelands [9], have been widely reported in Southern Africa [10 – 11]. Invader species can be found in different ecosystems, however, in South Africa, they are a significant environmental problem in terrestrial and freshwater ecosystems [12]. Bush encroachment and invasion on rangelands, therefore, have negative effects on rangeland biological and economic value. Thus, bush encroachment and invasion results in rangeland degradation, which leads to declination of rangeland functional capacity and subsequently on the increased food insecurity and poverty. Hence, introduction of woody plant cover in grasslands and their increase in savanna ecosystems is an indication of rangeland degradation [13]. The foregoing assertion is aligned with the definition of rangeland degradation, which states the reduction or loss of biological and economic productivity arising from inappropriate land use practices [13]. Therefore, if bush encroachment in rangeland is left unchecked, it progresses within grassland ecosystems until a closed canopy woodland thicket occurs [15], which influences vegetation species composition and in turn threatens the sustainability of livestock production as well as wildlife habitat [16] and grassland birds [17]. Thus, the increase in vegetation cover of encroaching species can significantly reduce grass productivity through competition, shading and allelo‐

260 Herbicides - Current Research and Case Studies in Use

Invasion phenomenon is becoming an increasing concern to land managers who are seeking cost-effective ways of combating the spread of invasive species [6]. It is important to acknowl‐ edge that factors causing invasion are complex [10, 19]. This is because of a large number of predisposing factors and that species behave differently at various environments. Therefore, any ecological and/or economic intervention in managing bush encroachment in rangelands should be anteceded by the comprehensive understanding of the drivers for this phenomenon. Nevertheless, bush encroachment is often associated with overgrazing [20]. This is because of a positive relationship between grazing pressure and woody vegetation cover [13]. There are other reported drivers of bush encroachment such as increased rainfall [21], fire suppression [22], and soil characteristics [23]. It is acknowledged, therefore, that bush encroachment threatens livestock production particularly, grazers [24] and in turn livelihoods of pastoral communities hence researchers, policy makers and practitioners need to understand bush encroachment dynamics and characteristics in order to adapt to live with or control it. Invasive plants in rangelands in the long-term affect livestock industry by lowering forage yield and quality, interfering with grazing accessibility and poisoning animals and subsequently

pathic effects.

Bush encroachment could be defined as an increase in woody plant abundance in grassland and savanna regions accompanied by changes in the herbaceous cover and composition of the natural vegetation [31 - 33]. This section addresses the question of whether bush encroachment and/or invasion are the problem in rangelands and if the phenomenon poses a challenge to natural ecosystems and human livelihoods. South Africa's natural ecosystems such as rangelands are under threat from invasive alien plants [12, 34], the scale of the problem facing mangers of invasive alien plants in South Africa is huge, and thus, about 10 million ha has been invaded [35]. There is some sort of cosmopolitan concern about the effects of bush encroachment and invasion on rangeland ecosystem productivity and sustainability. Thus, human communities and natural ecosystems worldwide are under siege from a growing number of destructive invasive alien species [36]. These species erode natural capital, com‐ promise ecosystem stability, and threaten economic productivity of rangeland ecosystems. Besides the effects of invasion in agriculture, forestry, and human health, biological invasions are also widely recognised as the second-largest global threat to biodiversity. The problem of invasion in rangelands is growing in severity and geographic extent as global trade and travel accelerate, and as human mediated disturbance and increased dissemination of propagules makes ecosystems more susceptible to invasion by alien species [36]. One of the remarkable characters of invasive alien plants is that few, if any, of them are invasive in their countries of origin. Thus, their ability to grow vigorously and produce copious amounts of seeds is kept in check by a host of co-evolved invertebrates and pathogens [6]. Some of these plant species, when transported to a new continent without the attendant enemies, they exhibit "ecological release." This phenomenon allows the introduced species to multiply rapidly in the absence of a host of attendant invertebrates and diseases, with associated tendencies to spread rapidly and to out-compete native species [6].

limiting factor for both grassy and woody plants growth. Based on this analogy, it is hypothe‐ sized that grasses use only topsoil moisture, and woody plants mostly use subsoil moisture [54]. Therefore, reduction of grass plant density and vigour through practices such as severe grazing, allows more water to percolate into the subsoil, where it is made available for woody plant growth. Subsequently, reduction of grassy vegetation has demonstrated an increase in shrub and tree abundance under heavy grazing [55, 56]. The two-layer model is still widely accepted to explain bush encroachment phenomenon, however, field data and other theoretical models have indicated the contravening evidence [20]. Thus, the release of trees from compe‐ tition with grass is not required for mass tree recruitment to occur; for example, encroachment of certain species such as *Prosopis glandulosa* is unrelated to herbaceous biomass or density [57]. Furthermore, a spatially explicit simulation model indicates that rooting niche separation might not be sufficient to warrant coexistence under a range of climatic situations [58].

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263

This indicates that the concepts of bush encroachment and invasion in rangelands are by far still complex in terms of causation and/or predisposition factors. There were great differences reported in a number of studies in the degree of niche separation. These variations depend on various abiotic factors, and plant species involved [59, 60, 61]. Therefore, the two mechanisms, heavy grazing and rooting niche separation, do not suffice to serve as the one-dimensionally exclusive explanations for bush encroachment. This is justified by the fact that at initiation of bush encroachment young trees use the same subsurface soil layer as grasses in the sensitive early stages of growth. In addressing the relationship between bush encroachment and grazing, bush encroachment has been reported in areas where grazing was not severe. Therefore, overgrazing in combination with rooting niche separation are not the solitary predisposing factors for bush encroachment; bush encroachment sometimes also occurs on soils too shallow to allow for root separation [62]. This further shows the complexity of comprehending the causes of bush encroachment in grasslands and savannas and that further translates to the complexity of controlling the problem. This, therefore, suggests that there is no panacea in addressing the bush encroachment; therefore, integration of bush encroachment control measures and practices could lead to a sustainable solution than accrediting one

There are a number of disturbances that have been mooted to be the major determinants of savanna vegetation structure, and savannas have been portrayed as inherently unstable ecosystems. Thus, they are considered to be oscillating in an intermediate state between those of stable grasslands and forests. This is because they are pushed back into the savanna state by frequent disturbances related to human impact, herbivory, fire [61], or drought, and spatial heterogeneities in water, nutrient, and seed distribution [58]. The disturbance hypotheses suggest that bush encroachment occurs as disturbances shift savannas from the open grassland towards the forest extreme of the environmental spectrum. Although disturbance theories may

Bush encroachment and invasion by alien plant species may further be, to a certain degree, attributed to climate change. Climate change causes a number of variations in the atmosphere, and such changes could positively or negatively affect vegetation growth performance. One of the effects of climate change is an accumulation of carbon dioxide (CO2) concentrations in

be valid for specific situations, however, they may lack generality [2].

method over others.

Mostly livestock and wildlife production depend on rangelands for sustenance as a source of feed and habitat. Rangelands are represented by a variety of ecosystems including desert and rich alluvial valleys, coastal and inland foothills, high mountain meadows and arid inland plains [25]. In the southern African context, the larger space of rangelands is represented by savanna and grassland ecosystems. Savannas are extensive, socioeconomically important ecosystems with a mixture of two life forms, thus, trees and grasses [37, 38, 39]. Whilst in Africa, savannas are the most important ecosystems for raising livestock [40]. Thus, domestic livestock, particularly *Bos* (cattle), *Ovis* (Sheep) and *Equus* (Horses) have grazed many of these areas for many years. As a result, the plant composition has changed greatly from the original ecosystems [41].

Factors and mechanisms regulating bush encroachment by invasive woody plants in range‐ land ecosystems are not fully apprehended [2, 42]. However, the dynamics and modalities of bush encroachment are mostly widespread in African [13, 20], Australian [43], and North American and Latin American rangelands [39]. The increase in the tree-grass ration in the savannas has been attributed to the replacement of indigenous herbivores by domestic grazing animals and the intense utilisation of the natural vegetation by domestic livestock [33, 44]. Furthermore, heavy grazing results in reduced fuel loads leading to less frequent and low intensity fire, which reduces the effectiveness of fire in the control of woody vegetation. This heavy grazing further leads to altered competitive interactions between the woody and herbaceous layers due to the removal of grasses [32]. However, a number of times, these phenomenon have been linked to climate change [45] or land use patterns [24] or combination of number of factors [13], both biotic and abiotic in nature. Thus, local climate and long-term climate change in conjunction with grazing effects and fire limitation have been identified as possible causes of bush encroachment [46, 47, 48]. Long-term prohibition of range fire, cultivation of bottomlands and continuous grazing on the remaining portion of the communal rangelands have been reported to have induced the invasion of bush encroachment to a level of more than 60%. This has resulted in reduced grass cover, poor range condition, and subsequently poor livestock productivity [13, 49, 50].

Although there are a myriad of explanations about bush encroachment and invasion in rangelands, the first attempt at a general explanation for bush encroachment was a two-layer hypothesis for tree-grass coexistence [2, 51, 52, 53]. In this model, water is assumed be the major limiting factor for both grassy and woody plants growth. Based on this analogy, it is hypothe‐ sized that grasses use only topsoil moisture, and woody plants mostly use subsoil moisture [54]. Therefore, reduction of grass plant density and vigour through practices such as severe grazing, allows more water to percolate into the subsoil, where it is made available for woody plant growth. Subsequently, reduction of grassy vegetation has demonstrated an increase in shrub and tree abundance under heavy grazing [55, 56]. The two-layer model is still widely accepted to explain bush encroachment phenomenon, however, field data and other theoretical models have indicated the contravening evidence [20]. Thus, the release of trees from compe‐ tition with grass is not required for mass tree recruitment to occur; for example, encroachment of certain species such as *Prosopis glandulosa* is unrelated to herbaceous biomass or density [57]. Furthermore, a spatially explicit simulation model indicates that rooting niche separation might not be sufficient to warrant coexistence under a range of climatic situations [58].

are also widely recognised as the second-largest global threat to biodiversity. The problem of invasion in rangelands is growing in severity and geographic extent as global trade and travel accelerate, and as human mediated disturbance and increased dissemination of propagules makes ecosystems more susceptible to invasion by alien species [36]. One of the remarkable characters of invasive alien plants is that few, if any, of them are invasive in their countries of origin. Thus, their ability to grow vigorously and produce copious amounts of seeds is kept in check by a host of co-evolved invertebrates and pathogens [6]. Some of these plant species, when transported to a new continent without the attendant enemies, they exhibit "ecological release." This phenomenon allows the introduced species to multiply rapidly in the absence of a host of attendant invertebrates and diseases, with associated tendencies to spread rapidly

Mostly livestock and wildlife production depend on rangelands for sustenance as a source of feed and habitat. Rangelands are represented by a variety of ecosystems including desert and rich alluvial valleys, coastal and inland foothills, high mountain meadows and arid inland plains [25]. In the southern African context, the larger space of rangelands is represented by savanna and grassland ecosystems. Savannas are extensive, socioeconomically important ecosystems with a mixture of two life forms, thus, trees and grasses [37, 38, 39]. Whilst in Africa, savannas are the most important ecosystems for raising livestock [40]. Thus, domestic livestock, particularly *Bos* (cattle), *Ovis* (Sheep) and *Equus* (Horses) have grazed many of these areas for many years. As a result, the plant composition has changed greatly from the original

Factors and mechanisms regulating bush encroachment by invasive woody plants in range‐ land ecosystems are not fully apprehended [2, 42]. However, the dynamics and modalities of bush encroachment are mostly widespread in African [13, 20], Australian [43], and North American and Latin American rangelands [39]. The increase in the tree-grass ration in the savannas has been attributed to the replacement of indigenous herbivores by domestic grazing animals and the intense utilisation of the natural vegetation by domestic livestock [33, 44]. Furthermore, heavy grazing results in reduced fuel loads leading to less frequent and low intensity fire, which reduces the effectiveness of fire in the control of woody vegetation. This heavy grazing further leads to altered competitive interactions between the woody and herbaceous layers due to the removal of grasses [32]. However, a number of times, these phenomenon have been linked to climate change [45] or land use patterns [24] or combination of number of factors [13], both biotic and abiotic in nature. Thus, local climate and long-term climate change in conjunction with grazing effects and fire limitation have been identified as possible causes of bush encroachment [46, 47, 48]. Long-term prohibition of range fire, cultivation of bottomlands and continuous grazing on the remaining portion of the communal rangelands have been reported to have induced the invasion of bush encroachment to a level of more than 60%. This has resulted in reduced grass cover, poor range condition, and

Although there are a myriad of explanations about bush encroachment and invasion in rangelands, the first attempt at a general explanation for bush encroachment was a two-layer hypothesis for tree-grass coexistence [2, 51, 52, 53]. In this model, water is assumed be the major

and to out-compete native species [6].

262 Herbicides - Current Research and Case Studies in Use

subsequently poor livestock productivity [13, 49, 50].

ecosystems [41].

This indicates that the concepts of bush encroachment and invasion in rangelands are by far still complex in terms of causation and/or predisposition factors. There were great differences reported in a number of studies in the degree of niche separation. These variations depend on various abiotic factors, and plant species involved [59, 60, 61]. Therefore, the two mechanisms, heavy grazing and rooting niche separation, do not suffice to serve as the one-dimensionally exclusive explanations for bush encroachment. This is justified by the fact that at initiation of bush encroachment young trees use the same subsurface soil layer as grasses in the sensitive early stages of growth. In addressing the relationship between bush encroachment and grazing, bush encroachment has been reported in areas where grazing was not severe. Therefore, overgrazing in combination with rooting niche separation are not the solitary predisposing factors for bush encroachment; bush encroachment sometimes also occurs on soils too shallow to allow for root separation [62]. This further shows the complexity of comprehending the causes of bush encroachment in grasslands and savannas and that further translates to the complexity of controlling the problem. This, therefore, suggests that there is no panacea in addressing the bush encroachment; therefore, integration of bush encroachment control measures and practices could lead to a sustainable solution than accrediting one method over others.

There are a number of disturbances that have been mooted to be the major determinants of savanna vegetation structure, and savannas have been portrayed as inherently unstable ecosystems. Thus, they are considered to be oscillating in an intermediate state between those of stable grasslands and forests. This is because they are pushed back into the savanna state by frequent disturbances related to human impact, herbivory, fire [61], or drought, and spatial heterogeneities in water, nutrient, and seed distribution [58]. The disturbance hypotheses suggest that bush encroachment occurs as disturbances shift savannas from the open grassland towards the forest extreme of the environmental spectrum. Although disturbance theories may be valid for specific situations, however, they may lack generality [2].

Bush encroachment and invasion by alien plant species may further be, to a certain degree, attributed to climate change. Climate change causes a number of variations in the atmosphere, and such changes could positively or negatively affect vegetation growth performance. One of the effects of climate change is an accumulation of carbon dioxide (CO2) concentrations in the atmosphere. These increased CO2 concentrations are likely to have an effect on tree-grass dynamics in savannas. This is because savanna trees and grasses have different photosynthetic pathways, which will respond differently to changes in atmospheric CO2 accumulations. It is predicted that atmospheric CO2 is exponentially increasing and will likely double to 700 parts per million (ppm) within the next century [62]. This has a further potential beneficial effect on plant life; the benefit is attributed to the fact that plants take up CO2 via photosynthesis and use it in photosynthesis to produce carbohydrates. Thus, the higher CO2 concentration could significantly increase the capacity of plants to absorb and temporarily store excess carbon. The efficiency of plants in the savanna to utilise the high CO2 concentrations will be influenced to a larger extend by the photosynthetic pathways of different plant species and, therefore, that will influence plant species composition and ecosystem structure. For example, *Acacia* trees have the C3 photosynthetic pathway, which is less efficient, hence, they have a lower net photosynthetic rate at current atmospheric CO2 levels than the C4 pathway used by most of savanna grasses [62]. However, at the higher atmospheric CO2 levels than currently experi‐ enced, C3 plants will have a higher net photosynthetic rate than C4 plants. Thus, C3 plants should show increases in yield of 20 – 35% with a doubling of atmospheric CO2, while C4 plants such as grasses should only experience a 10% increase in yield. Furthermore, the increased CO2 concentrations will improve the competitive ability of trees against grasses. Thus, *Acacia* trees will have more carbon to invest in carbon-based defences against herbivory such as condensed tannins [63, 64].

**2.2. Spatial distribution of encroaching and invasive plant species in rangelands**

and Succulent Karoo [71] and the thicket biome in the Eastern Cape [12].

their invasive potential prior to their introduction to a region [75, 76].

There are a number of species introduced from other continents and can cause significant problems on rangelands. The temporal and spatial spread of an invading organism including plants generally follows a sigmoid curve [72, 73]. Thus, the initial expansion is slow as the founder colony expands and starts new colonies, decreasing again as the potential habitat (invadable area) becomes fully occupied. The increase of invasive species on the given space and time leads to significant changes on the ecosystem integrity. Thus, invasive plants in the new region lead to profound changes in ecosystem processes, community structure, and displacing native species [74]. Therefore, it is fundamental to determine the spread of invading species in terms of time and space prior to development of a plan to control them. Several attempts have been made to prioritize alien species according to their invasive potential in different parts of the world. However, most attention has been given to screening species for

The ranking of Weeds of National Significance was developed for Australia based on expert scoring of four criteria [77]. These are grounded on their invasiveness, impacts, potential for spread, and socio-economic and environmental values. In South Africa, invasive species were prioritized based on their potential invasiveness, spatial characteristics, potential impacts, and conflicts of interest [78]. The Southern African Plant Invaders Atlas (SAPIA) database contains records for over 500 species of invasive alien plants in South Africa, Lesotho, and Swaziland, with information on their distribution, abundance, and habitat types [79]. There are two lists of invasive alien plants, classified into group species based on similarities in their distribution, abundance, and/or biological traits [80]. The first list contains those species that have already had a substantial impact on natural and semi-natural ecosystems such as rangeland in South Africa. Species demonstrating high value for any of the three components was considered to

Several estimates have been made of the spatial extent of alien plant invasions in South Africa [36]. The rapid reconnaissance in 1996/97 [35] suggested that about 10 million hectares of South Africa has been invaded by the approximately 180 species that were mapped. In South Africa, there are a number of invading species; however, the principal invaders are trees and shrubs in the genera *Acacia*, *Hakea* and *Pinus*. However, the majority of invasive and/or encroaching species in rangelands are in the Fabaceae family, which are normally nitrogen-fixing legumes [70]. Localization of invading species distribution is influenced by the landscape formation gradient, thus, there are dense invasions in the mountains and lowlands and along the major river systems [12].The susceptibility of rangelands to bush encroachment and/or invasion varies between the vegetation types. Thus, vegetation types such as grassland and savanna biomes are extensively invaded mostly by species such as Australian wattles (*Acacia* species), other tree species, and a variety of woody scramblers (notably, triffid weed, *Chromolaena odorata*, and brambles, *Rubus* species). Invading trees such as jacaranda (*Jacaranda mimosifolia*) and syringe (*Melia azedarach*) have spread into semi-arid savanna by spreading along perennial rivers. In the Nama Karoo, woody invaders, notably mesquite (*Prosopis* species), have invaded large areas of alluvial plains and seasonal and ephemeral watercourses. Several cacti (*Opuntia* species) and saltbushes (*Atriplex* species) have invaded large areas of the Nama Karoo

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In an attempt to further explain bush encroachment phenomenon in semi arid and arid environments, it is hypothesised that it is a natural phenomenon occurring in ecological systems governed by patch-dynamic processes [65]. This hypothesis has been based on field observations gained on the spatial distribution of *Acacia reficiens* trees in arid central Namibia. It is argued that encroachment of *A. reficiens* along rainfall gradient increases with increasing rainfall in spite of a relatively constant level of grazing [65]. However, any form of vegetation disturbance in rangelands (grazing, fire, etc.) can create space, and thus, making water and nutrients available for tree establishment due to reduced competition. However, under low soil nitrogen conditions, the nitrogen-fixing trees have a competitive advantage over other plants and, given enough rainfall, may germinate as a group in the bare patches created by the disturbances. The mechanism underlying this hypothesis, which demonstrates how it may be used to explain this phenomenon are such that both tree-grass coexistence and bush encroachment occur in a patch-dynamic system with stochastic rainfall patterns [2]. Nevertheless, it was suggested that in arid and semi-arid savanna ecosystems, woody vegetation needs above-average precipitation for germination and subsequent establishment [66]. To keep the soil moist for a period sufficient for germination and survival through the sensitive early stages of seedling de‐ velopment, several rain events close in succession are necessary [67]. However, in a sav‐ anna ecosystem, rainfall is often patchily distributed, in terms of both time and space [46, 68, 69]. Therefore, the spatial overlap of several rainfall events of high frequency in a sin‐ gle year is a rare occurrence in semi-arid and arid ecosystems. In addition to local seed availability, this rainfall frequency is a necessary condition for the creation of a bush en‐ croachment patch.

### **2.2. Spatial distribution of encroaching and invasive plant species in rangelands**

the atmosphere. These increased CO2 concentrations are likely to have an effect on tree-grass dynamics in savannas. This is because savanna trees and grasses have different photosynthetic pathways, which will respond differently to changes in atmospheric CO2 accumulations. It is predicted that atmospheric CO2 is exponentially increasing and will likely double to 700 parts per million (ppm) within the next century [62]. This has a further potential beneficial effect on plant life; the benefit is attributed to the fact that plants take up CO2 via photosynthesis and use it in photosynthesis to produce carbohydrates. Thus, the higher CO2 concentration could significantly increase the capacity of plants to absorb and temporarily store excess carbon. The efficiency of plants in the savanna to utilise the high CO2 concentrations will be influenced to a larger extend by the photosynthetic pathways of different plant species and, therefore, that will influence plant species composition and ecosystem structure. For example, *Acacia* trees have the C3 photosynthetic pathway, which is less efficient, hence, they have a lower net photosynthetic rate at current atmospheric CO2 levels than the C4 pathway used by most of savanna grasses [62]. However, at the higher atmospheric CO2 levels than currently experi‐ enced, C3 plants will have a higher net photosynthetic rate than C4 plants. Thus, C3 plants should show increases in yield of 20 – 35% with a doubling of atmospheric CO2, while C4 plants such as grasses should only experience a 10% increase in yield. Furthermore, the increased CO2 concentrations will improve the competitive ability of trees against grasses. Thus, *Acacia* trees will have more carbon to invest in carbon-based defences against herbivory such as

In an attempt to further explain bush encroachment phenomenon in semi arid and arid environments, it is hypothesised that it is a natural phenomenon occurring in ecological systems governed by patch-dynamic processes [65]. This hypothesis has been based on field observations gained on the spatial distribution of *Acacia reficiens* trees in arid central Namibia. It is argued that encroachment of *A. reficiens* along rainfall gradient increases with increasing rainfall in spite of a relatively constant level of grazing [65]. However, any form of vegetation disturbance in rangelands (grazing, fire, etc.) can create space, and thus, making water and nutrients available for tree establishment due to reduced competition. However, under low soil nitrogen conditions, the nitrogen-fixing trees have a competitive advantage over other plants and, given enough rainfall, may germinate as a group in the bare patches created by the disturbances. The mechanism underlying this hypothesis, which demonstrates how it may be used to explain this phenomenon are such that both tree-grass coexistence and bush encroachment occur in a patch-dynamic system with stochastic rainfall patterns [2]. Nevertheless, it was suggested that in arid and semi-arid savanna ecosystems, woody vegetation needs above-average precipitation for germination and subsequent establishment [66]. To keep the soil moist for a period sufficient for germination and survival through the sensitive early stages of seedling de‐ velopment, several rain events close in succession are necessary [67]. However, in a sav‐ anna ecosystem, rainfall is often patchily distributed, in terms of both time and space [46, 68, 69]. Therefore, the spatial overlap of several rainfall events of high frequency in a sin‐ gle year is a rare occurrence in semi-arid and arid ecosystems. In addition to local seed availability, this rainfall frequency is a necessary condition for the creation of a bush en‐

condensed tannins [63, 64].

264 Herbicides - Current Research and Case Studies in Use

croachment patch.

Several estimates have been made of the spatial extent of alien plant invasions in South Africa [36]. The rapid reconnaissance in 1996/97 [35] suggested that about 10 million hectares of South Africa has been invaded by the approximately 180 species that were mapped. In South Africa, there are a number of invading species; however, the principal invaders are trees and shrubs in the genera *Acacia*, *Hakea* and *Pinus*. However, the majority of invasive and/or encroaching species in rangelands are in the Fabaceae family, which are normally nitrogen-fixing legumes [70]. Localization of invading species distribution is influenced by the landscape formation gradient, thus, there are dense invasions in the mountains and lowlands and along the major river systems [12].The susceptibility of rangelands to bush encroachment and/or invasion varies between the vegetation types. Thus, vegetation types such as grassland and savanna biomes are extensively invaded mostly by species such as Australian wattles (*Acacia* species), other tree species, and a variety of woody scramblers (notably, triffid weed, *Chromolaena odorata*, and brambles, *Rubus* species). Invading trees such as jacaranda (*Jacaranda mimosifolia*) and syringe (*Melia azedarach*) have spread into semi-arid savanna by spreading along perennial rivers. In the Nama Karoo, woody invaders, notably mesquite (*Prosopis* species), have invaded large areas of alluvial plains and seasonal and ephemeral watercourses. Several cacti (*Opuntia* species) and saltbushes (*Atriplex* species) have invaded large areas of the Nama Karoo and Succulent Karoo [71] and the thicket biome in the Eastern Cape [12].

There are a number of species introduced from other continents and can cause significant problems on rangelands. The temporal and spatial spread of an invading organism including plants generally follows a sigmoid curve [72, 73]. Thus, the initial expansion is slow as the founder colony expands and starts new colonies, decreasing again as the potential habitat (invadable area) becomes fully occupied. The increase of invasive species on the given space and time leads to significant changes on the ecosystem integrity. Thus, invasive plants in the new region lead to profound changes in ecosystem processes, community structure, and displacing native species [74]. Therefore, it is fundamental to determine the spread of invading species in terms of time and space prior to development of a plan to control them. Several attempts have been made to prioritize alien species according to their invasive potential in different parts of the world. However, most attention has been given to screening species for their invasive potential prior to their introduction to a region [75, 76].

The ranking of Weeds of National Significance was developed for Australia based on expert scoring of four criteria [77]. These are grounded on their invasiveness, impacts, potential for spread, and socio-economic and environmental values. In South Africa, invasive species were prioritized based on their potential invasiveness, spatial characteristics, potential impacts, and conflicts of interest [78]. The Southern African Plant Invaders Atlas (SAPIA) database contains records for over 500 species of invasive alien plants in South Africa, Lesotho, and Swaziland, with information on their distribution, abundance, and habitat types [79]. There are two lists of invasive alien plants, classified into group species based on similarities in their distribution, abundance, and/or biological traits [80]. The first list contains those species that have already had a substantial impact on natural and semi-natural ecosystems such as rangeland in South Africa. Species demonstrating high value for any of the three components was considered to have high impact and species with high values for all three components have the highest impact. These species are perceived to constitute the prime concern for managers and, therefore, are referred to as the major invaders. Therefore, the presence and abundance of this species could be regarded to be above the economic threshold and warrant economic and ecological attention. Thus, the projects aimed at the prevention and/or control of these species should receive the largest proportion of available funding over the next few decades.

**Rangeabundance**

Very widespreadabundant

Very widespreadcommon

Widespreadabundant

**Scientific name Common name No of**

*Prosopis glandulosa* var*. Torreyana/velutina*

*Ageratum colyzoides/ houstonianum*

*Campuloclinium macrocephalum*

*halicacabum*

*laevigatum*

*Cestrum aurantiacum/*

*Cardiospermum grandiflorum/*

*Argemone mexicana* Yello–flowered Mexican

poppy

*Acacia mearnsii* Black wattle 432 28 Both 2 *Poplars alba/canescens* White and grey poplars 557 20 Riparian 2

*Eucalyptus spp.* Gum trees 506 4 Both 1

*Nicotiana glauca* Wild tobacco 396 3 Both 3 *Opuntia ficus-indica* Sweet prickly pear 863 4 Landscape 1 *Ricinus communis* Castor-oil plant 471 7 Riparian 2 *Salix babylonica* Weeping willow 475 12 Riparian 2

*Acacia cyclops* Red eye 167 29 Both 2 *Acacia dealbata* Silver wattle 256 24 Riparian 1/2 *Acacia longifolia* Long-leaved wattle 95 24 Both 1 *Acacia saligna* Port Jackson willow 160 28 Both 2 *Ageratina adenophora* Crofton weed 11 19 Riparian 1

*Atriplex lindleyi spp. inflata* Sponge-fruit saltbush 164 43 Landscape 3 *Azolla filiculoides* Red water fern 206 36 Riparian 1 *Caesalpina decapetala* Mauritius thorn 128 19 Both 1

*Chromolaena odorata* Triffid weed 96 36 Both 1 *Eichlomia crassipes* Water hyacinth 95 22 Riparian 1 *Lantana camara* Lantana 261 27 Both 1 *Pinus pinaster* Cluster pine 86 26 Landscape 2

*Melia azedarach* Seringa 558 7 Both

*Agave americana* American agave 433 1 Landscape Proposed *Arundo donax* Giant reed 377 14 Riparian proposed

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

Honey mesquite/ prosopis

**grids-cells**

Invading ageratum 74 26 Riparian 1

Pompom weed 17 25 Both 1

Balloon vines 63 22 Both 1

Inkberry 80 24 Both 1

29 18 Riparian 1

**%Gridcells abundant**

453 15 Both 2

**Riparian or landscape**

http://dx.doi.org/10.5772/56182

**CARA category** 267

The second list contains those species that currently have a lower impact on natural or seminatural ecosystems in South Africa. Thus, these species exhibit a lower product of range, abundance, and effect, but appear to have the capacity to exercise greater influence in the future. They are, therefore, termed "emerging invaders," and are currently afforded lower priority in management. However, some of these species are likely to become more important in the future, and could become targets for pre-emptive action such as biocontrol. These species should be carefully monitored to ensure that they do not become major problems. There are 117 major invaders identified in South Africa, and black wattle (*Acacia mearnsii*), white and grey poplars (*Populus alba*/*canescens*) and mesquite (*Prosopis glandulosa* var. *Torreyana/velutina*) are the three species/species-groups falling within the 'very wide spread-abundant' category [80]. The distribution pattern of these 'very widespread/widespread-abundant' species corresponds to the areas where high overall numbers of invasive alien plants were recorded. Most of the major invaders are found within the 'widespread common' and localised abundant categories. The highest numbers of species in the 'localized-abundant' category are restricted to Western Cape and Natal coasts, and northeastern Mpumalanga and Gauteng (Table 1). A list of 84 emerging invaders identified in South Africa was also presented; a majority (60%) of these species have been listed by the regulations under the Conservation of Agricultural Resources Act (CARA). Emerging invaders account for approximately 2500 records, or 5%, of the SAPIA database, and those species added from other sources [81, 82] and expert knowl‐ edge. Almost 20% of the emerging species are classified as riparian species according to expert opinion. A further 17% of these species are estimated to have the potential of expanding over a large part of the country if unmanaged (categories 'large habitat–large propagule pool', 'large habitat–moderate propagule pool' and 'large habitat–small propagule pool'), and almost 80% of species falling in these categories have been afforded legal status [80]. These species are distributed along the eastern coast and northeastern interior, but have not yet been recorded in the Northern Cape and Western Cape.

Most of the emerging invaders (61%) are estimated to have a moderate amount of invasible habitat available within South Africa (categories 'moderate habitat–large propagule pool' and 'moderate habitat– moderate propagule pool'). These categories show a slight difference in species distribution; distribution patterns of the 'moderate habitat–large propagule pool' category are similar to the 'localized–abundant' category of major weeds, whilst distribution patterns for the 'moderate habitat-moderate propagule pool' category show a lower incidence of fynbos invaders. The emerging invaders that are estimated to have a small amount of invasible habitat available but a large current propagule pool size (Table 2) show a very similar distribution pattern to the species which fall into the 'moderate habitat–large propagule pool' category.

have high impact and species with high values for all three components have the highest impact. These species are perceived to constitute the prime concern for managers and, therefore, are referred to as the major invaders. Therefore, the presence and abundance of this species could be regarded to be above the economic threshold and warrant economic and ecological attention. Thus, the projects aimed at the prevention and/or control of these species

The second list contains those species that currently have a lower impact on natural or seminatural ecosystems in South Africa. Thus, these species exhibit a lower product of range, abundance, and effect, but appear to have the capacity to exercise greater influence in the future. They are, therefore, termed "emerging invaders," and are currently afforded lower priority in management. However, some of these species are likely to become more important in the future, and could become targets for pre-emptive action such as biocontrol. These species should be carefully monitored to ensure that they do not become major problems. There are 117 major invaders identified in South Africa, and black wattle (*Acacia mearnsii*), white and grey poplars (*Populus alba*/*canescens*) and mesquite (*Prosopis glandulosa* var. *Torreyana/velutina*) are the three species/species-groups falling within the 'very wide spread-abundant' category [80]. The distribution pattern of these 'very widespread/widespread-abundant' species corresponds to the areas where high overall numbers of invasive alien plants were recorded. Most of the major invaders are found within the 'widespread common' and localised abundant categories. The highest numbers of species in the 'localized-abundant' category are restricted to Western Cape and Natal coasts, and northeastern Mpumalanga and Gauteng (Table 1). A list of 84 emerging invaders identified in South Africa was also presented; a majority (60%) of these species have been listed by the regulations under the Conservation of Agricultural Resources Act (CARA). Emerging invaders account for approximately 2500 records, or 5%, of the SAPIA database, and those species added from other sources [81, 82] and expert knowl‐ edge. Almost 20% of the emerging species are classified as riparian species according to expert opinion. A further 17% of these species are estimated to have the potential of expanding over a large part of the country if unmanaged (categories 'large habitat–large propagule pool', 'large habitat–moderate propagule pool' and 'large habitat–small propagule pool'), and almost 80% of species falling in these categories have been afforded legal status [80]. These species are distributed along the eastern coast and northeastern interior, but have not yet been recorded

Most of the emerging invaders (61%) are estimated to have a moderate amount of invasible habitat available within South Africa (categories 'moderate habitat–large propagule pool' and 'moderate habitat– moderate propagule pool'). These categories show a slight difference in species distribution; distribution patterns of the 'moderate habitat–large propagule pool' category are similar to the 'localized–abundant' category of major weeds, whilst distribution patterns for the 'moderate habitat-moderate propagule pool' category show a lower incidence of fynbos invaders. The emerging invaders that are estimated to have a small amount of invasible habitat available but a large current propagule pool size (Table 2) show a very similar distribution pattern to the species which fall into the 'moderate habitat–large propagule pool'

should receive the largest proportion of available funding over the next few decades.

in the Northern Cape and Western Cape.

266 Herbicides - Current Research and Case Studies in Use

category.



*Opuntia stricta* Australian pest pear 108 10 Landscape 1 *Pinus halepensis* Aleppo pine 85 3 Landscape 2 *Pinus patula* Patula pine 90 12 Both 2 *Pinus radiata* Radiata pine 71 12 Landscape 2 *Pinus spp.* Pine trees 126 9 Landscape *Pyracantha angustifolia* Yellow fire thorn 143 1 Both 3 *Robinia pseudoacacia* Black locus 110 9 Both 2

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

*Schinus molle* Pepper tree 232 1 Both Proposed

40 6 Both 1

http://dx.doi.org/10.5772/56182

269

Tamarisk 92 4 Riparian 1/3

*Senna didymobotrya* Peanut butter cassia 142 13 Both 3

*Sesbania punicea* Red sesbania 325 13 Riparian 1 *Solanum seaforthianum* Potato creeper 33 7 Both 1

*Sorghum halepense* Johnson grass 44 4 Riparian 2

*Verbena bonariensis* Purple top 58 5 Riparian *Verbena tenuisecta* Fine-leaved verbena 14 4 riparian *Xanthium strumarium* Large cocklebur 151 12 Both 1

*Zinnia peruviana* Redstar Zinnia 4 0 Both

*Acacia baileyana* Bailey's wattle 87 0 Both 3

*Acacia pycnantha* Golden wattle 35 25 Landscape 1 *Albizia lebbeck* Lebbeck tree 5 33 No data 1 *Azolla pinnata var. imbricata* Mosquito fern 3 25 Riparian *Colocasia esculenta* Elephant's ear 10 21 Riparian *Echinopsis spachiana* Torch cactus 57 3 Landscape 1 *Eucalyptus lehmannii* Spider gum 41 13 Landscape 1/2 *Flaveria bidentis* Smelter's bush 19 26 Riparian *Hakea drupacea* Sweet hakea 28 7 Landscape 1 *Hakea gibbosa* Rock hakea 18 11 Landscape 1 *Harrisia martinii* Moon cactus 21 43 Both 1 *Hedychium coccineum* Red ginger lily 3 20 Riparian 1 *Hedychium flavescens* Yellow ginger lily 5 40 Both 1 *Hedychium spp.* Ginger lilies 7 25 Riparian 1

*Populus nigra var. italica* Lombardy poplar 90 0 Riparian Proposed

*Senna occidentalis* Wild coffee 56 8 Both

*Solanum sisymbriifolium* Dense-thorned bitter

*Tamarix spp. (T. chinensis/T.*

*ramosissima)*

Widespreadscarce

Localizedabundant

apple

*Psidium guajava* Guava 167 17 Both 2 *Rubus cuneifolius* American bramble 75 34 Both 1 *Rubus fruticosus* Europian blackberry 89 20 Both 2 *Salix fragilis* Crack willow 75 22 Riparian 2 *Solanum mauritianum* Bugweed 268 21 Both 1

*Acacia decurrens* Green wattle 101 21 Both 2 *Acacia melanoxylon* Australian blackwood 138 15 Both 2 *Achyranthes aspera* Burweed 77 4 Both 1 *Ailanthus altissima* Tree-of-heaven 32 5 Both 3 *Anredera cordifolia* Bridal wreath 24 8 Both 1 *Araujia sericifera* Moth catcher 36 2 Both 1

*Bidens formosa* Cosmos 48 11 Riparian *Cardiospermum halicacaburn* Heart pea 30 0 Riparian *Casuarina equisetifolia* Horsetail tree 24 3 Both 2 *Cereus jamacaru* Queen of the night 127 9 Landscape 1 *Conyza bonariensis* Flax-leaf fleabane 5 0 Riparian

*Cuscuta campestris* Common dodder 82 1 Both 1

*Eucalytus camaldulensis* Red river gum 123 15 Riparian 2 *Hakea sericea* Silky hakea 78 12 Landscape 1 *Ipomoea alba* Moonflower 23 3 Riparian 1 *Ipomoea indica/purpurea* Morning glories 98 8 Both 1 *Jacaranda mimosifolia* Jacaranda 201 6 Both 3

*Mirabilis jalapa* Four-o'clock 7 0 Landscape Proposed

*Opuntia aurantiaca* Jointed cactus 61 5 Landscape 1 *Opuntia imbricata* Imbricate cactus 131 10 Landscape 1 *Opuntia monacantha* Cochineal pricky pear 48 1 Both 1 *Opuntia robusta* Blue-leaf cactus 225 1 Landscape

Old-man saltbush 173 7 Both 2

Bird flower 18 0 Both Proposed

44 14 Both 1

130 4 Riparian 3

Thorn apples 84 1 Riparian 1

Widespreadcommon

Widespreadcommon

*Atriplex nummularia spp.*

*Crotalaria agatiflora subsp.*

*Datura spp (D. Ferox/ D. Inoxia/D. Stramonium)*

*Echium plantagineum/vulgare* Patterson's curse/blue

*Morus alba* White or common

echium

mulberry

*nummularia*

268 Herbicides - Current Research and Case Studies in Use

*imperialis*



**3. Effects of bush encroachment and invasion on rangelands**

It is important to establish an understanding of ecological effects of bush encroachment on rangeland ecosystems prior to embarking on any bush encroachment intervention. Thus, the degree of invasion should be quantified to help justify the need for, and deter‐ mine the type of intervention. It is fundamental to characterise invasion and these could be in terms of identification of invading species (morphology, phenology, anatomy, phys‐ iology, mode of spread), plant population density, spatial localization (along the land‐ scape, vegetation types, soil type, water distribution), seasonal distribution, their impact on the ecosystem stability (soil cover and biodiversity) and productivity (primary and secondary). The global reviews of plant invasions suggest that the most damaging species transform ecosystems by using excessive amounts of resources, notably, water, light, and oxygen. Invading species achieve these by adding resources such as nitrogen, promoting or suppressing fire, stabilising sand movement, and/or promoting erosion, accumulating litter and accumulating or redistributing salt [82]. Such changes potentially alter the flow, availability, or quality of nutrient resources in biogeochemical cycles. They further modi‐ fy tropic resources within the food web and alter physical resources such as living space or habitat, sediment, light and water. In addition, invaders are most likely to have sub‐ stantial effects on ecosystems by rapidly changing the disturbance regime [36]. Thus, dense stands of alien trees and shrubs in rangelands can rapidly reduce abundance and

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

http://dx.doi.org/10.5772/56182

271

Different invading species have similar or specific effects on rangeland ecosystem dynam‐ ics. Thus, invasion of black wattle (*Acacia mearnsii*) in South African rangeland ecosys‐ tems has negative ecological impacts [8]. These impacts include reduction of surface stream flow, loss of biodiversity, increase in fire hazard, and increases in soil erosion, de‐ stabilisation of riverbanks, and loss of recreational opportunities, aesthetic costs, and ni‐ trogen pollution and subsequently loss of grazing potential. An increase in the height and biomass of vegetation increase rainfall interception and transpiration, and decreases stream flow [8]. Alien trees and shrubs increase above ground biomass and evapotranspi‐ ration and thereby decrease both surface water runoff and ground water recharge [84]. The reduction of surface water runoff as a result of current invasions was estimated to be

, which is about 7% of the national total [35], most of which is coming from

the fynbos and grassland biomes [85]. The increased biomass and evapotranspiration rates associated with invasive alien plants arise because of their greater height, root depth, and senescence, compared to the native species that they replace [86]. Invasive plants may influence native ecosystems by exerting resource competition on native plants to altering fire dynamics [87]. Thus, the increased biomass that accompanies plant inva‐ sions also result in more intense fires [8, 36, 70] due to an accumulation of fuel loads. On the other hand, the dense stands of invasive trees hamper access for fire management

**3.1. Ecological impact**

diversity of native plants [83].

3 300 mm3

N.B: Major invaders grouped according to categories. 'No. grid-cells' is the number of grid-cells where the species has been recorded in the Southern African Plant Invaders Atlas (SAPIA) database; '% grid-cells abundant' is the percentage of grid-cells in South Africa where the species is recorded as very abundant or abundant in the SAPIA database (note: where more than one record with the same species and abundance code occurred within a grid-cell, it was counted as one record); 'Riparian or landscape' is the classification given to a species if more than 75% of its records in the SAPIA database fell into the respective category (if neither the landscape nor riparian records exceeded 75% then the species was classified as 'both'); and 'CARA category' lists the species regulated by the Conservation of Agricultural Resources Act (Act 43 of 1983), where 1 refers to Category 1 prohibited weeds that must be controlled in all situations; 2 includes Category 2 plants with commercial value that may be planted in demarcated areas subject to a permit, provided that steps are taken to control spread; 3 includes Category 3 ornamental plants that may no longer be planted or traded, but may remain in place provided a permit is obtained and steps taken to control their spread; and 'proposed' includes those species that were proposed for listing under the Conservation of Agricultural Resources Act, but require further investigation before they can be included.

**Table 1.** Major invaders plants species in South Africa according to their categories (Source: [80])

## **3. Effects of bush encroachment and invasion on rangelands**

#### **3.1. Ecological impact**

*Helianthus annuus* Sunflower 5 17 No data *Leptospermum laevigatum* Australian mrytle 38 30 Landscape 1 *Ligustrum vulgare* Common privet 3 20 Riparian 3 *Lilium formosanum* Formosa lily 16 21 Landscape 3 *Litsea glutinosa* Indian laurel 8 44 Both 1 *Macfadyena unguis-cati* Cat's claw creeper 27 27 Both 1 *Melilotus alba* White sweet clover 15 40 Riparian

2 25 Riparian 3

11 17 Landscape 1

11 21 Landscape 1

bottlebrush

cactus

Small round-leaved prickly pear

N.B: Major invaders grouped according to categories. 'No. grid-cells' is the number of grid-cells where the species has been recorded in the Southern African Plant Invaders Atlas (SAPIA) database; '% grid-cells abundant' is the percentage of grid-cells in South Africa where the species is recorded as very abundant or abundant in the SAPIA database (note: where more than one record with the same species and abundance code occurred within a grid-cell, it was counted as one record); 'Riparian or landscape' is the classification given to a species if more than 75% of its records in the SAPIA database fell into the respective category (if neither the landscape nor riparian records exceeded 75% then the species was classified as 'both'); and 'CARA category' lists the species regulated by the Conservation of Agricultural Resources Act (Act 43 of 1983), where 1 refers to Category 1 prohibited weeds that must be controlled in all situations; 2 includes Category 2 plants with commercial value that may be planted in demarcated areas subject to a permit, provided that steps are taken to control spread; 3 includes Category 3 ornamental plants that may no longer be planted or traded, but may remain in place provided a permit is obtained and steps taken to control their spread; and 'proposed' includes those species that were proposed for listing under the Conservation of Agricultural Resources Act, but require further

**Table 1.** Major invaders plants species in South Africa according to their categories (Source: [80])

*Myriophyllum aquaticum* Parrot's feather 48 19 Riparian 1 *Nassella trichotoma* Nassella tussock 12 21 Landscape 1 *Nerium oleander* Oleander 24 6 Riparian 1

*Paraserianthes lophantha* Stinkbean 54 10 Both 1 *Parthenium hysterophorus* Parthenium weed 24 37 Riparian 1 *Paspalum dilatatum* Common Paspalum 6 33 Riparian *Pennisetum villosum* Feathertop 22 21 Landscape 1 *Pinus elliottii* Slash pine 34 15 Landscape 2 *Pistia stratiotes* Water lettuce 27 17 Riparian 1 *Pittosporum undulatum* Australian cheesewood 3 0 Both 1 *Rumex usambarensis* Rumex 4 20 Landscape *Salvinia molesta* Salvinia 33 20 Riparian 1 *Schinus terebinthifolius* Brazilian pepper tree 32 16 Both 1

*Metrosideros excelsa* New Zealand

*Opuntia lindheimeri/Opunia engelmannii var. linderheimeri*

270 Herbicides - Current Research and Case Studies in Use

investigation before they can be included.

*Opuntia fulgida* Chainfruit-cholla/rosea

It is important to establish an understanding of ecological effects of bush encroachment on rangeland ecosystems prior to embarking on any bush encroachment intervention. Thus, the degree of invasion should be quantified to help justify the need for, and deter‐ mine the type of intervention. It is fundamental to characterise invasion and these could be in terms of identification of invading species (morphology, phenology, anatomy, phys‐ iology, mode of spread), plant population density, spatial localization (along the land‐ scape, vegetation types, soil type, water distribution), seasonal distribution, their impact on the ecosystem stability (soil cover and biodiversity) and productivity (primary and secondary). The global reviews of plant invasions suggest that the most damaging species transform ecosystems by using excessive amounts of resources, notably, water, light, and oxygen. Invading species achieve these by adding resources such as nitrogen, promoting or suppressing fire, stabilising sand movement, and/or promoting erosion, accumulating litter and accumulating or redistributing salt [82]. Such changes potentially alter the flow, availability, or quality of nutrient resources in biogeochemical cycles. They further modi‐ fy tropic resources within the food web and alter physical resources such as living space or habitat, sediment, light and water. In addition, invaders are most likely to have sub‐ stantial effects on ecosystems by rapidly changing the disturbance regime [36]. Thus, dense stands of alien trees and shrubs in rangelands can rapidly reduce abundance and diversity of native plants [83].

Different invading species have similar or specific effects on rangeland ecosystem dynam‐ ics. Thus, invasion of black wattle (*Acacia mearnsii*) in South African rangeland ecosys‐ tems has negative ecological impacts [8]. These impacts include reduction of surface stream flow, loss of biodiversity, increase in fire hazard, and increases in soil erosion, de‐ stabilisation of riverbanks, and loss of recreational opportunities, aesthetic costs, and ni‐ trogen pollution and subsequently loss of grazing potential. An increase in the height and biomass of vegetation increase rainfall interception and transpiration, and decreases stream flow [8]. Alien trees and shrubs increase above ground biomass and evapotranspi‐ ration and thereby decrease both surface water runoff and ground water recharge [84]. The reduction of surface water runoff as a result of current invasions was estimated to be 3 300 mm3 , which is about 7% of the national total [35], most of which is coming from the fynbos and grassland biomes [85]. The increased biomass and evapotranspiration rates associated with invasive alien plants arise because of their greater height, root depth, and senescence, compared to the native species that they replace [86]. Invasive plants may influence native ecosystems by exerting resource competition on native plants to altering fire dynamics [87]. Thus, the increased biomass that accompanies plant inva‐ sions also result in more intense fires [8, 36, 70] due to an accumulation of fuel loads. On the other hand, the dense stands of invasive trees hamper access for fire management purposes [36], which makes it difficult for fire control in rangelands. The increase in fire intensity due to accumulation of sufficient fuel load subsequently damages vegetation and soil [70], which in turn leads to excessive soil erosion due to soil water repellency caused by fire [36].

**Habitat– propagule pool size**

Large–moderate *Celtis sinensis/* Chinese nettle

*Pennisetum purpureum*

Moderate–large *Acacia elata* Peppertree

*Cinnamomum camphora*

*Cotoneaster franchetii* Orange

*Cotoneaster pannosus* Silver-leaf

*Celtis occidentalis*/ Common

*Celtis australis* European

**Scientific name Common name Impact Weediness Biocontrol % Weedy**

*Pinus taeda* Loblolly pine 10 1 10 4 87 2 *Tecoma stans* Yellow bells 5 1 10 3 69 1 *Tipuana tipu* Tipu tree 5 1 10 10 73 3

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

*Cytisus scoparius* Scotch broom 5 5 10 4 86 1

*Pereskia aculeata* Pereskia 10 1 10 2 87 1 *Rosa rubiginosa* Eglantine 10 3 10 3 96 1 *Toona ciliata* Toon tree 5 1 10 2 64 3 *Ulex europaeus* European gorse 5 5 10 1 80 1

*Pueraria lobata* Kudzu vine 5 3 10 5 76 1 *Triplaris americana* Triplaris 5 0 10 1 62 1

*Acacia podalyriifolia* Pearl acacia 5 1 10 3 67 3 *Ardisia crenata* Coralberry tree 5 1 10 0 66 1

*Eucalyptus cladocalyx* Sugar gum 5 1 10 2 68 2

*Eugenia uniflora* Surinam cherry 5 2 10 0 68 1

*Eucalyptus saligna* Saligna gum 5 1 10 2 66

Large–small *Acacia paradoxa* Kangaroo thorn 5 2 10 3 69 1

Large–large *Bromus diandrus* Ripgut brome 0 2 10 5 53

tree/

hackberry/

hackberry

wattle

cotoneaster

cotoneaster

**relatives**

0 1 10 1 45 Proposed

Elephant grass 10 3 10 2 95 Proposed

5 2 10 3 69 3

5 2 10 1 69 3

5 2 10 1 69 3

Camphor tree 10 2 10 0 90 1/3

**Combined Score**

http://dx.doi.org/10.5772/56182

**CARA category** 273

Therefore, it suffices to indicate that the alien invasive plants reduce the functional ca‐ pacity of rangeland ecosystems such as support for livestock and wildlife [36, 70]. This is among others due to competition between invasive plants and grasses that are important for grazing. This competition leads to reduction on performance of a number of ecosys‐ tem functions such as grass cover, which subsequently contributes to loss of grazing po‐ tential [36]. There is also a significant loss of biodiversity due to competition [70], resulting from the displacement of species-rich indigenous plant communities by singlespecies stands, and disruption of important ecosystem processes [8]. On the other hand, invasion of riverbanks causes deep channelling followed by slumping during floods and that result in destabilized riverbanks. Subsequently, the invasion along the riverbanks leads to loss of recreational opportunities due to reduction of access for anglers, canoe‐ ists, white-water rafters, and swimmers. Invasive plants further detract from the wilder‐ ness character of many rural landscapes and conservation areas and that imposes reduction of the aesthetic value of ecosystems. An increase in soil nitrogen levels in nu‐ trient-poor environments can make habitats unsuitable for indigenous plants and more susceptible to invasion by other species, and, in turn, reducing biodiversity.

In order to develop the effective invasion control in rangelands, it is significant to under‐ stand the mechanisms that are employed by the invader species to survive and colonise the new ecosystems. There are a number of ways through which invasive plants survive and outcompete the indigenous species in rangelands; one of the mechanisms is their ability to grow rapidly compared to indigenous plants. Thus, invasive alien plants typi‐ cally grow more rapidly, often increasing the proportion of biomass contributed by alien plants. The large biomass contributed by invasive plants is composed of leaves, bark, seed, flowers, and twigs that become 'terrestrial litter' after abscission [88]. Such litter en‐ ters and is retained in water bodies where its rate of breakdown by invertebrate feeding as well as decomposition through fungal and bacterial activity differs from that of inputs from indigenous plants [89]. The often large differences in litter inputs from invasive ali‐ en plants relative to indigenous species leads to reduced decomposition rate and dramati‐ cally alters the nutrient cycle in rangeland ecosystem [90]. Additions in the biomass contributed by alien plants can increase the amount of metabolised nutrients, which in turn escalates natural eutrophication processes [91] as well as free-floating and rooted aquatic macrophyte invasions [92]. Thus, eutrophication leads to gradual changes in the plant and animal populations and the development of potentially toxic algal blooms and, therefore, a slow decline in water and habitat quality [91]. The level of impact that litter from invasive alien plants has on nutrient cycles is determined by vegetative spread, plant structure, phenology, plant water and nutrient uptake efficiency, photosynthesis type, presence of symbionts and nitrogen fixation, phosphorus content and tissue chemis‐ try such as allelopathy [93].

purposes [36], which makes it difficult for fire control in rangelands. The increase in fire intensity due to accumulation of sufficient fuel load subsequently damages vegetation and soil [70], which in turn leads to excessive soil erosion due to soil water repellency

Therefore, it suffices to indicate that the alien invasive plants reduce the functional ca‐ pacity of rangeland ecosystems such as support for livestock and wildlife [36, 70]. This is among others due to competition between invasive plants and grasses that are important for grazing. This competition leads to reduction on performance of a number of ecosys‐ tem functions such as grass cover, which subsequently contributes to loss of grazing po‐ tential [36]. There is also a significant loss of biodiversity due to competition [70], resulting from the displacement of species-rich indigenous plant communities by singlespecies stands, and disruption of important ecosystem processes [8]. On the other hand, invasion of riverbanks causes deep channelling followed by slumping during floods and that result in destabilized riverbanks. Subsequently, the invasion along the riverbanks leads to loss of recreational opportunities due to reduction of access for anglers, canoe‐ ists, white-water rafters, and swimmers. Invasive plants further detract from the wilder‐ ness character of many rural landscapes and conservation areas and that imposes reduction of the aesthetic value of ecosystems. An increase in soil nitrogen levels in nu‐ trient-poor environments can make habitats unsuitable for indigenous plants and more

susceptible to invasion by other species, and, in turn, reducing biodiversity.

In order to develop the effective invasion control in rangelands, it is significant to under‐ stand the mechanisms that are employed by the invader species to survive and colonise the new ecosystems. There are a number of ways through which invasive plants survive and outcompete the indigenous species in rangelands; one of the mechanisms is their ability to grow rapidly compared to indigenous plants. Thus, invasive alien plants typi‐ cally grow more rapidly, often increasing the proportion of biomass contributed by alien plants. The large biomass contributed by invasive plants is composed of leaves, bark, seed, flowers, and twigs that become 'terrestrial litter' after abscission [88]. Such litter en‐ ters and is retained in water bodies where its rate of breakdown by invertebrate feeding as well as decomposition through fungal and bacterial activity differs from that of inputs from indigenous plants [89]. The often large differences in litter inputs from invasive ali‐ en plants relative to indigenous species leads to reduced decomposition rate and dramati‐ cally alters the nutrient cycle in rangeland ecosystem [90]. Additions in the biomass contributed by alien plants can increase the amount of metabolised nutrients, which in turn escalates natural eutrophication processes [91] as well as free-floating and rooted aquatic macrophyte invasions [92]. Thus, eutrophication leads to gradual changes in the plant and animal populations and the development of potentially toxic algal blooms and, therefore, a slow decline in water and habitat quality [91]. The level of impact that litter from invasive alien plants has on nutrient cycles is determined by vegetative spread, plant structure, phenology, plant water and nutrient uptake efficiency, photosynthesis type, presence of symbionts and nitrogen fixation, phosphorus content and tissue chemis‐

caused by fire [36].

272 Herbicides - Current Research and Case Studies in Use

try such as allelopathy [93].



**Habitat– propagule pool size**

> *Cytisus monspessulanus*

*Leucaena leucocephala*

*Pyracantha crenulata* Himalayan

*Sophora japonica* Japanese pagoda tree

*Tithonia diversifolia* Mexican

*Verbena brasiliensis* Slender wild

*Senna pendula* var. *glabrata*

*Sesbania bispinosa* var. *bispinosa*

firethorn

sunflower

verbena

**Scientific name Common name Impact Weediness Biocontrol % Weedy**

*Duranta erecta* Forget-me-not 0 1 10 1 44 Proposed

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

Leucaena 5 3 4 3 52 1

Rambling cassia 5 2 10 1 68 3

0 0 10 2 42

0 1 10 2 45

0 1 10 3 48 1

Spiny sesbania 0 0 10 4 45

*Eriobotrya japonica* Loquat 0 2 10 0 50 3

*Gleditsia triacanthos* Honey locust 5 2 10 1 68 2

*Mangifera indica* Mango 0 1 10 0 46 1

*Passiflora edulis* Passion fruit 0 2 10 1 50 1

*Senna bicapsularis* Rambling cassia 5 0 10 1 62 3

*Syzygium cumini* Jambolan 5 1 10 0 66 3 *Syzygium jambos* Rose apple 5 1 10 0 66 3

*Ulmus parvifolia* Chinese elm 0 0 10 5 46

*Canna* x *generalis* Garden canna 5 1 10 10 72

Riparian–large *Canna indica* Indian shot 5 2 10 10 79 1

*Ficus carica* Fig 0 2 10 0 50

*Montanoa hibiscifolia* Tree daisy 0 1 10 1 44

*Passiflora subpeltata* Granadina 0 1 10 1 46 *Physalis peruviana* Cape gooseberry 0 2 10 5 54 *Phytolacca octandra* Forest inkberry 0 2 10 6 55

Montpellier broom

**relatives**

5 0 10 4 66 1

5 1 10 8 73 3

**Combined Score**

http://dx.doi.org/10.5772/56182

**CARA category** 275


**Habitat– propagule pool size**

> *Hedychium coronarium*

274 Herbicides - Current Research and Case Studies in Use

*Hedychium gardnerianum*

*Myoporum tenuifolium* ssp. *montanum*

*Syzygium paniculatum*

*Anacardium occidentale*

*Callistemon rigidus* Sitt-

*Catharanthus roseus* Madagascar

*Cynodon nlemfuensis* East African

Moderate– moderate

*Ligustrum japonicum* Japanese wax-

*Ligustrum lucidum* Chinese wax-

*Lonicera japonica* Japanese

leaved privet

leaved privet

honeysuckle

Australian water

leavedbottlebrus

periwinkle

couch

h

pear

**Scientific name Common name Impact Weediness Biocontrol % Weedy**

**relatives**

White ginger lily 10 2 10 1 87 1

Kahili ginger lily 10 3 10 1 92 1

*Ligustrum ovalifolium* Californian privet 5 1 10 3 68 3 *Ligustrum sinense* Chinese privet 5 4 10 3 80 3

*Myoporum serratum* Manatoka 5 0 10 2 84 3

*Nephrolepis exaltata* Sword fern 10 0 10 3 82 1

*Spartium junceum* Spanish broom 5 3 10 10 82 1

*Albizia procera* False lebbeck 5 1 10 2 64 1

*Alhagi maurorum* Camelthorn bush 5 2 10 10 79 11

*Cestrum parqui* Chilean cestrum 10 3 10 1 91 1

Cashew nut 5 1 10 1 63

*Pyracantha coccinea* Red firethorn 5 0 10 8 61

Manatoka 5 0 10 2 69

5 0 10 0 61

0 2 10 3 51

5 2 10 10 76

5 1 10 3 66 3

5 4 10 3 78 3

5 6 10 1 83 Proposed

0 1 10 1 45 Proposed

**Combined Score**

**CARA category**


The majority of invasive and/or encroaching species in rangelands is dominated by the genus *Acacia,* which is the second largest with over 900 species [70]. Australian acacias are important invaders of South African rangeland areas [94]. In the fynbos ecosystems where soil nutrients are generally poor, the invasion by nitrogen-fixing acacias increases nitrogen inputs, and subsequently leads to an increase in soil fertility. Therefore, the massive increase in soil fertility permits acacia species to propagate and outcompete indigenous species [90]. There are a number of *acacia* species found in rangelands and their ability to fix nitrogen has been widely reported; these include *Acacia cyclops*, *A. dealbata*, *A. mearnsii* and *A. saligna* [90, 95]. The

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

concentrations compared to groundwater in natural ecosystems [94]. The presence of *A. saligna*, as well as the nutrient leaching that occurred after its removal, result in seasonal nitrogen concentrations that are higher than the water quality targets for domestic use (NOx < 6 mg/l) [94, 96]. Therefore, the removal of alien plants would be beneficial from both a water

In natural communities, plants compete in different ways; one of these ways is chemical interactions in the form of allelopathy [87, 97]. Invasive plants interfere with other plants by releasing allelochemicals into the environment and that negatively affects surrounding plants, thus giving the producer a competitive advantage. Invasive plants possess physiological traits that enable them to exploit ecological opportunities. The word allelopathy comes from the Latin words *allelon,* which means of each other and *pathos,* which means to suffer, which is commonly associated with the chemical inhibition of one species of plants by another [98]. Allelopathy is the process through which invasive plants such as *eucalyptu*s, *Pinus*, *Chromo‐ laena* and *Lantana* produce biochemicals that influence the growth, survival, and reproduction of indigenous species. However, it is important to note that most of the plant species naturally produce number of allelopathic substances such as monoterpenes and phenols [97]. Phenolics and volatile compounds can be released from eucalyptus foliage. These biochemicals can act

Although it has not been evaluated, the impacts of allelochemicals may subsequently influence water quality through soil erosion or surface runoff processes [70]. Allelochemicals are believed to be present in almost all plant tissues such as leaves, flowers, fruits, stems, roots, rhizomes, seeds, and pollen where they may be released from plants into the environment by means of volatilization, leaching, root exudation, and decomposition of plant residues [99, 100]. Invasive plants use the mechanism of allelopathy to outcompete other plants [87]. Allelochemicals can be found present in litter and on the soil surface where plants grow. Rain assists with the leaching of allelopathic substances into the soil, where they may affect the germination and growth of other plants [97, 101]. Allelopathic substances might play a role in shaping plant community structure in semi-arid and arid environments [97]. Thus, allelopathic substances inhibit plant growth depending on the concentration, leachability, season, and age of the plants [101]. Phytotoxins can persist in the soil and litter layer for long after allelopathic plants senesce, thereby reducing the establishment potential of an area. Allelopathic substan‐ ces can be present in the soil and often determined by a number of important factors [97]. These factors include the density at which the leaves fall, the rate at which this material decomposes,


http://dx.doi.org/10.5772/56182

and NO2


277

groundwater on places that were invaded by *A. saligna* has shown elevated NO3

quantity as well as water quality perspective [94].

as antibiotics in certain soils, possibly affecting nitrogen cycles.

N. B: Scores for 'Impact', 'Weediness', Biocontrol' and 'Weedy relatives' are standardized by dividing the maximum score for that criterion and multiplying by 10. Scores for these four criteria were weighted, with 'Impact', 'Weediness' and Biocontrol' receiving an equal weight‐ ing of four, and 'Weedy relatives' receiving a lower weighting of one. The weighted criteria were summed to obtain the 'Combined score' for each species. 'CARA category' lists the species regulated by the Conservation of Agricultural Resources Act (Act 43 of 1983), where 1 refers to Category 1 prohibited weeds that must be controlled in all situations; 2 includes Category 2 plants with commercial value that may be planted in demarcated areas subject to a permit, provided that steps are taken to control spread; 3 includes Category 3 ornamen‐ tal plants that may no longer be planted or traded, but may remain in place provided a permit is obtained and steps taken to control their spread; and 'proposed' includes those species that were proposed for listing under the Conservation of Agricultural Resources Act, but require further investigation before they can be included.

**Table 2.** Emerging invaders grouped according to categories (Source: [80])

The majority of invasive and/or encroaching species in rangelands is dominated by the genus *Acacia,* which is the second largest with over 900 species [70]. Australian acacias are important invaders of South African rangeland areas [94]. In the fynbos ecosystems where soil nutrients are generally poor, the invasion by nitrogen-fixing acacias increases nitrogen inputs, and subsequently leads to an increase in soil fertility. Therefore, the massive increase in soil fertility permits acacia species to propagate and outcompete indigenous species [90]. There are a number of *acacia* species found in rangelands and their ability to fix nitrogen has been widely reported; these include *Acacia cyclops*, *A. dealbata*, *A. mearnsii* and *A. saligna* [90, 95]. The groundwater on places that were invaded by *A. saligna* has shown elevated NO3 and NO2 concentrations compared to groundwater in natural ecosystems [94]. The presence of *A. saligna*, as well as the nutrient leaching that occurred after its removal, result in seasonal nitrogen concentrations that are higher than the water quality targets for domestic use (NOx < 6 mg/l) [94, 96]. Therefore, the removal of alien plants would be beneficial from both a water quantity as well as water quality perspective [94].

**Habitat– propagule pool size**

> *Casuarina cunninghamiana*

276 Herbicides - Current Research and Case Studies in Use

*Eucalyptus microtheca*

*Myriophyllum spicatum*

*Oenothera glazioviana*

*Mimosa pigra* Giant sensitive

*Oenothera jamesii* Giant evening

*Oenothera laciniata* Cutleaf evening

*Oenothera tetraptera* White evening

*Grevillea robusta* Australian silky

require further investigation before they can be included.

oak

**Table 2.** Emerging invaders grouped according to categories (Source: [80])

plant

Spiked watermilfoil

primrose

primrose

primrose

**Scientific name Common name Impact Weediness Biocontrol % Weedy**

**relatives**

Beefwood 5 1 10 4 69 2

*Cortaderia jubata* Purple Pampas 5 3 10 2 75 1 *Cortaderia selloana* Pampas grass 5 5 10 2 81 1

*Populus deltoides* Match poplar Proposed

Coolabah 0 0 10 2 42

Evening primrose 5 2 10 4 72

5 4 10 1 76 3

5 4 10 3 80 1

5 0 10 4 64

5 1 10 4 67

5 0 10 4 66

5 2 10 0 67 3

*Oenothera biennis* Evening primrose 5 1 10 4 67

*Oenothera indecora* Evening primrose 5 1 10 4 68

*Parkinsonia aculeata* Jerusalem thorn 5 1 10 0 66

*Quercus robur* English oak 5 1 10 1 67

N. B: Scores for 'Impact', 'Weediness', Biocontrol' and 'Weedy relatives' are standardized by dividing the maximum score for that criterion and multiplying by 10. Scores for these four criteria were weighted, with 'Impact', 'Weediness' and Biocontrol' receiving an equal weight‐ ing of four, and 'Weedy relatives' receiving a lower weighting of one. The weighted criteria were summed to obtain the 'Combined score' for each species. 'CARA category' lists the species regulated by the Conservation of Agricultural Resources Act (Act 43 of 1983), where 1 refers to Category 1 prohibited weeds that must be controlled in all situations; 2 includes Category 2 plants with commercial value that may be planted in demarcated areas subject to a permit, provided that steps are taken to control spread; 3 includes Category 3 ornamen‐ tal plants that may no longer be planted or traded, but may remain in place provided a permit is obtained and steps taken to control their spread; and 'proposed' includes those species that were proposed for listing under the Conservation of Agricultural Resources Act, but

Small–large *Alpinia zerumbet* Shell ginger 5 0 10 0 62

**Combined Score**

**CARA category**

> In natural communities, plants compete in different ways; one of these ways is chemical interactions in the form of allelopathy [87, 97]. Invasive plants interfere with other plants by releasing allelochemicals into the environment and that negatively affects surrounding plants, thus giving the producer a competitive advantage. Invasive plants possess physiological traits that enable them to exploit ecological opportunities. The word allelopathy comes from the Latin words *allelon,* which means of each other and *pathos,* which means to suffer, which is commonly associated with the chemical inhibition of one species of plants by another [98]. Allelopathy is the process through which invasive plants such as *eucalyptu*s, *Pinus*, *Chromo‐ laena* and *Lantana* produce biochemicals that influence the growth, survival, and reproduction of indigenous species. However, it is important to note that most of the plant species naturally produce number of allelopathic substances such as monoterpenes and phenols [97]. Phenolics and volatile compounds can be released from eucalyptus foliage. These biochemicals can act as antibiotics in certain soils, possibly affecting nitrogen cycles.

> Although it has not been evaluated, the impacts of allelochemicals may subsequently influence water quality through soil erosion or surface runoff processes [70]. Allelochemicals are believed to be present in almost all plant tissues such as leaves, flowers, fruits, stems, roots, rhizomes, seeds, and pollen where they may be released from plants into the environment by means of volatilization, leaching, root exudation, and decomposition of plant residues [99, 100]. Invasive plants use the mechanism of allelopathy to outcompete other plants [87]. Allelochemicals can be found present in litter and on the soil surface where plants grow. Rain assists with the leaching of allelopathic substances into the soil, where they may affect the germination and growth of other plants [97, 101]. Allelopathic substances might play a role in shaping plant community structure in semi-arid and arid environments [97]. Thus, allelopathic substances inhibit plant growth depending on the concentration, leachability, season, and age of the plants [101]. Phytotoxins can persist in the soil and litter layer for long after allelopathic plants senesce, thereby reducing the establishment potential of an area. Allelopathic substan‐ ces can be present in the soil and often determined by a number of important factors [97]. These factors include the density at which the leaves fall, the rate at which this material decomposes,

the distance from other plants and, finally, rainfall [101, 102, 103]. Phenolics signify the main allelopathic compounds that inhibit seed germination, plant growth and other physiological processes that result in changes of floristic composition within a plant community.

However, all extracts, except the one obtained from the leaves of *E*. *tomentosa* significantly inhibited the germination of lettuce seed and appeared to stunt the growth of roots and shoots

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

http://dx.doi.org/10.5772/56182

279

There are different allelochemicals exuded by invasive plants; these may have direct and indirect effects on germination and establishment of native species. However, phenolics are widely recognized for their allelopathic potential in plants, and can be found in a variety of tissues. Phytotoxic activity of allelochemicals in soil has been considered as plant-to-plant interaction, which is mediated by chemicals released from the plants [99]. Indirect effects of allelochemicals include its influence on the availability of nutrients in the soil, which may cause changes in soil chemical characteristics [110]. Allelochemicals might inhibit the growth of nitrifying bacteria, which would decrease N-availability at the plant level [111]. Additionally, chemical compounds produced in the process of litter decomposition are inhibitory for both heterotrophic and autotrophic bacteria and fungi [110,111] and, thus, rates of mineralization may be reduced. Allelochemicals such as phenolic acids are considered to have an important influence on nutrient cycling in terrestrial ecosystems [110]. The allelochemicals can produce some changes in the resource exploitation competition in such way that allelochemicals affect the mycorrhizae that allow the plant to absorb the nutrients, which leads to decrease in the soil productivity [106, 112]. Soil microorganisms are affected by root exudates that eventually affect other plant roots. Some chaparral species produce substances, which accumulate on the soil surface and make the soil less wettable [111]. The allelochemicals affect availability and accumulation of inorganic ions, although their activities are influenced by ecological factors

such as nutrient limitation, light regime and soil moisture deficiency [106].

Allelochemicals, such as phenolics and terpenoids, play an important role in the inhibition of nitrification and, thus, influence soil productivity of a plant community [113]. Thus, any influence on nutrient dynamics may ultimately affect the growth of plants in the community, which will lead to the increase of invasive plants. Reduced soil fertility may enhance the production of allelochemicals from invasive plants [106]. The addition of plant litter to soil may influence nutrient mobilization and soil pH, which can further influence nutrient immobilization and microbial activity [114]. Therefore, litter can alter the chemistry of the soil in such a way that it inhibits germination of other plants [106]. Chemicals released into the environment by a plant may not necessarily have direct effects on community structure but abiotic soil factors can influence these chemicals. Many phenolic acids have potential to influence microbial population, cause a shift in the microbial community, and eventually affect soil productivity of the area [106]. The soil microflora is directly responsible for decomposition and mineralisation processes and soil fauna is of considerable importance in regulating these processes through influencing the growth and activity of soil microbes [115]. Allelochemicals exuded from roots of invasive plants and residue decomposition play an important role in inhibiting plant pathogens particularly those borne in soil [116]. However, amended soils with allelopathic residues tend to be rich in organic matter [117]. Electrical conductivity (EC) of the amended soils increased as compared to the control and all nutrients were significantly more [117]. Although, earlier reports show that inclusion of plant litter, in addition to releasing putative phytotoxins into the soil medium, alters the soil nutrient dynamics and, thus, affects

of germinants [97].

Competition between plants can lead to the allelopathic inhibition of germination or growth via phytotoxic chemical releases, which are caused by competing species. However, allelop‐ athy can be extremely difficult to demonstrate in the field due to difficulties in differentiating allelopathic effects from resource competition [87, 99]. Allelochemical compounds are in fact released into the soil and accumulate to levels of toxicity, which leads to inhibition of germi‐ nation [100]. Allelochemicals released by invasive plants may affect native plant survival and production in a number of ways. These include the modification of the soil microbiota [74, 104], and enhancement of growth of beneficial microbes in their rhizosphere leading to an establishment of positive feedbacks that can contribute to the decrease of native biodiversity [74]. Allelochemicals are further known to inhibit absorption of ions [105]. Other than allelo‐ pathic effects, invasive plants exert competition of resource especially through light [87]. Therefore, allelopathy and resource competition operate simultaneously influencing each other and, in the meantime, they are influencing plant community structure [106].

Allelochemicals, as soon as released into the soil, may inhibit germination, shoot, and root growth of other plants, which will affect nutrient uptake thereby destroying the plant's usable source of nutrients [107]. Allelopathy of invasive plants delays the germination and growth of seedlings of other species and eventually hinders their growth completely. Therefore, degree of inhibition due to allelopathy is largely dependent on the concentration of the extracts and, to a lesser extent, on the species from which they were derived [101, 108]. The effects of allelopathy on germination and growth of plants occur through a variety of mechanisms including reduced mitotic activity in roots and hypocotyls, suppressed hormone activity, reduced rate of ion uptake, inhibited photosynthesis, and respiration, inhibit protein forma‐ tion, decreased permeability of cell membranes and/or inhibition of enzyme action [97]. Plants that germinate at slower rates are often smaller; thereby, this may seriously influence their chances of competing with neighbouring plants for resources such as water [109]. Indirectly, allelopathic effects of invasive species on germination and growth of native species determine their competitive ability against them [97]. The roots of *Aloe ferox* have allelopathic inhibition on tomato seed germination [97]. Accumulation of allelochemicals in the rhizosphere because of root and microbial exudates and/or metabolism may affect the germination. However, under arid conditions germination will be less affected since microbial activities are very low due to low availability of soil moisture [101]. The effects of allelochemicals on the root growth are due to cell division destruction [105]. *L. maackii* also exudes allelopathic compounds from its leaves or roots that inhibit germination and growth of species that grow on the same site [87]. Allelochemicals could be found on any part of the plant; however, the concentration varies with plant parts. The leaf extracts of *L. maackii* appeared to have a more negative effect on seed germination than root extracts [87]. Generally, leaf extract concentrations have a stronger effect on germination of seeds of other plants [87]. However, it is important to note that allelopathic chemicals from one plant can hinder germination of seeds of the same plant. For example, chenopod seed germination can also be inhibited by extracts generated from its leaves [97]. However, all extracts, except the one obtained from the leaves of *E*. *tomentosa* significantly inhibited the germination of lettuce seed and appeared to stunt the growth of roots and shoots of germinants [97].

the distance from other plants and, finally, rainfall [101, 102, 103]. Phenolics signify the main allelopathic compounds that inhibit seed germination, plant growth and other physiological

Competition between plants can lead to the allelopathic inhibition of germination or growth via phytotoxic chemical releases, which are caused by competing species. However, allelop‐ athy can be extremely difficult to demonstrate in the field due to difficulties in differentiating allelopathic effects from resource competition [87, 99]. Allelochemical compounds are in fact released into the soil and accumulate to levels of toxicity, which leads to inhibition of germi‐ nation [100]. Allelochemicals released by invasive plants may affect native plant survival and production in a number of ways. These include the modification of the soil microbiota [74, 104], and enhancement of growth of beneficial microbes in their rhizosphere leading to an establishment of positive feedbacks that can contribute to the decrease of native biodiversity [74]. Allelochemicals are further known to inhibit absorption of ions [105]. Other than allelo‐ pathic effects, invasive plants exert competition of resource especially through light [87]. Therefore, allelopathy and resource competition operate simultaneously influencing each

processes that result in changes of floristic composition within a plant community.

278 Herbicides - Current Research and Case Studies in Use

other and, in the meantime, they are influencing plant community structure [106].

Allelochemicals, as soon as released into the soil, may inhibit germination, shoot, and root growth of other plants, which will affect nutrient uptake thereby destroying the plant's usable source of nutrients [107]. Allelopathy of invasive plants delays the germination and growth of seedlings of other species and eventually hinders their growth completely. Therefore, degree of inhibition due to allelopathy is largely dependent on the concentration of the extracts and, to a lesser extent, on the species from which they were derived [101, 108]. The effects of allelopathy on germination and growth of plants occur through a variety of mechanisms including reduced mitotic activity in roots and hypocotyls, suppressed hormone activity, reduced rate of ion uptake, inhibited photosynthesis, and respiration, inhibit protein forma‐ tion, decreased permeability of cell membranes and/or inhibition of enzyme action [97]. Plants that germinate at slower rates are often smaller; thereby, this may seriously influence their chances of competing with neighbouring plants for resources such as water [109]. Indirectly, allelopathic effects of invasive species on germination and growth of native species determine their competitive ability against them [97]. The roots of *Aloe ferox* have allelopathic inhibition on tomato seed germination [97]. Accumulation of allelochemicals in the rhizosphere because of root and microbial exudates and/or metabolism may affect the germination. However, under arid conditions germination will be less affected since microbial activities are very low due to low availability of soil moisture [101]. The effects of allelochemicals on the root growth are due to cell division destruction [105]. *L. maackii* also exudes allelopathic compounds from its leaves or roots that inhibit germination and growth of species that grow on the same site [87]. Allelochemicals could be found on any part of the plant; however, the concentration varies with plant parts. The leaf extracts of *L. maackii* appeared to have a more negative effect on seed germination than root extracts [87]. Generally, leaf extract concentrations have a stronger effect on germination of seeds of other plants [87]. However, it is important to note that allelopathic chemicals from one plant can hinder germination of seeds of the same plant. For example, chenopod seed germination can also be inhibited by extracts generated from its leaves [97].

There are different allelochemicals exuded by invasive plants; these may have direct and indirect effects on germination and establishment of native species. However, phenolics are widely recognized for their allelopathic potential in plants, and can be found in a variety of tissues. Phytotoxic activity of allelochemicals in soil has been considered as plant-to-plant interaction, which is mediated by chemicals released from the plants [99]. Indirect effects of allelochemicals include its influence on the availability of nutrients in the soil, which may cause changes in soil chemical characteristics [110]. Allelochemicals might inhibit the growth of nitrifying bacteria, which would decrease N-availability at the plant level [111]. Additionally, chemical compounds produced in the process of litter decomposition are inhibitory for both heterotrophic and autotrophic bacteria and fungi [110,111] and, thus, rates of mineralization may be reduced. Allelochemicals such as phenolic acids are considered to have an important influence on nutrient cycling in terrestrial ecosystems [110]. The allelochemicals can produce some changes in the resource exploitation competition in such way that allelochemicals affect the mycorrhizae that allow the plant to absorb the nutrients, which leads to decrease in the soil productivity [106, 112]. Soil microorganisms are affected by root exudates that eventually affect other plant roots. Some chaparral species produce substances, which accumulate on the soil surface and make the soil less wettable [111]. The allelochemicals affect availability and accumulation of inorganic ions, although their activities are influenced by ecological factors such as nutrient limitation, light regime and soil moisture deficiency [106].

Allelochemicals, such as phenolics and terpenoids, play an important role in the inhibition of nitrification and, thus, influence soil productivity of a plant community [113]. Thus, any influence on nutrient dynamics may ultimately affect the growth of plants in the community, which will lead to the increase of invasive plants. Reduced soil fertility may enhance the production of allelochemicals from invasive plants [106]. The addition of plant litter to soil may influence nutrient mobilization and soil pH, which can further influence nutrient immobilization and microbial activity [114]. Therefore, litter can alter the chemistry of the soil in such a way that it inhibits germination of other plants [106]. Chemicals released into the environment by a plant may not necessarily have direct effects on community structure but abiotic soil factors can influence these chemicals. Many phenolic acids have potential to influence microbial population, cause a shift in the microbial community, and eventually affect soil productivity of the area [106]. The soil microflora is directly responsible for decomposition and mineralisation processes and soil fauna is of considerable importance in regulating these processes through influencing the growth and activity of soil microbes [115]. Allelochemicals exuded from roots of invasive plants and residue decomposition play an important role in inhibiting plant pathogens particularly those borne in soil [116]. However, amended soils with allelopathic residues tend to be rich in organic matter [117]. Electrical conductivity (EC) of the amended soils increased as compared to the control and all nutrients were significantly more [117]. Although, earlier reports show that inclusion of plant litter, in addition to releasing putative phytotoxins into the soil medium, alters the soil nutrient dynamics and, thus, affects the plant growth [106, 112, 116]. A similar increase in electrical conductivity of the soil incorporated with residues of allelopathic plants was reported [118]. In fact, the behaviour of the allelopathic compounds present in soil remains unclear [119].

been observed on the respiration of the plants which affect oxygen absorption capacity [127], eventually inhibit photosynthesis by reducing the chlorophyll content which affect photosynthesis rate [98, 112, 126]. There is an inhibition of the activity of hydroxyphenyl‐ pyruvate dioxygenase (HPPD) enzyme due to isoxaflutole, which results in the inhibition of meristmatic tissue, which leads to inhibition of shoot growth [126]. Therefore, the modes of action of most allelochemicals and phytotoxins are complex and are not clearly

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The active compound or compounds must be isolated in an amount adequate for identification and for further characterisation in bioassays [110]. Screening of fractions of plant extracts or leachates for their effects on seed germination of various plant species are frequently used to identify phytotoxic compounds [110]. The identification of an active phytotoxic compound from a suspected allelopathic plant does not establish that this is the only compound involved in allelopathy. The release of allelochemicals of different chemical classes from allelopathic plant species has been documented including tannins, cyanogenic glycosides, several flavo‐ noids and phenolic acids [129]. The most clearly identified compounds can be divided into four groups: phenolic acids, hydroxamic acids, alkaloids, and quinones. In the study of allelopathy, plants are identified based on the allelochemical release [120]. Most studies utilized some parts of the plants such as roots, leaves and leaves plus stem to establish the

Rangelands contribute to the economy of Southern Africa in a number of ways. They provide agricultural commodities that can be valued in the market such as wool, meat, milk etc. These are the major source of forage for grazing animal which in turn influence animal production. Rangelands further provide benefits that, are not directly related to the agricultural sector, such as wildlife habitat, however, have an impact on the economy through activities that make use of them [130]. Increases in the density of woody plants worldwide are a major threat to livestock production [13, 131], and rangeland biodiversity. Invasive species pose problems for managers of rangelands because they reduce the land's usefulness for grazing activities. In addition, they interfere with other non-agricultural functions that rangelands provide, such as acreage of wildlife habitat and watershed quality. Therefore, in order to realise the impact of invasion on rangelands, it is important to understand the total economic loss that invasive plant infestations create on the economy in relation to both its agricultural and non-agricultural

Economic impact of invasive species could be defined as the product of a species' range, abundance and per capita [36, 80, 132]. Although the invasive plants have an ecological implication they also have some economic implications; these could be either positive or negative. Species such as *Acacia mearnsii* (Black wattle) are highly invasive and have spread over an area of almost 2.5 million ha in South Africa [133]. It has significant negative impacts on water resources, biodiversity, and the stability and integrity of riparian ecosystems [8]. These two features, a commercial value on the one hand, and an invasive, damaging ability on the other, give rise to a classic conflict of interests, where the benefits accrue to a number

existence of allelochemicals on the identified plants [107, 128].

understood [126].

**3.2. Economic impacts**

products of the ecosystems [130].

The modes of release of the allelopathic compounds are not specific because they vary from plant to plant [120]. Thus, allelochemicals are released into the environment by root exudation, leaching from aboveground parts, volatilisation, and decomposition of plant material and ultimately enter into the soil [99, 110, 121]. Therefore, allelochemicals may reach other plants through transport such as root exudates into the soil and may induce the inhibitory activity on the other plants. The behaviour of allelochemicals in soil is run by the physicochemical properties including soil organic matter and organisms [99]. The model that has assumptions such as "allelochemicals are released into the soil from living plants and degraded into non-allelopathic substances was developed. Therefore, rate of the release is proportional to the amount of allelochemicals in living plants and rate of al‐ lelochemicals degradation is proportional to the amount of allelochemicals released [121]. However, the soil microorganisms were also reported to produce and release allelochemi‐ cals [112]. The release of allelochemicals by mature shrubs may inhibit plant germination, survival or growth [111]. Allelopathic content of a plant varies according to its maturity [122]. Allelopathic compounds released from different plant parts can be either released continuously within specific periods such as specific developmental stages or influenced by external factors such as precipitation [123]. The synthesis and exudation of allelochem‐ icals via roots is usually enhanced by stress conditions that the plant encounters such as extreme temperature, drought, and ultraviolet exposure [124].

The visible effects of allelopathy frequently observed are inhibited or delayed seed germi‐ nation or reduced seedling growth. The diversity of structure among allelochemicals sug‐ gests that they have no common mode of action [110]. Plant exudates can also have an indirect effect on the surrounding environment and reduce neighbouring plant germina‐ tion or growth, independent of toxicity [111]. Allelopathic activities are more pronounced when allelopathic potential species grow under water stress [125]. Phenolic acids that were tested had a similar mode of action such as inhibition of nutrient uptake by roots of plants [126]. In most cases, various allelochemicals take action as growth regulators by in‐ hibiting growth and changing development [112]. The common mode of action of allelo‐ chemicals is quite related to the membrane destruction [126]. It was discovered that allelochemicals affect plants on cell division, cell elongation, cell structure, cell wall, ultrastructure of the cell [112, 127]. Phenolic allelochemicals can also lead to increased cell membrane permeability; cell contents spill which lead to the increase of lipid peroxida‐ tion, and eventually, slow growth or death of plant tissue occurs [112, 126, 127]. Further‐ more, nutrient uptake can be affected negatively by allelochemicals. This occurs when these allelochemicals inhibit nutrient absorption of the plant [127]. The mode of action of benzoic acid involved the inhibition of nutrient uptake by plant roots, which resulted in growth inhibition [126]. The radicle elongation was significantly reduced by the extract of leaves, and leaves and stem at the three concentrations of *Acacia mearnsii*, which signifies that *A. mearnsii* has allelopathic potential [128]. The impact of allelochemicals also have been observed on the respiration of the plants which affect oxygen absorption capacity [127], eventually inhibit photosynthesis by reducing the chlorophyll content which affect photosynthesis rate [98, 112, 126]. There is an inhibition of the activity of hydroxyphenyl‐ pyruvate dioxygenase (HPPD) enzyme due to isoxaflutole, which results in the inhibition of meristmatic tissue, which leads to inhibition of shoot growth [126]. Therefore, the modes of action of most allelochemicals and phytotoxins are complex and are not clearly understood [126].

The active compound or compounds must be isolated in an amount adequate for identification and for further characterisation in bioassays [110]. Screening of fractions of plant extracts or leachates for their effects on seed germination of various plant species are frequently used to identify phytotoxic compounds [110]. The identification of an active phytotoxic compound from a suspected allelopathic plant does not establish that this is the only compound involved in allelopathy. The release of allelochemicals of different chemical classes from allelopathic plant species has been documented including tannins, cyanogenic glycosides, several flavo‐ noids and phenolic acids [129]. The most clearly identified compounds can be divided into four groups: phenolic acids, hydroxamic acids, alkaloids, and quinones. In the study of allelopathy, plants are identified based on the allelochemical release [120]. Most studies utilized some parts of the plants such as roots, leaves and leaves plus stem to establish the existence of allelochemicals on the identified plants [107, 128].

#### **3.2. Economic impacts**

the plant growth [106, 112, 116]. A similar increase in electrical conductivity of the soil incorporated with residues of allelopathic plants was reported [118]. In fact, the behaviour of

The modes of release of the allelopathic compounds are not specific because they vary from plant to plant [120]. Thus, allelochemicals are released into the environment by root exudation, leaching from aboveground parts, volatilisation, and decomposition of plant material and ultimately enter into the soil [99, 110, 121]. Therefore, allelochemicals may reach other plants through transport such as root exudates into the soil and may induce the inhibitory activity on the other plants. The behaviour of allelochemicals in soil is run by the physicochemical properties including soil organic matter and organisms [99]. The model that has assumptions such as "allelochemicals are released into the soil from living plants and degraded into non-allelopathic substances was developed. Therefore, rate of the release is proportional to the amount of allelochemicals in living plants and rate of al‐ lelochemicals degradation is proportional to the amount of allelochemicals released [121]. However, the soil microorganisms were also reported to produce and release allelochemi‐ cals [112]. The release of allelochemicals by mature shrubs may inhibit plant germination, survival or growth [111]. Allelopathic content of a plant varies according to its maturity [122]. Allelopathic compounds released from different plant parts can be either released continuously within specific periods such as specific developmental stages or influenced by external factors such as precipitation [123]. The synthesis and exudation of allelochem‐ icals via roots is usually enhanced by stress conditions that the plant encounters such as

The visible effects of allelopathy frequently observed are inhibited or delayed seed germi‐ nation or reduced seedling growth. The diversity of structure among allelochemicals sug‐ gests that they have no common mode of action [110]. Plant exudates can also have an indirect effect on the surrounding environment and reduce neighbouring plant germina‐ tion or growth, independent of toxicity [111]. Allelopathic activities are more pronounced when allelopathic potential species grow under water stress [125]. Phenolic acids that were tested had a similar mode of action such as inhibition of nutrient uptake by roots of plants [126]. In most cases, various allelochemicals take action as growth regulators by in‐ hibiting growth and changing development [112]. The common mode of action of allelo‐ chemicals is quite related to the membrane destruction [126]. It was discovered that allelochemicals affect plants on cell division, cell elongation, cell structure, cell wall, ultrastructure of the cell [112, 127]. Phenolic allelochemicals can also lead to increased cell membrane permeability; cell contents spill which lead to the increase of lipid peroxida‐ tion, and eventually, slow growth or death of plant tissue occurs [112, 126, 127]. Further‐ more, nutrient uptake can be affected negatively by allelochemicals. This occurs when these allelochemicals inhibit nutrient absorption of the plant [127]. The mode of action of benzoic acid involved the inhibition of nutrient uptake by plant roots, which resulted in growth inhibition [126]. The radicle elongation was significantly reduced by the extract of leaves, and leaves and stem at the three concentrations of *Acacia mearnsii*, which signifies that *A. mearnsii* has allelopathic potential [128]. The impact of allelochemicals also have

the allelopathic compounds present in soil remains unclear [119].

280 Herbicides - Current Research and Case Studies in Use

extreme temperature, drought, and ultraviolet exposure [124].

Rangelands contribute to the economy of Southern Africa in a number of ways. They provide agricultural commodities that can be valued in the market such as wool, meat, milk etc. These are the major source of forage for grazing animal which in turn influence animal production. Rangelands further provide benefits that, are not directly related to the agricultural sector, such as wildlife habitat, however, have an impact on the economy through activities that make use of them [130]. Increases in the density of woody plants worldwide are a major threat to livestock production [13, 131], and rangeland biodiversity. Invasive species pose problems for managers of rangelands because they reduce the land's usefulness for grazing activities. In addition, they interfere with other non-agricultural functions that rangelands provide, such as acreage of wildlife habitat and watershed quality. Therefore, in order to realise the impact of invasion on rangelands, it is important to understand the total economic loss that invasive plant infestations create on the economy in relation to both its agricultural and non-agricultural products of the ecosystems [130].

Economic impact of invasive species could be defined as the product of a species' range, abundance and per capita [36, 80, 132]. Although the invasive plants have an ecological implication they also have some economic implications; these could be either positive or negative. Species such as *Acacia mearnsii* (Black wattle) are highly invasive and have spread over an area of almost 2.5 million ha in South Africa [133]. It has significant negative impacts on water resources, biodiversity, and the stability and integrity of riparian ecosystems [8]. These two features, a commercial value on the one hand, and an invasive, damaging ability on the other, give rise to a classic conflict of interests, where the benefits accrue to a number

**4. Management of rangelands for bush encroachment and invasion**

Bush encroachment forms dense infestations that rapidly deplete soil moisture, preventing the establishment of other species. As it displaces native vegetation, it reduces wildlife habitat and ecosystem diversity, and suppresses production of nutritious, palatable forage for wildlife and livestock, which leads to a reduction in grazing and wildlife carrying capacity. Soil and water conservation benefits of the regions rangelands also decline; watershed quality declines in

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Bush encroachment is considered a threat to forage production, which is the feed for the grazing livestock [42]. The threat to the pastoral economy by bush encroachment and invasion is often the main reason for the control of bush encroachment [136]. Bush encroachment control is a disturbance that reduces the threat of bush encroachment by disrupting the invasive woody plant community structure through transformations of biotic environments and habitat conditions in which colonization of the disturbed microhabitat takes place. Bush control methods shift the rangeland vegetation from dominance by woody vegetation to dominance by herbaceous vegetation. This control of the bush is aimed at creating suitable habitat for grazers [137, 138]. Thus, forage production of herbaceous vegetation increases with reduction of woody species. The principle of bush encroachment control is based on the ability of the control method to shift the competition between desired and undesired species. Encroaching species have the higher competitive ability over the native species, which is why they colonise. They build up this competitive advantage by modifying the environment in such a way that growing conditions will suit their needs through a number of ways. These include release of chemical substances that suppresses germination and growth of their competitors (Allelop‐ athy) and modification of soil fertility in the case of acacias through higher nitrogen inputs, which in turn favours their growth. Encroaching species also impose competition for light and through shading and subsequently growth for native species becomes negatively affected. There is also a competition for soil moisture and soil nutrient; in this manner, most of the invasive plants win because of their deeper root systems. Other invasive species produce large numbers of seeds, which normally are dispersed faster, have a shorter dormant time before germination, and colonise. Invasive plants use one or a combination of these mechanisms for survival. Therefore, bush encroachment control reduces the ability of invasive plants to exhibit these survival mechanisms. The use of selective herbicides is aimed at reducing the competitive ability of invasive species through killing them and, in that, species that are not affected by this herbicide gain an advantage. Mechanical methods such as hand clearing targets unwanted plants and create a competitive space for desired plants, thus, without this clearing the invasive species are more competitive. Use of fire to control invasive woody plants is justified by the fact that when woody plants are burned they do not recover or they take a longer time to recover which gives the herbaceous species time to grow with minimal or no competition. In the biological control method, use of herbivores such as goats to selectively-browse on the encroaching species or use of invertebrates that feed on the seed of invading species also

**4.1. Bush encroachment control**

areas where the weeds have advanced [135].

reduces the competition against native plants.

**Figure 1.** Hypothetical flow chart indicating economic impact of bush encroachment in rangelands (Source [80]).

of people, while the society at large bears the external costs. Furthermore, there are larger reductions of water resulting from the presence and densities of invasive plants. Thus, the potential water reductions in South Africa would be more than 8 times greater if invasive alien plants were to occupy the full extent of their potential range [85]. These invasions come at a significant cost to the economy, estimated at about R6.5 billion per annum, which is about 0.3% of South Africa's GDP of around R 2 000 billion, and with potential to rise to > 5% of GDP if invasive plants were to be allowed to invade all of the suitable habitat [134]. Economic of bush encroachment in rangelands can be divided into agricultural and non-agricultural, direct and indirect impacts, and, further, into primary and secondary impacts (Figure 1). Economic impacts of plant invasions may be related to a decline in cattle carrying capacity (agricultural impact), wildlife carrying capacity, and watershed quality (non-agricultural impacts). Reduc‐ tions in cattle grazing outlays may account for the direct agricultural costs. In addition, economic impacts may be estimated as reductions in wildland-associated recreation expendi‐ tures and increases in expenditures to mitigate damages from runoff and soil erosion to account for the non-agricultural losses. These estimated losses are incorporated into an input– output model of economy to compute total (direct plus secondary) economic costs incurred due to the invasion of noxious weeds [130]. Secondary economic effects of bush encroachment include indirect and induced losses on the economy. Indirect losses are linked to economic sectors not necessarily directly affected by the infestations, but these sectors supply inputs needed by directly affected industries. Induced effects represent changes in household spending patterns, caused by changes in employment that the direct and indirect effects generate.

### **4. Management of rangelands for bush encroachment and invasion**

#### **4.1. Bush encroachment control**

of people, while the society at large bears the external costs. Furthermore, there are larger reductions of water resulting from the presence and densities of invasive plants. Thus, the potential water reductions in South Africa would be more than 8 times greater if invasive alien plants were to occupy the full extent of their potential range [85]. These invasions come at a significant cost to the economy, estimated at about R6.5 billion per annum, which is about 0.3% of South Africa's GDP of around R 2 000 billion, and with potential to rise to > 5% of GDP if invasive plants were to be allowed to invade all of the suitable habitat [134]. Economic of bush encroachment in rangelands can be divided into agricultural and non-agricultural, direct and indirect impacts, and, further, into primary and secondary impacts (Figure 1). Economic impacts of plant invasions may be related to a decline in cattle carrying capacity (agricultural impact), wildlife carrying capacity, and watershed quality (non-agricultural impacts). Reduc‐ tions in cattle grazing outlays may account for the direct agricultural costs. In addition, economic impacts may be estimated as reductions in wildland-associated recreation expendi‐ tures and increases in expenditures to mitigate damages from runoff and soil erosion to account for the non-agricultural losses. These estimated losses are incorporated into an input– output model of economy to compute total (direct plus secondary) economic costs incurred due to the invasion of noxious weeds [130]. Secondary economic effects of bush encroachment include indirect and induced losses on the economy. Indirect losses are linked to economic sectors not necessarily directly affected by the infestations, but these sectors supply inputs needed by directly affected industries. Induced effects represent changes in household spending patterns, caused by changes in employment that the direct and indirect effects

**Figure 1.** Hypothetical flow chart indicating economic impact of bush encroachment in rangelands (Source [80]).

282 Herbicides - Current Research and Case Studies in Use

generate.

Bush encroachment forms dense infestations that rapidly deplete soil moisture, preventing the establishment of other species. As it displaces native vegetation, it reduces wildlife habitat and ecosystem diversity, and suppresses production of nutritious, palatable forage for wildlife and livestock, which leads to a reduction in grazing and wildlife carrying capacity. Soil and water conservation benefits of the regions rangelands also decline; watershed quality declines in areas where the weeds have advanced [135].

Bush encroachment is considered a threat to forage production, which is the feed for the grazing livestock [42]. The threat to the pastoral economy by bush encroachment and invasion is often the main reason for the control of bush encroachment [136]. Bush encroachment control is a disturbance that reduces the threat of bush encroachment by disrupting the invasive woody plant community structure through transformations of biotic environments and habitat conditions in which colonization of the disturbed microhabitat takes place. Bush control methods shift the rangeland vegetation from dominance by woody vegetation to dominance by herbaceous vegetation. This control of the bush is aimed at creating suitable habitat for grazers [137, 138]. Thus, forage production of herbaceous vegetation increases with reduction of woody species. The principle of bush encroachment control is based on the ability of the control method to shift the competition between desired and undesired species. Encroaching species have the higher competitive ability over the native species, which is why they colonise. They build up this competitive advantage by modifying the environment in such a way that growing conditions will suit their needs through a number of ways. These include release of chemical substances that suppresses germination and growth of their competitors (Allelop‐ athy) and modification of soil fertility in the case of acacias through higher nitrogen inputs, which in turn favours their growth. Encroaching species also impose competition for light and through shading and subsequently growth for native species becomes negatively affected. There is also a competition for soil moisture and soil nutrient; in this manner, most of the invasive plants win because of their deeper root systems. Other invasive species produce large numbers of seeds, which normally are dispersed faster, have a shorter dormant time before germination, and colonise. Invasive plants use one or a combination of these mechanisms for survival. Therefore, bush encroachment control reduces the ability of invasive plants to exhibit these survival mechanisms. The use of selective herbicides is aimed at reducing the competitive ability of invasive species through killing them and, in that, species that are not affected by this herbicide gain an advantage. Mechanical methods such as hand clearing targets unwanted plants and create a competitive space for desired plants, thus, without this clearing the invasive species are more competitive. Use of fire to control invasive woody plants is justified by the fact that when woody plants are burned they do not recover or they take a longer time to recover which gives the herbaceous species time to grow with minimal or no competition. In the biological control method, use of herbivores such as goats to selectively-browse on the encroaching species or use of invertebrates that feed on the seed of invading species also reduces the competition against native plants.

It is important to mention that the shift towards herbaceous species dominance, in turn, may induce shifts in herbaceous species that tolerate bush cover and such species might decline in numbers [139]. The changes could cause partial or total reduction of plant biomass [140] by shifting vegetation structure and composition [141]. Furthermore, disturbance can produce changes in the life history strategies of individual species in response to intensities of disturb‐ ance forces [140] and the created micro-environmental conditions [142]. Although livestockforage production of rangelands may support removal of encroaching species to enhance forage production, it is important to note that bush encroachment control methods are management systems [137] that might have varied policy implications for bush control [143]. Therefore, understanding the potential role of different bush encroachment control methods for promoting herbaceous species composition requires recognition of the objectives of resource users and policymakers [144]. Thus, the intended ecosystem status is dependant of the functional characteristics of such an ecosystem.

or poison livestock (e.g. *Delphinium* spp., *Astragalus* spp., and *Amsinckia menziesii* var. *interme‐ dia*). One of the challenges of managing invasive species is that there is no particular life cycle typical to noxious weeds of rangelands reported [151]. Thus, noxious rangeland weeds can be annuals (e.g. *Centaurea soltitialis, Crupina vulgaris, Bromus tectorum*), biennials (e.g. *Carduus nutans, Conium maculatum, Onopordum acanthium*), long-lived herbaceous perennials (e.g. *Convolvulus arvensis, Centaurea maculosa, Cirsium arvense*), shrubs (e.g. *Gutierrezia* spp., *Artemisia tridentata*), or trees (e.g. *Juniperus* spp., *Prosopis glandulosa*). Although several plant families represent these species, the largest number of noxious species belongs to the Astere‐

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Effective rangeland management requires sound ecological data about the land being man‐ aged; however, obtaining such data is not sufficient to ensure the implementation of restoration practices by land users. Thus, rational decisions at the farm or community, regional and national levels, depend on researchers providing not only ecologically sound but also eco‐ nomical, effective alternatives for land use [152]. In addition, because natural resource depletion and recovery compound over time, it is necessary to assess the sustainability of management alternatives over decadal periods [153]. Furthermore, to determine the true advantage of restoration management, it is necessary to compare the benefits of changing management practices with the cost of not changing current practices, which, rather than maintaining productivity, may lead to loss of production through shifts in plant species

Chemical control methods are usually expensive to apply and should be considered only under specific circumstances. Thus, their nature are suited primarily to the initial thinning of bush at high density, where there is poor fuel load to support fire, where trees are above the browse line, where the bush is unacceptable to animals and where the herbicide is intended to selectively kill a specific plant [154]. However, herbicides can sometimes be used in follow up operations such as after fire where there is a need for pre-emergence herbicide application intended to kill the seedlings of a target plant in soil. Herbicides have been applied extensively on rangelands to reduce forbs that were considered undesirable, which have been assumed to lead to an increase in grass production and ultimately to an improvement in livestock performance [155]. Herbicides are the primary method of weed control in most rangeland systems [151]. In South Africa, there is a considerable effort taken by the government to address the negative impact of alien invading species on the natural and environmental resources of

Herbicides vary in their chemical properties, that make them vary more with their mode of action under different climatic and soil conditions, and they further vary in their methods of application and their effect on the ecosystems. There are two broad groups of herbicides used in rangelands. The first type is composed of the herbicides that are applied on the soil surface and are absorbed by the roots; these are the herbicides that are based on tebuthiuron, ethidi‐ muron or bromacil as their active ingredient [154]. The second group of herbicides is sprayed onto the plant and absorbed directly by the foliage and other above ground parts of the plants;

composition, accelerated soil erosion, and loss of biodiversity.

ceae (sunflower) family.

*4.2.2. Chemical — Herbicides*

the country [8].

#### **4.2. Bush encroachment management methods**

#### *4.2.1. Rangeland management practices*

Grazing management entails management of livestock and vegetation resources. The main livestock decisions made by farmers both in the commercial and communal areas are con‐ cerned with livestock type, number and seasonal pattern of movement [145]. Commercial and communal livestock farming are generally regarded as the rangeland management systems and they are distinct in grazing management practices. Thus, communal grazing areas are generally characterised by continuous grazing, which is perceived by most of the scientists to be the root cause of the often-reported land degradation in this system. On the other hand, commercial livestock farming is characterised by structured and objective grazing manage‐ ment practices such as assigning the correct livestock units in proportion to the carrying capacity of the land. These would be done in rotation to give vegetation in grazed areas time to recover such that the rested areas can be grazed again. Understanding the dynamics of bush encroachment in relation to rangeland management systems over a broad range of environ‐ ments is essential for sustainable management of rangeland ecosystems [146]. Although rangelands are complex ecosystems varying at multiple scales in time and space [147, 148], most management usually intends to maintain or enhance livestock production by reducing plant community variability in space and time [149, 150]. This is usually accomplished by promoting spatially uniform dominance of a few productive forage species. Although it is generally believed that improper grazing practices leading to overgrazing are responsible for bush encroachment, it is not attributed to heavy grazing alone, but is strongly influenced by seasonality, which is a characteristic of arid and semi-arid environments [42]. In combination with seasonality, the ban on fire and exclusion of browsing animals such as goats and camels may also contribute to the invasion of bush encroachment.

Rangeland management practices, particularly fire suppression and overgrazing, have been reported to increase the proportion of some native species [70]. These natives can reduce overall forage quality or quantity (e.g. *Juniperus* spp., *Artemisia tridentata*, and *Gutierrezia* spp.) or poison livestock (e.g. *Delphinium* spp., *Astragalus* spp., and *Amsinckia menziesii* var. *interme‐ dia*). One of the challenges of managing invasive species is that there is no particular life cycle typical to noxious weeds of rangelands reported [151]. Thus, noxious rangeland weeds can be annuals (e.g. *Centaurea soltitialis, Crupina vulgaris, Bromus tectorum*), biennials (e.g. *Carduus nutans, Conium maculatum, Onopordum acanthium*), long-lived herbaceous perennials (e.g. *Convolvulus arvensis, Centaurea maculosa, Cirsium arvense*), shrubs (e.g. *Gutierrezia* spp., *Artemisia tridentata*), or trees (e.g. *Juniperus* spp., *Prosopis glandulosa*). Although several plant families represent these species, the largest number of noxious species belongs to the Astere‐ ceae (sunflower) family.

Effective rangeland management requires sound ecological data about the land being man‐ aged; however, obtaining such data is not sufficient to ensure the implementation of restoration practices by land users. Thus, rational decisions at the farm or community, regional and national levels, depend on researchers providing not only ecologically sound but also eco‐ nomical, effective alternatives for land use [152]. In addition, because natural resource depletion and recovery compound over time, it is necessary to assess the sustainability of management alternatives over decadal periods [153]. Furthermore, to determine the true advantage of restoration management, it is necessary to compare the benefits of changing management practices with the cost of not changing current practices, which, rather than maintaining productivity, may lead to loss of production through shifts in plant species composition, accelerated soil erosion, and loss of biodiversity.

#### *4.2.2. Chemical — Herbicides*

It is important to mention that the shift towards herbaceous species dominance, in turn, may induce shifts in herbaceous species that tolerate bush cover and such species might decline in numbers [139]. The changes could cause partial or total reduction of plant biomass [140] by shifting vegetation structure and composition [141]. Furthermore, disturbance can produce changes in the life history strategies of individual species in response to intensities of disturb‐ ance forces [140] and the created micro-environmental conditions [142]. Although livestockforage production of rangelands may support removal of encroaching species to enhance forage production, it is important to note that bush encroachment control methods are management systems [137] that might have varied policy implications for bush control [143]. Therefore, understanding the potential role of different bush encroachment control methods for promoting herbaceous species composition requires recognition of the objectives of resource users and policymakers [144]. Thus, the intended ecosystem status is dependant of

Grazing management entails management of livestock and vegetation resources. The main livestock decisions made by farmers both in the commercial and communal areas are con‐ cerned with livestock type, number and seasonal pattern of movement [145]. Commercial and communal livestock farming are generally regarded as the rangeland management systems and they are distinct in grazing management practices. Thus, communal grazing areas are generally characterised by continuous grazing, which is perceived by most of the scientists to be the root cause of the often-reported land degradation in this system. On the other hand, commercial livestock farming is characterised by structured and objective grazing manage‐ ment practices such as assigning the correct livestock units in proportion to the carrying capacity of the land. These would be done in rotation to give vegetation in grazed areas time to recover such that the rested areas can be grazed again. Understanding the dynamics of bush encroachment in relation to rangeland management systems over a broad range of environ‐ ments is essential for sustainable management of rangeland ecosystems [146]. Although rangelands are complex ecosystems varying at multiple scales in time and space [147, 148], most management usually intends to maintain or enhance livestock production by reducing plant community variability in space and time [149, 150]. This is usually accomplished by promoting spatially uniform dominance of a few productive forage species. Although it is generally believed that improper grazing practices leading to overgrazing are responsible for bush encroachment, it is not attributed to heavy grazing alone, but is strongly influenced by seasonality, which is a characteristic of arid and semi-arid environments [42]. In combination with seasonality, the ban on fire and exclusion of browsing animals such as goats and camels

Rangeland management practices, particularly fire suppression and overgrazing, have been reported to increase the proportion of some native species [70]. These natives can reduce overall forage quality or quantity (e.g. *Juniperus* spp., *Artemisia tridentata*, and *Gutierrezia* spp.)

the functional characteristics of such an ecosystem.

may also contribute to the invasion of bush encroachment.

**4.2. Bush encroachment management methods**

*4.2.1. Rangeland management practices*

284 Herbicides - Current Research and Case Studies in Use

Chemical control methods are usually expensive to apply and should be considered only under specific circumstances. Thus, their nature are suited primarily to the initial thinning of bush at high density, where there is poor fuel load to support fire, where trees are above the browse line, where the bush is unacceptable to animals and where the herbicide is intended to selectively kill a specific plant [154]. However, herbicides can sometimes be used in follow up operations such as after fire where there is a need for pre-emergence herbicide application intended to kill the seedlings of a target plant in soil. Herbicides have been applied extensively on rangelands to reduce forbs that were considered undesirable, which have been assumed to lead to an increase in grass production and ultimately to an improvement in livestock performance [155]. Herbicides are the primary method of weed control in most rangeland systems [151]. In South Africa, there is a considerable effort taken by the government to address the negative impact of alien invading species on the natural and environmental resources of the country [8].

Herbicides vary in their chemical properties, that make them vary more with their mode of action under different climatic and soil conditions, and they further vary in their methods of application and their effect on the ecosystems. There are two broad groups of herbicides used in rangelands. The first type is composed of the herbicides that are applied on the soil surface and are absorbed by the roots; these are the herbicides that are based on tebuthiuron, ethidi‐ muron or bromacil as their active ingredient [154]. The second group of herbicides is sprayed onto the plant and absorbed directly by the foliage and other above ground parts of the plants; these herbicides have picloram as the active ingredient. The second group may also have ingredients such as 2, 4-D and 2, 4, 5-T. Soil applied formulations are marketed as granules, wettable powders or as liquid with active ingredients ranging in concentration between 20% and 70%. Granular products can be applied by hand, with some suited to aerial application. Wettable and liquid products are mixed with water and applied on the soil surface adjacent to the stem of the plant. The application rates of soil formulations vary according to clay content, organic matter and pH of the soil. These herbicides remain in the soil inactive until it rains such that the active ingredient can dissolve in water so that the roots can absorb it. Herbicides applied directly to the plant normally have an oil or water base and are applied to either the stem or the leaves of the plant.

wildlife habitat [165] and livestock diets [166]. Some forbs are foraged by animals especially during the seasons when forage is scarce. Therefore, reducing forbs with herbicide might influence ecosystems across trophic levels and potentially alter ecosystem function. Further‐ more, biodiversity has been proposed as a source of stability in managed ecosystems [167, 168]. Therefore, decreasing forb diversity with the use of phenoxy herbicides like 2,4-D alters arthropod habitat and reduces arthropod diversity, which influences higher trophic levels [149,169]. The decrease in forb abundance and diversity beyond normal temporal dynamics could be detrimental to wildlife because forbs also comprise key structural, vegetative, and

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

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287

Although herbicides are considered effective in controlling weeds, they are often facing the challenge with evolution of resistant weed populations [170, 171]. Thus, depending on both the population's genetic background and ecological scenario, apart from expressing herbicide resistance, weed species adapt to herbicides by phenological changes [172, 173]. Comparisons of herbicide-resistant and susceptible biotypes have shown that populations can vary not only in morphological traits but also in developmental responses, such as relative growth rate, photosynthetic rate or germination rate [174, 175]. Adjusting seed germination time and rate has been considered as one of the potential mechanisms by which annual weeds can improve their competitive ability in agricultural scenarios [173, 176]. Hence, success of annual weed species in cropping systems may be assessed through the degree of synchronization of germination (determined by factors controlling exit from dormancy), ability to germinate at high rates (determined by seed response to environmental factors, mainly temperature), and seed longevity (determined by genotype and seed response to environmental factors promot‐

On the other hand, herbicides have some effects on the environment, thus, some plants and animals, which are not targeted are also exposed. The environmental fate of herbi‐ cides is related to chemical and physical properties of the products, amount, and frequen‐ cy of use, methods of application, abiotic and biotic characteristics of the environment, and meteorological conditions [177]. At the recommended rates of use in agriculture, the half-life of herbicides ranges from up to 1 month (e.g. 2, 4-D), to 3-12 months (e.g. atra‐ zine, trifluralin, metsulphuron methyl), to more than 1 year for picloram, tebuthiuron, pendimethalin, chlorsulphuron, and ethametsulphuron methyl [178, 179]. Persistence can be extended under certain use conditions, for example, high pH soils, and low soil mois‐ ture [179]. Residues can accumulate to toxic concentrations with consecutive treatments, and products and their metabolites such as atrazine and chlorsulphuron can exhibit per‐

Mechanical control options include the physical felling or uprooting of plants, often in combination with burning [180]. Mechanical control is labour-intensive and thus expensive to

use in extensive and dense infestations, or in remote or rugged areas.

nutritional elements [165].

ing ageing).

sistent and toxic properties [179].

*4.2.3. Mechanical*

In South Africa, of particular note are herbicides containing bromacil (5 – Bromo -3- sec – butyl – 6 – methyluracil) as the active ingredient (a. i.) which are used to control encroaching species. These herbicides include Bushwacker SC (Enviro Weed Comtrol Systems (Pty – Ltd), Bush‐ wacker GG (Enviro Weed Control Systems (Pty Ltd) and Rinkals 400 PA (Dow AgroSciences LLC) e.t.c [156]. These herbicides vary primarily in their bromacil concentration, thus, Bushwacker SC contains 500 g of bromacil per litre, Bushwacker GG contains 200 g of bromacil per kilogram and Rinkals 400 PA contains 400 g of bromacil per kilogram. These herbicides are usually selective within certain application rates, environmental conditions, and methods of application. Bromacil works by interfering with the photosynthetic pathway of plants [157]. Its application is usually done just before the active growth stage of plants, thus, before the wet season stabilizes. It quickly dissolves in soil water and may stay in the soil for several years [157]. Bromacil is readily absorbed through the root system [158] and is a specific powerful mobile inhibitor of photosynthesis [159]. The target plant must be undergoing active photo‐ synthesis for the herbicide to be effective. It inhibits photosynthesis by blocking the photosystem II reaction, thereby, preventing the conversion of sunlight into chemical energy, thus, it blocks the photosynthetic electron transport [159]. Bromacil blocks electron transport from QA to QB in the chloroplast thylakoid membranes by binding to the D-1 protein at the QB binding niche. The electrons that are blocked from passing through photosystem II are transferred through a series of reactions to other reactive toxic compounds. These compounds disrupt cell membranes and cause chloroplast swelling, membrane leakage, and ultimately cellular destruction [160]. Inhibition of photosynthesis thus results in slow starvation of the target plant and eventual death. It is translocated upward via the xylem to foliage and interferes with light-harvesting complexes [159]. In the soil, there is little adsorption of bromacil to soil colloids, therefore, it moves (leaches) through the soil and it can contaminate groundwater [157]; however, it is highly susceptible to microbial degradation [161]. When used as a selective herbicide, it can persist in the soil for one year; however, if it is applied at high concentrations, it can persist for more than one year [161].

The herbicide 2,4-D [(2, 4-dichlorophenoxy) acetic acid] is also a commonly used herbicide in the rangeland vegetation management [162]. Combined estimates of 2,4-D use annually on cropland, pastureland, and rangeland could range from 12.7 to 14.9 million kg [163]. Native and exotic dicots are primary targets of many rangeland herbicide applications [162, 164]. However, these plants also contribute key structural, vegetation, and nutritional elements to wildlife habitat [165] and livestock diets [166]. Some forbs are foraged by animals especially during the seasons when forage is scarce. Therefore, reducing forbs with herbicide might influence ecosystems across trophic levels and potentially alter ecosystem function. Further‐ more, biodiversity has been proposed as a source of stability in managed ecosystems [167, 168]. Therefore, decreasing forb diversity with the use of phenoxy herbicides like 2,4-D alters arthropod habitat and reduces arthropod diversity, which influences higher trophic levels [149,169]. The decrease in forb abundance and diversity beyond normal temporal dynamics could be detrimental to wildlife because forbs also comprise key structural, vegetative, and nutritional elements [165].

Although herbicides are considered effective in controlling weeds, they are often facing the challenge with evolution of resistant weed populations [170, 171]. Thus, depending on both the population's genetic background and ecological scenario, apart from expressing herbicide resistance, weed species adapt to herbicides by phenological changes [172, 173]. Comparisons of herbicide-resistant and susceptible biotypes have shown that populations can vary not only in morphological traits but also in developmental responses, such as relative growth rate, photosynthetic rate or germination rate [174, 175]. Adjusting seed germination time and rate has been considered as one of the potential mechanisms by which annual weeds can improve their competitive ability in agricultural scenarios [173, 176]. Hence, success of annual weed species in cropping systems may be assessed through the degree of synchronization of germination (determined by factors controlling exit from dormancy), ability to germinate at high rates (determined by seed response to environmental factors, mainly temperature), and seed longevity (determined by genotype and seed response to environmental factors promot‐ ing ageing).

On the other hand, herbicides have some effects on the environment, thus, some plants and animals, which are not targeted are also exposed. The environmental fate of herbi‐ cides is related to chemical and physical properties of the products, amount, and frequen‐ cy of use, methods of application, abiotic and biotic characteristics of the environment, and meteorological conditions [177]. At the recommended rates of use in agriculture, the half-life of herbicides ranges from up to 1 month (e.g. 2, 4-D), to 3-12 months (e.g. atra‐ zine, trifluralin, metsulphuron methyl), to more than 1 year for picloram, tebuthiuron, pendimethalin, chlorsulphuron, and ethametsulphuron methyl [178, 179]. Persistence can be extended under certain use conditions, for example, high pH soils, and low soil mois‐ ture [179]. Residues can accumulate to toxic concentrations with consecutive treatments, and products and their metabolites such as atrazine and chlorsulphuron can exhibit per‐ sistent and toxic properties [179].

#### *4.2.3. Mechanical*

these herbicides have picloram as the active ingredient. The second group may also have ingredients such as 2, 4-D and 2, 4, 5-T. Soil applied formulations are marketed as granules, wettable powders or as liquid with active ingredients ranging in concentration between 20% and 70%. Granular products can be applied by hand, with some suited to aerial application. Wettable and liquid products are mixed with water and applied on the soil surface adjacent to the stem of the plant. The application rates of soil formulations vary according to clay content, organic matter and pH of the soil. These herbicides remain in the soil inactive until it rains such that the active ingredient can dissolve in water so that the roots can absorb it. Herbicides applied directly to the plant normally have an oil or water base and are applied to

In South Africa, of particular note are herbicides containing bromacil (5 – Bromo -3- sec – butyl – 6 – methyluracil) as the active ingredient (a. i.) which are used to control encroaching species. These herbicides include Bushwacker SC (Enviro Weed Comtrol Systems (Pty – Ltd), Bush‐ wacker GG (Enviro Weed Control Systems (Pty Ltd) and Rinkals 400 PA (Dow AgroSciences LLC) e.t.c [156]. These herbicides vary primarily in their bromacil concentration, thus, Bushwacker SC contains 500 g of bromacil per litre, Bushwacker GG contains 200 g of bromacil per kilogram and Rinkals 400 PA contains 400 g of bromacil per kilogram. These herbicides are usually selective within certain application rates, environmental conditions, and methods of application. Bromacil works by interfering with the photosynthetic pathway of plants [157]. Its application is usually done just before the active growth stage of plants, thus, before the wet season stabilizes. It quickly dissolves in soil water and may stay in the soil for several years [157]. Bromacil is readily absorbed through the root system [158] and is a specific powerful mobile inhibitor of photosynthesis [159]. The target plant must be undergoing active photo‐ synthesis for the herbicide to be effective. It inhibits photosynthesis by blocking the photosystem II reaction, thereby, preventing the conversion of sunlight into chemical energy, thus, it blocks the photosynthetic electron transport [159]. Bromacil blocks electron transport from QA to QB in the chloroplast thylakoid membranes by binding to the D-1 protein at the QB binding niche. The electrons that are blocked from passing through photosystem II are transferred through a series of reactions to other reactive toxic compounds. These compounds disrupt cell membranes and cause chloroplast swelling, membrane leakage, and ultimately cellular destruction [160]. Inhibition of photosynthesis thus results in slow starvation of the target plant and eventual death. It is translocated upward via the xylem to foliage and interferes with light-harvesting complexes [159]. In the soil, there is little adsorption of bromacil to soil colloids, therefore, it moves (leaches) through the soil and it can contaminate groundwater [157]; however, it is highly susceptible to microbial degradation [161]. When used as a selective herbicide, it can persist in the soil for one year; however, if it is applied at high

The herbicide 2,4-D [(2, 4-dichlorophenoxy) acetic acid] is also a commonly used herbicide in the rangeland vegetation management [162]. Combined estimates of 2,4-D use annually on cropland, pastureland, and rangeland could range from 12.7 to 14.9 million kg [163]. Native and exotic dicots are primary targets of many rangeland herbicide applications [162, 164]. However, these plants also contribute key structural, vegetation, and nutritional elements to

either the stem or the leaves of the plant.

286 Herbicides - Current Research and Case Studies in Use

concentrations, it can persist for more than one year [161].

Mechanical control options include the physical felling or uprooting of plants, often in combination with burning [180]. Mechanical control is labour-intensive and thus expensive to use in extensive and dense infestations, or in remote or rugged areas.

#### *4.2.3.1. Rangeland burning*

Fire is regarded as the natural factor of the southern African environment; it is thought to have occurred from time immemorial, and therefore, it is part of ecosystems. Rangeland burning is an important ecological management tool in the maintenance and productivity of grasslands in Southern Africa region [181]. The burning in rangelands is practiced for a number of reasons; one of these reasons is to control bush encroachment. To use fire effectively in rangelands, it is important to understand how it behaves and to develop an insight into the way in which various factors influence such behaviour. Fire intensity is one of the important components of the fire regime [182]. Fire regime can be defined as season and frequency of burning together with type and intensity of fire [18]. The effect of fire on natural ecosystems arises from a response of living organisms to the release of heat energy generated by the combustion of plant material. Thus, it is an oxidation process involving a chain reaction during which the solar energy originally converted into carbon compounds by photosynthesis is released as heat during fire [183]. The effect of fire on vegetation, therefore, depends upon the amount of heat energy, and upon the rate and vertical level at which it is released [184]. The rate of fire is measured in terms of time taken to burn a given unit area, it is affected by a number of factors including fuel load and moisture. The vertical level at which heat energy is released during fire determines the height at which plants will be burned. The plant (tree) height is one of the important factors determining the effect of fire on bushes, thus, as the bushes become taller, the fire intensity required to cause a topkill of the stems and braches become critical. Thus, as the plant height increases, the bushes become resistant to fire [182].

increased, which is positive to livestock production. Where fire is used as a regular manage‐ ment tool, it changes species composition, thus, species that are adapted to fire tend to dominate while species that are not favouring fire do not persist. Thus, in South Africa, frequent burning in the False Thornveld of Eastern Cape, favours species such as *Themeda triandra* and has a negative effect on the abundance of *Cymbopogon plurinodes* [185]. Similar results have been observed at the Tall Grassveld of Kwazulu Natal, where *Tristachya leucothrix, Cymbopogon excuvatus* and *cymbopogon validus* became dominant with burning frequency [183]. Furthermore, where higher frequency of fire is used, for example where burning is annual, the bush will be controlled but that has an effect on the basal cover of herbaceous plants, thus, the basal cover becomes poor due to effects of fire on plant vigour. That, in turn, renders the soil susceptible to soil erosion, which is another environmental disaster. Fire remains the cheapest form of management available to conserve and perpetuate natural plant communities. However, its effectiveness is based on clear and objective application of a fire regime, thus frequency, season and intensity may be used effectively to retain the natural element and

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

http://dx.doi.org/10.5772/56182

289

Manual and mechanical techniques such as pulling, cutting, and otherwise damaging plants, are used to control some invasive plants, particularly if the population is relatively small. These techniques can be extremely specific, and therefore, minimizing damage to desirable plants. However, manual techniques are generally labour and time intensive. These techniques are effective if the treatments are administered several times to prevent the weed from reestablishing. In the process, labourers and machines may severely trample vegetation and disturb the soil, thus, providing prime conditions for re-invasion by the same or other invasive

Bush encroachment reduces grass growth in rangeland as discussed in the previous sections and that results in reduced biomass production, which subsequently affects forage production. The approach that has been used to address the negative impacts of invading species in South Africa has been predominantly physical by clearing alien plants [187]. Clearing of the bush in encroached areas results in an increased dry matter yield and basal cover of herbaceous vegetation [184], which are good indicators for rangeland health if the functional characteristic of such an ecosystem is forage production. Furthermore, species richness of herbaceous plants and relative abundance of few of the species among the initial population that is intolerant of bush cover increase with tree cutting [142]. As a result, the reduction of bush cover can restore herbaceous plant productivity and biodiversity in rangelands [188]. However, there are herbaceous species that have a positive relationship with certain trees, and removal of such trees negatively leads to reduction of these herbaceous species. This decline indicates the shifts in the microenvironment due to the removal of ecologically important trees, thus exposing

It is important, however, to note that although bush cutting has positive results on forage productivity, it has high costs involved [142]. Therefore, it is more applicable on the smaller scale. On the larger scale, where bush clearing is done with heavy implements such as a

control the invasive elements in the flora of natural ecosystems [186].

sensitive herbaceous species to increased light intensity.

*4.2.3.2. Manual/Physical cutting/clearing*

species.

Since the effectiveness of fire in rangeland to control bush encroachment depends largely on the fire intensity, which, in turn, depends on fuel characteristics such as fuel load. It is important to note that fire cannot be applied at all times, thus, there should be considerations on the suitability of the ecosystem to support fire. The high intensity fire is required to control bush encroachment at all phases, thus, controlling coppice growth and bush seedlings or maintaining bush at an available height and in an acceptable state for browsing animals [184]. Use of fire as a control method for bush encroachment, therefore, has higher potential in higher rainfall areas where the soil moisture available is reliable and sufficient to produce fuel load that can support regular fires. The use of fire has to be sustained in order to get good results; this is because the bush can recover through coppice regrowth and seedling recruitment after burning, therefore, there should periodic follow up burn. In moist areas, the frequency of burning required to control bush encroachment depends on the rate at which the bush recovers. The recommended type of fire used in controlling bush encroachment is generally head fire (burning towards the direction of wind); this will mostly occur in the form of surface fire except in extreme conditions where it can develop into crown fire in more densely wooded areas with more flammable foliage. The season of burning should be during the early spring, after the first spring rain. This will ensure the intense fire but with minimal undue deleterious effects on the grass sward. Fire should be applied close to the commencement of the growing season as possible to minimise the length of soil exposure to potential soil erosion.

Reduction of bush encroachment with fire has positive results on herbaceous vegetation biomass production, thus, biomass production is enhanced, and therefore, forage production increased, which is positive to livestock production. Where fire is used as a regular manage‐ ment tool, it changes species composition, thus, species that are adapted to fire tend to dominate while species that are not favouring fire do not persist. Thus, in South Africa, frequent burning in the False Thornveld of Eastern Cape, favours species such as *Themeda triandra* and has a negative effect on the abundance of *Cymbopogon plurinodes* [185]. Similar results have been observed at the Tall Grassveld of Kwazulu Natal, where *Tristachya leucothrix, Cymbopogon excuvatus* and *cymbopogon validus* became dominant with burning frequency [183]. Furthermore, where higher frequency of fire is used, for example where burning is annual, the bush will be controlled but that has an effect on the basal cover of herbaceous plants, thus, the basal cover becomes poor due to effects of fire on plant vigour. That, in turn, renders the soil susceptible to soil erosion, which is another environmental disaster. Fire remains the cheapest form of management available to conserve and perpetuate natural plant communities. However, its effectiveness is based on clear and objective application of a fire regime, thus frequency, season and intensity may be used effectively to retain the natural element and control the invasive elements in the flora of natural ecosystems [186].

#### *4.2.3.2. Manual/Physical cutting/clearing*

*4.2.3.1. Rangeland burning*

288 Herbicides - Current Research and Case Studies in Use

Fire is regarded as the natural factor of the southern African environment; it is thought to have occurred from time immemorial, and therefore, it is part of ecosystems. Rangeland burning is an important ecological management tool in the maintenance and productivity of grasslands in Southern Africa region [181]. The burning in rangelands is practiced for a number of reasons; one of these reasons is to control bush encroachment. To use fire effectively in rangelands, it is important to understand how it behaves and to develop an insight into the way in which various factors influence such behaviour. Fire intensity is one of the important components of the fire regime [182]. Fire regime can be defined as season and frequency of burning together with type and intensity of fire [18]. The effect of fire on natural ecosystems arises from a response of living organisms to the release of heat energy generated by the combustion of plant material. Thus, it is an oxidation process involving a chain reaction during which the solar energy originally converted into carbon compounds by photosynthesis is released as heat during fire [183]. The effect of fire on vegetation, therefore, depends upon the amount of heat energy, and upon the rate and vertical level at which it is released [184]. The rate of fire is measured in terms of time taken to burn a given unit area, it is affected by a number of factors including fuel load and moisture. The vertical level at which heat energy is released during fire determines the height at which plants will be burned. The plant (tree) height is one of the important factors determining the effect of fire on bushes, thus, as the bushes become taller, the fire intensity required to cause a topkill of the stems and braches become critical. Thus, as

Since the effectiveness of fire in rangeland to control bush encroachment depends largely on the fire intensity, which, in turn, depends on fuel characteristics such as fuel load. It is important to note that fire cannot be applied at all times, thus, there should be considerations on the suitability of the ecosystem to support fire. The high intensity fire is required to control bush encroachment at all phases, thus, controlling coppice growth and bush seedlings or maintaining bush at an available height and in an acceptable state for browsing animals [184]. Use of fire as a control method for bush encroachment, therefore, has higher potential in higher rainfall areas where the soil moisture available is reliable and sufficient to produce fuel load that can support regular fires. The use of fire has to be sustained in order to get good results; this is because the bush can recover through coppice regrowth and seedling recruitment after burning, therefore, there should periodic follow up burn. In moist areas, the frequency of burning required to control bush encroachment depends on the rate at which the bush recovers. The recommended type of fire used in controlling bush encroachment is generally head fire (burning towards the direction of wind); this will mostly occur in the form of surface fire except in extreme conditions where it can develop into crown fire in more densely wooded areas with more flammable foliage. The season of burning should be during the early spring, after the first spring rain. This will ensure the intense fire but with minimal undue deleterious effects on the grass sward. Fire should be applied close to the commencement of the growing

season as possible to minimise the length of soil exposure to potential soil erosion.

Reduction of bush encroachment with fire has positive results on herbaceous vegetation biomass production, thus, biomass production is enhanced, and therefore, forage production

the plant height increases, the bushes become resistant to fire [182].

Manual and mechanical techniques such as pulling, cutting, and otherwise damaging plants, are used to control some invasive plants, particularly if the population is relatively small. These techniques can be extremely specific, and therefore, minimizing damage to desirable plants. However, manual techniques are generally labour and time intensive. These techniques are effective if the treatments are administered several times to prevent the weed from reestablishing. In the process, labourers and machines may severely trample vegetation and disturb the soil, thus, providing prime conditions for re-invasion by the same or other invasive species.

Bush encroachment reduces grass growth in rangeland as discussed in the previous sections and that results in reduced biomass production, which subsequently affects forage production. The approach that has been used to address the negative impacts of invading species in South Africa has been predominantly physical by clearing alien plants [187]. Clearing of the bush in encroached areas results in an increased dry matter yield and basal cover of herbaceous vegetation [184], which are good indicators for rangeland health if the functional characteristic of such an ecosystem is forage production. Furthermore, species richness of herbaceous plants and relative abundance of few of the species among the initial population that is intolerant of bush cover increase with tree cutting [142]. As a result, the reduction of bush cover can restore herbaceous plant productivity and biodiversity in rangelands [188]. However, there are herbaceous species that have a positive relationship with certain trees, and removal of such trees negatively leads to reduction of these herbaceous species. This decline indicates the shifts in the microenvironment due to the removal of ecologically important trees, thus exposing sensitive herbaceous species to increased light intensity.

It is important, however, to note that although bush cutting has positive results on forage productivity, it has high costs involved [142]. Therefore, it is more applicable on the smaller scale. On the larger scale, where bush clearing is done with heavy implements such as a bulldozer blade, the trees are removed with their roots, which minimises resprouting of encroaching species. However, the soil disturbance generally severely affects the grass layer, but the grasses will often re-establish themselves [154]. The re-establishment of grasses will be following the secondary succession trend, thus the first colonisers are likely to be annual pioneers, which have little forage value. Furthermore, severe soil disturbance may encourage the establishment of a large number of seedlings of some woody plants. This may lead to establishment of a woody community that is denser than the original community.

(KNP). The impact of *S. rufinasus* on *A. filiculoides* within the Kruger National Park (KNP) has been exceptionally good. Thus, 100% clearing of the weed was achieved in a few months after release and the infestation has been maintained at that level. The insects are able to survive for long periods in the vicinity and re-establish themselves should the area become re-infested.

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Biological control agents such as *Neochetina eichhorniae, Cercospora rodmanii, Orthogalumna terebrantis, Niphograpta albiguttalis, Neochetina bruchi, Eccritotarsus catarinensi* have been used to control invasion by *Eichhornia crassipes* (Water hyacinth). Although proven in many other instances elsewhere to be effective, the agents released within the KNP have had little impact in terms of bringing the infestation under control. This little impact has been ascribed to frequent low level flooding as well as major floods that have repeatedly washed the infestation away, and therefore, preventing large numbers of insects to build up [194]. *Lantana camara* has been cited to be one of the invasive plants at KNP and other areas of South Africa. Two biological control agents viz. *Octotoma scabripennis* (leaf-mining hispine beetle) and *Falconia intermedia* (Lantana sapsucker) have been introduced at KNP. However, *O. scabripennis* failed to establish and the initial trial site for *Falconia intermedia* was reported to have been destroyed by the floods and therefore, both agents have provided insignificant impact on *L. camara* [194].

*Opuntia stricta* (Sour prickly pear) has been identified as one of the invasive species at KNP and therefore, it was one of the species that were controlled. In an attempt to control this species, two agents have been introduced against it, the first of which being *Cactoblastis cactorum* (phycitid moth) in 1988 [195] and subsequently *Dactylopius opuntiae* (cochineal) in 1996 [196]. The structure of infestations of *O. stricta* changed after the introduction of *C. cactorum* where large plants were replaced by high densities of smaller plants. However, fruit production did not decline and therefore *C. cactorum* failed to provide the degree of control that was expected [195]. Predation and parasitism, especially ant predation of eggs, has a definite impact on the distribution and abundance of *C. cactorum*. *Dactylopius opuntiae*, which had been instrumental in the control against *O. ficusindica*, was released on at least three occasions between 1990 and 1995 yet failed to establish due to the biotype that was used. The Plant Protection Research Institute (PPRI) sourced a different biotype of *D. opuntiae* from Australia, which established well and is reported to be currently destroying large stands in

*Pistia stratiotes* (Water lettuce) was determined to be one of the invasive species within the Kruger National Park. The snout weevil (*Neohydronomus affinis*) was introduced to control the weeds. The impact of *N. Affinis* on *P. Stratiotes* varied at different infestations throughout the KNP. The other biocontrol agent *Cyrtobagous salviniae* (snout beetle) was released to control *Salvinia molesta* (Kariba weed). The infestations of *S. molesta* at the three areas where the agent was released and established were brought under complete control and have been maintained at that level. *Trichapion lativentre, Rhyssomatus marginatus* and *Neodiplogrammus quadrivattatus* were used to control *Sesbania punicea* (Red Sesbania) at Kruger National Park. The impact of the three agents on plants has been reported to be exceptionally good [194]. The three weevil species have reduced the problem to such an extent that *S. punicea* is under complete control in the area, thereby requiring no further action to be taken. The biological control of *S.*

*punicea* remains the best example of an invasive tree species control.

the Skukuza region in South Africa [196].

#### *4.2.4. Biological control of encroaching and invasive species*

Biological control has been defined as the use of living organisms to reduce the vigour, reproductive capacity, or effects of weeds [189]. Biological control (biocontrol) involves the deliberate introduction of invertebrates or diseases, and is aimed at reducing the effects of ecological release. Biocontrol is aimed at arriving at a situation where the plant is returned to the status of a non-invasive naturalized alien, that is an alien plant that is able to survive, and even reproduce, but does not invade aggressively in its new habitat [6]. Biological control could be regarded as the only sustainable mechanism to prevent the spread of invasive alien species in the long term [190]. Biocontrol is potentially very cost-effective, and environmentally benign. Despite concerns to the contrary [191], the modern practice of using carefully screened and host-specific biocontrol agents is safe, and "host shifts" have not occurred in the over 350 recorded cases where weed biocontrol agents have been used worldwide [192].

Although there are some inconsistencies in terms of when biocontrol practices were establish‐ ed in South Africa, at least there is an agreement in that biocontrol agents have been released against 47 weed species. The disagreement in literature is such that Olckers and Hill (1999) indicated that in South Africa, biocontrol has been practiced since 1910, and that to date, 103 biocontrol agents (including invertebrates and pathogens) have been released against 47 weed species. Whilst on the other side, it has been suggested that the biological control of weeds has been practiced since 1913 and since then some 47 weed species have been subjected to the effects of approximately 85 species of biocontrol agents [190]. Therefore, based on the cited literature, there is an uncertainty about the years of establishment of biocontrol in South Africa and for this chapter the assumption will be that the biocontrol was adopted for use between 1910 and 1913. Although in South Africa physical methods of controlling the alien species are mostly used, biological control using species-specific invertebrates and pathogens from the plant's country of origin is also a control option; however, there has been a considerable resistance to its use [180]. The seed-feeding weevil is one of the agents that have been released against *Acacia mearnsii* in areas where the wattle is not grown commercially [8]. Nevertheless, plant-attacking agents could potentially be used; however, these compared with seedattacking agents such as weevils could kill the target plant and therefore, impact severely on commercial prospects. The impact of biological control agents on controlling invasive species vary with species controlled, biological agents introduced, mode of operation of agents and many other factors. The use of biological control measures on invasive plants have been reported in South Africa with varying rates of success. The elaborate example where the invasive plants were controlled with biological control agents was at Kruger National Park (KNP). The impact of *S. rufinasus* on *A. filiculoides* within the Kruger National Park (KNP) has been exceptionally good. Thus, 100% clearing of the weed was achieved in a few months after release and the infestation has been maintained at that level. The insects are able to survive for long periods in the vicinity and re-establish themselves should the area become re-infested.

bulldozer blade, the trees are removed with their roots, which minimises resprouting of encroaching species. However, the soil disturbance generally severely affects the grass layer, but the grasses will often re-establish themselves [154]. The re-establishment of grasses will be following the secondary succession trend, thus the first colonisers are likely to be annual pioneers, which have little forage value. Furthermore, severe soil disturbance may encourage the establishment of a large number of seedlings of some woody plants. This may lead to

Biological control has been defined as the use of living organisms to reduce the vigour, reproductive capacity, or effects of weeds [189]. Biological control (biocontrol) involves the deliberate introduction of invertebrates or diseases, and is aimed at reducing the effects of ecological release. Biocontrol is aimed at arriving at a situation where the plant is returned to the status of a non-invasive naturalized alien, that is an alien plant that is able to survive, and even reproduce, but does not invade aggressively in its new habitat [6]. Biological control could be regarded as the only sustainable mechanism to prevent the spread of invasive alien species in the long term [190]. Biocontrol is potentially very cost-effective, and environmentally benign. Despite concerns to the contrary [191], the modern practice of using carefully screened and host-specific biocontrol agents is safe, and "host shifts" have not occurred in the over 350

Although there are some inconsistencies in terms of when biocontrol practices were establish‐ ed in South Africa, at least there is an agreement in that biocontrol agents have been released against 47 weed species. The disagreement in literature is such that Olckers and Hill (1999) indicated that in South Africa, biocontrol has been practiced since 1910, and that to date, 103 biocontrol agents (including invertebrates and pathogens) have been released against 47 weed species. Whilst on the other side, it has been suggested that the biological control of weeds has been practiced since 1913 and since then some 47 weed species have been subjected to the effects of approximately 85 species of biocontrol agents [190]. Therefore, based on the cited literature, there is an uncertainty about the years of establishment of biocontrol in South Africa and for this chapter the assumption will be that the biocontrol was adopted for use between 1910 and 1913. Although in South Africa physical methods of controlling the alien species are mostly used, biological control using species-specific invertebrates and pathogens from the plant's country of origin is also a control option; however, there has been a considerable resistance to its use [180]. The seed-feeding weevil is one of the agents that have been released against *Acacia mearnsii* in areas where the wattle is not grown commercially [8]. Nevertheless, plant-attacking agents could potentially be used; however, these compared with seedattacking agents such as weevils could kill the target plant and therefore, impact severely on commercial prospects. The impact of biological control agents on controlling invasive species vary with species controlled, biological agents introduced, mode of operation of agents and many other factors. The use of biological control measures on invasive plants have been reported in South Africa with varying rates of success. The elaborate example where the invasive plants were controlled with biological control agents was at Kruger National Park

establishment of a woody community that is denser than the original community.

recorded cases where weed biocontrol agents have been used worldwide [192].

*4.2.4. Biological control of encroaching and invasive species*

290 Herbicides - Current Research and Case Studies in Use

Biological control agents such as *Neochetina eichhorniae, Cercospora rodmanii, Orthogalumna terebrantis, Niphograpta albiguttalis, Neochetina bruchi, Eccritotarsus catarinensi* have been used to control invasion by *Eichhornia crassipes* (Water hyacinth). Although proven in many other instances elsewhere to be effective, the agents released within the KNP have had little impact in terms of bringing the infestation under control. This little impact has been ascribed to frequent low level flooding as well as major floods that have repeatedly washed the infestation away, and therefore, preventing large numbers of insects to build up [194]. *Lantana camara* has been cited to be one of the invasive plants at KNP and other areas of South Africa. Two biological control agents viz. *Octotoma scabripennis* (leaf-mining hispine beetle) and *Falconia intermedia* (Lantana sapsucker) have been introduced at KNP. However, *O. scabripennis* failed to establish and the initial trial site for *Falconia intermedia* was reported to have been destroyed by the floods and therefore, both agents have provided insignificant impact on *L. camara* [194].

*Opuntia stricta* (Sour prickly pear) has been identified as one of the invasive species at KNP and therefore, it was one of the species that were controlled. In an attempt to control this species, two agents have been introduced against it, the first of which being *Cactoblastis cactorum* (phycitid moth) in 1988 [195] and subsequently *Dactylopius opuntiae* (cochineal) in 1996 [196]. The structure of infestations of *O. stricta* changed after the introduction of *C. cactorum* where large plants were replaced by high densities of smaller plants. However, fruit production did not decline and therefore *C. cactorum* failed to provide the degree of control that was expected [195]. Predation and parasitism, especially ant predation of eggs, has a definite impact on the distribution and abundance of *C. cactorum*. *Dactylopius opuntiae*, which had been instrumental in the control against *O. ficusindica*, was released on at least three occasions between 1990 and 1995 yet failed to establish due to the biotype that was used. The Plant Protection Research Institute (PPRI) sourced a different biotype of *D. opuntiae* from Australia, which established well and is reported to be currently destroying large stands in the Skukuza region in South Africa [196].

*Pistia stratiotes* (Water lettuce) was determined to be one of the invasive species within the Kruger National Park. The snout weevil (*Neohydronomus affinis*) was introduced to control the weeds. The impact of *N. Affinis* on *P. Stratiotes* varied at different infestations throughout the KNP. The other biocontrol agent *Cyrtobagous salviniae* (snout beetle) was released to control *Salvinia molesta* (Kariba weed). The infestations of *S. molesta* at the three areas where the agent was released and established were brought under complete control and have been maintained at that level. *Trichapion lativentre, Rhyssomatus marginatus* and *Neodiplogrammus quadrivattatus* were used to control *Sesbania punicea* (Red Sesbania) at Kruger National Park. The impact of the three agents on plants has been reported to be exceptionally good [194]. The three weevil species have reduced the problem to such an extent that *S. punicea* is under complete control in the area, thereby requiring no further action to be taken. The biological control of *S. punicea* remains the best example of an invasive tree species control.

The use of mammals such as goats in agricultural areas to control bush encroachment has been reported in South Africa [197]. Apart from tree seedlings, which can be affected by smaller browsers, the use of browsers to execute control on woody plants largely excludes wild game [154]. However, elephants have also been reported to be effective in controlling bush en‐ croachment [198, 199]. Nevertheless, their use is confined to large game reserves or game farms where their population should be large enough to make an appreciable impact on the woody vegetation, which could, in turn, lead to serious management problems. The control of bush encroachment by use of mammals such as goats is dependent firstly on the acceptability of plant species that are controlled to these mammals for use as browse, and secondly availability of the browse material. The acceptability relates to the palatability and nutritional value of a browse material to the browser. Browse availability relates to the height at which browse material can be accessed by browsing animals, the browse line for goats is approximately 1.5 m. Boer goats are well suited to controlling woody plants because the intensity and frequency with which they utilise the browse can be controlled. Furthermore, the Boer goats are relatively insensitive to chemical deterrents, such as high tannin levels present in many woody species [154]. Boer goats cannot be used to control dense stands of woody plants whose canopies extend above the browse line of approximately 1.5 m.

The increasing invasion of non-indigenous species has become one of the top causes of global biodiversity loss and environmental change [200, 201]. Therefore, there is a need for develop‐ ment of intensive mechanisms to control these invasions in the ecosystems before the natural value of ecosystems is lost permanently. As part of a comprehensive remedial effort to control invasions, assessment and characterisation of invader species will serve as a foundation towards integration of efforts to control invaders species. There is urgency for more rigorous and comprehensive assessments of the impacts and risks associated with plant invasions [202]. Thus, prevention and control strategies can be targeted appropriately if sufficient assessments are conducted [203]. In the approaches toward the control of invasive alien weeds, any intervention needs to be aligned with the different stages of spread and characteristics of a desired ecosystem. The stages of spread can be divided into four broad phases: (i) arrival or entry phase; (ii) adaptation and establishment phase; (iii) an exponential growth phase; and (iv) dominance phase. It is in the exponential growth stage of weed spread that integrated control programmes find a logical relevance. Prevention, and early detection and eradication, are more appropriate for the first two stages, while options may be severely limited once weed

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Plant invasions are interdisciplinary both by their impacts and by utility and therefore, assessments should recognize the interdisciplinary nature of the problem of species invasions. Thus, the ecosystem characteristics determine whether the appropriate conditions allow for the establishment of the invasive species, and on the other hand, economic systems affect the state of the ecosystem through its use, and through the prevention and control measures implemented to stop the invasions. Hence, accounting for the economic and ecological links and feedbacks is critical in invasion assessments [204]. It is fundamental to have a clear understanding on different functions of ecosystems, thus, an assessment of rangeland area in terms of its ability to achieve its ecosystem functions. Natural resource managers and farmers at all levels require full knowledge of ecosystem functions. This could be achieved through collating results from experiments in different fields or locations within the context of a more encompassing systems management framework that treats the rangeland ecosystem as a complete bio-economic unit. Therefore, in order to improve decision making, farmers need answers to questions at the systems level, including the biological and economic elements of

Most often, a single method is not always effective to achieve sustainable control of the rangeland weeds. This is because of among other reasons some methods can only control bush encroachment at a certain stage and some could leave areas that are treated vulnerable to other forms of landscape hazards. For example, use of fire in rangelands depending on the intensity will burn shoots of woody plants; however, the seeds in the soil could be left to germinate and furthermore, some seeds may be stimulated to germinate by fire. It is also difficult to ascertain a complete kill of unwanted species with fire because normally the basal buds of certain trees remain unburned and therefore resprout. It is for these reasons that the introduction of biological control agents becomes important especially where complete removal of the invading species is anticipated. Use of herbivores works effectively where the intention is to maintain the current stand of encroaching species especially in the savanna where there is

populations reach the final stage of total ecosystem domination.

the rangeland production entities they are attempting to manage.

#### *4.2.5. Integrated bush encroachment and invasion management*

Integrated weed control usually involves a combination of at least three of the primary elements of control - mechanical, chemical and biological [180]. Integrated weed management (IWM) could be defined as a system for the planning and implementation of programs, using an interdisciplinary approach, to select a method for controlling undesirable plant species or group of species using all available methods. These methods generally vary between preven‐ tative and restorative domains. The success of preventative encroachment measures mostly depends on the understanding of the causes of encroachment and identification of barriers for natural recovery. Restorative measures depend on the rangeland ecosystem structure and functional characteristics to be restored. Integrated bush encroachment control is a multidis‐ ciplinary, ecological approach to managing unwanted plant species in rangeland ecosystems.

However, it is important to note that the decision to use a certain method to control the bush encroachment is informed by the cost of using that method against the benefit. Bush encroach‐ ment control methods are management systems [137] that might have varied policy implica‐ tions for bush control [193]. Therefore, understanding the potential role of different bush encroachment control methods for promoting herbaceous species composition requires recognition of the objectives of resource users and policymakers [142]. The failure to recognise the long-term intended ecosystem status could lead to a subsequent failure to achieve bush encroachment control objectives and that could further lead to land use practice and policy controversy. Thus, the resource users are interested in livestock production through increased plant productivity, while the goal of policymakers is environmental preservation. Therefore, the land use practice imperatives and policy directives should be harmonised to permit both forage production and biodiversity conservation functional characteristics of the ecosystem to thrive.

The increasing invasion of non-indigenous species has become one of the top causes of global biodiversity loss and environmental change [200, 201]. Therefore, there is a need for develop‐ ment of intensive mechanisms to control these invasions in the ecosystems before the natural value of ecosystems is lost permanently. As part of a comprehensive remedial effort to control invasions, assessment and characterisation of invader species will serve as a foundation towards integration of efforts to control invaders species. There is urgency for more rigorous and comprehensive assessments of the impacts and risks associated with plant invasions [202]. Thus, prevention and control strategies can be targeted appropriately if sufficient assessments are conducted [203]. In the approaches toward the control of invasive alien weeds, any intervention needs to be aligned with the different stages of spread and characteristics of a desired ecosystem. The stages of spread can be divided into four broad phases: (i) arrival or entry phase; (ii) adaptation and establishment phase; (iii) an exponential growth phase; and (iv) dominance phase. It is in the exponential growth stage of weed spread that integrated control programmes find a logical relevance. Prevention, and early detection and eradication, are more appropriate for the first two stages, while options may be severely limited once weed populations reach the final stage of total ecosystem domination.

The use of mammals such as goats in agricultural areas to control bush encroachment has been reported in South Africa [197]. Apart from tree seedlings, which can be affected by smaller browsers, the use of browsers to execute control on woody plants largely excludes wild game [154]. However, elephants have also been reported to be effective in controlling bush en‐ croachment [198, 199]. Nevertheless, their use is confined to large game reserves or game farms where their population should be large enough to make an appreciable impact on the woody vegetation, which could, in turn, lead to serious management problems. The control of bush encroachment by use of mammals such as goats is dependent firstly on the acceptability of plant species that are controlled to these mammals for use as browse, and secondly availability of the browse material. The acceptability relates to the palatability and nutritional value of a browse material to the browser. Browse availability relates to the height at which browse material can be accessed by browsing animals, the browse line for goats is approximately 1.5 m. Boer goats are well suited to controlling woody plants because the intensity and frequency with which they utilise the browse can be controlled. Furthermore, the Boer goats are relatively insensitive to chemical deterrents, such as high tannin levels present in many woody species [154]. Boer goats cannot be used to control dense stands of woody plants whose canopies

Integrated weed control usually involves a combination of at least three of the primary elements of control - mechanical, chemical and biological [180]. Integrated weed management (IWM) could be defined as a system for the planning and implementation of programs, using an interdisciplinary approach, to select a method for controlling undesirable plant species or group of species using all available methods. These methods generally vary between preven‐ tative and restorative domains. The success of preventative encroachment measures mostly depends on the understanding of the causes of encroachment and identification of barriers for natural recovery. Restorative measures depend on the rangeland ecosystem structure and functional characteristics to be restored. Integrated bush encroachment control is a multidis‐ ciplinary, ecological approach to managing unwanted plant species in rangeland ecosystems.

However, it is important to note that the decision to use a certain method to control the bush encroachment is informed by the cost of using that method against the benefit. Bush encroach‐ ment control methods are management systems [137] that might have varied policy implica‐ tions for bush control [193]. Therefore, understanding the potential role of different bush encroachment control methods for promoting herbaceous species composition requires recognition of the objectives of resource users and policymakers [142]. The failure to recognise the long-term intended ecosystem status could lead to a subsequent failure to achieve bush encroachment control objectives and that could further lead to land use practice and policy controversy. Thus, the resource users are interested in livestock production through increased plant productivity, while the goal of policymakers is environmental preservation. Therefore, the land use practice imperatives and policy directives should be harmonised to permit both forage production and biodiversity conservation functional characteristics of the ecosystem to

extend above the browse line of approximately 1.5 m.

292 Herbicides - Current Research and Case Studies in Use

thrive.

*4.2.5. Integrated bush encroachment and invasion management*

Plant invasions are interdisciplinary both by their impacts and by utility and therefore, assessments should recognize the interdisciplinary nature of the problem of species invasions. Thus, the ecosystem characteristics determine whether the appropriate conditions allow for the establishment of the invasive species, and on the other hand, economic systems affect the state of the ecosystem through its use, and through the prevention and control measures implemented to stop the invasions. Hence, accounting for the economic and ecological links and feedbacks is critical in invasion assessments [204]. It is fundamental to have a clear understanding on different functions of ecosystems, thus, an assessment of rangeland area in terms of its ability to achieve its ecosystem functions. Natural resource managers and farmers at all levels require full knowledge of ecosystem functions. This could be achieved through collating results from experiments in different fields or locations within the context of a more encompassing systems management framework that treats the rangeland ecosystem as a complete bio-economic unit. Therefore, in order to improve decision making, farmers need answers to questions at the systems level, including the biological and economic elements of the rangeland production entities they are attempting to manage.

Most often, a single method is not always effective to achieve sustainable control of the rangeland weeds. This is because of among other reasons some methods can only control bush encroachment at a certain stage and some could leave areas that are treated vulnerable to other forms of landscape hazards. For example, use of fire in rangelands depending on the intensity will burn shoots of woody plants; however, the seeds in the soil could be left to germinate and furthermore, some seeds may be stimulated to germinate by fire. It is also difficult to ascertain a complete kill of unwanted species with fire because normally the basal buds of certain trees remain unburned and therefore resprout. It is for these reasons that the introduction of biological control agents becomes important especially where complete removal of the invading species is anticipated. Use of herbivores works effectively where the intention is to maintain the current stand of encroaching species especially in the savanna where there is coexistence between grasses and trees. There are species which are not preferred by animals for foraging, and use of biological control through herbivores would not be effective; therefore, introduction of invertebrates could be used. However, most of the invertebrates are not readily available in Southern Africa for use at the farm or landscape level. It is impractical to burn certain areas that are encroached; this is sometimes due to poor fuel load that can support high fire intensity needed to burn the woody species. Encroachment in some of these areas cannot be controlled with the use of herbivores (goats) and herbicides could be useful.

control and other control measures may provide effective solutions to the problem, and various methods therefore, should not be used in contradiction to one another. All available knowledge surrounding a particular invasive plant problem needs to be considered when developing such integrated programmes. No single method is likely to prevent either distribution or densifi‐ cation of the plant from or in its current range. Combination of the biological control and herbicides can bring remarkable results; while herbicides are used to contain the infestation to its present range, biological control (invertebrates) is being released into dense stands where it is proving destructively effective in controlling the plants. Goats used in the system that allows coppice growth to be used frequently and severely strongly influence woody plants, that is, provided that their canopies be below the browse line. Where the plants are above browse line, fire can be used to reduce plant height where fuel load is sufficient; however, where fuel load is not sufficient chemical or physical control can be used and, in both cases,

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In this chapter, integration in the control of invasive species is not limited to control methods themselves in isolation but in all the processes relating to bush encroachment management. Primarily, it is important as the initial stage of integration to identify and characterise invasion/ encroachment of species. This should include establishment of their origin, mode of establish‐ ment and spread (seeds, cuttings etc), their phenological and morphological characteristics and assessing their favourable growth conditions. It is further important to determine the degree of invasion/encroachment, which will help setting economic and ecological thresholds of invasion. The analysis of the ecological and economic impact of invasion/encroachment in the environment should be carried out prior to any intervention. That will help in determining whether there is a need for intervention and magnitude of such intervention. The need for intervention should be assessed against the set thresholds for invasion. Setting objectives for invasion/encroachment management is very fundamental because the objectives will be used

A number of factors will guide selection of the approach to control bush encroachment. These factors include species to be controlled, the stage of invasion and landscape of an area. The approach to be selected would be chemical, mechanical and biological depending on the approach suited to the species to be controlled, the major landscape on which the invasion has occurred and the stage of invasion. The method that is ecologically and economically sound and practical should be selected. Integrated bush encroachment approaches may be practiced in combinations that could either be used simultaneously, alternatively or sequentially. In simultaneous integration of bush control methods, more than one method that could comple‐ ment each other under the prospects of chemical, biological or physical methods used together. Some methods cannot be used simultaneously because of the danger that they can cause on other organisms and environment. For example, the methods that can be integrated simulta‐ neously could be manual clearance and use of goats as browsers. The alternative integration could be executed through turns, thus, one method first and then the other. The alternate integration can be practiced in rotation if planned properly, for example, use fire with a given period in between goat treatment. Thus, burning can be applied every three years while goat use is continued. Sequential integration is executed in succession of methods where one

goats can be used as follow up control.

as the yardstick for the control.

All this, therefore, suggests that there are areas and bush encroachment situations where a single method can be used; however, a combination of different methods could be used simultaneously or alternatively in subsequent approaches. Nevertheless, it is important that prior to the implementation of any selected method or any combination or any sequence to develop post encroachment treatment management plan. This is because removal of bush with any technique can leave the land vulnerable to soil erosion or further encroachment of the same or new species. Therefore, a successful long-term management program should be designed to include combinations of mechanical, biological, and chemical control techniques. Numerous mechanical and cultural options have been developed to manage noxious range‐ land weeds, including mowing, prescribed burning, timely grazing, and perennial grass reseeding or inter-seeding. Furthermore, several herbicides are registered for use on range‐ lands and most biological control programs focus on noxious rangeland weed control. Successful management of noxious weeds on rangeland will require the development of a long-term strategic plan incorporating prevention programs, education materials and activi‐ ties, economical and sustainable multi-year integrated approaches that improve degraded rangeland communities, enhance the utility of the ecosystem, and prevent reinvasion or encroachment by other noxious weed species [151].

There are a number of factors to consider in selection of the bush control method; however, the dominant consideration is the cost of the method. However, there are furthermore considerations beyond the cost of the method. The use of fire in controlling bush encroachment in rangelands is determined by a threshold amount of flammable fine fuel needed to carry fire that is sufficiently intensive to reduce woody plants. Furthermore, to effectively control woody plants with burning, fire must be applied regularly. Many rangelands occur in semi-arid environments in which forage-based livestock production is the primary agricultural activity and intermittent droughts are inevitable [205]). Therefore, accumulating sufficient fine fuel to carry fires in such environments requires the reduction in livestock numbers compared to areas where fire is not used. Hence, sustainable utilisation of semi-arid rangelands depends on complex management of animal species, stocking rates, and the vegetation composition, structure, phenology and quality [129].

The integration of bio-control agents and herbicides in a scientifically sound and rigorous management plan is the first step in a long-term approach to weed management. Such management plans should aim to maximise the benefits of all the respective control options and thereby ensure the infestation is contained and the density reduced to acceptable thresh‐ olds. Biological control is used as an important, long-term management solution to numerous weeds worldwide. When carefully integrated into management plans the combination of biocontrol and other control measures may provide effective solutions to the problem, and various methods therefore, should not be used in contradiction to one another. All available knowledge surrounding a particular invasive plant problem needs to be considered when developing such integrated programmes. No single method is likely to prevent either distribution or densifi‐ cation of the plant from or in its current range. Combination of the biological control and herbicides can bring remarkable results; while herbicides are used to contain the infestation to its present range, biological control (invertebrates) is being released into dense stands where it is proving destructively effective in controlling the plants. Goats used in the system that allows coppice growth to be used frequently and severely strongly influence woody plants, that is, provided that their canopies be below the browse line. Where the plants are above browse line, fire can be used to reduce plant height where fuel load is sufficient; however, where fuel load is not sufficient chemical or physical control can be used and, in both cases, goats can be used as follow up control.

coexistence between grasses and trees. There are species which are not preferred by animals for foraging, and use of biological control through herbivores would not be effective; therefore, introduction of invertebrates could be used. However, most of the invertebrates are not readily available in Southern Africa for use at the farm or landscape level. It is impractical to burn certain areas that are encroached; this is sometimes due to poor fuel load that can support high fire intensity needed to burn the woody species. Encroachment in some of these areas cannot

All this, therefore, suggests that there are areas and bush encroachment situations where a single method can be used; however, a combination of different methods could be used simultaneously or alternatively in subsequent approaches. Nevertheless, it is important that prior to the implementation of any selected method or any combination or any sequence to develop post encroachment treatment management plan. This is because removal of bush with any technique can leave the land vulnerable to soil erosion or further encroachment of the same or new species. Therefore, a successful long-term management program should be designed to include combinations of mechanical, biological, and chemical control techniques. Numerous mechanical and cultural options have been developed to manage noxious range‐ land weeds, including mowing, prescribed burning, timely grazing, and perennial grass reseeding or inter-seeding. Furthermore, several herbicides are registered for use on range‐ lands and most biological control programs focus on noxious rangeland weed control. Successful management of noxious weeds on rangeland will require the development of a long-term strategic plan incorporating prevention programs, education materials and activi‐ ties, economical and sustainable multi-year integrated approaches that improve degraded rangeland communities, enhance the utility of the ecosystem, and prevent reinvasion or

There are a number of factors to consider in selection of the bush control method; however, the dominant consideration is the cost of the method. However, there are furthermore considerations beyond the cost of the method. The use of fire in controlling bush encroachment in rangelands is determined by a threshold amount of flammable fine fuel needed to carry fire that is sufficiently intensive to reduce woody plants. Furthermore, to effectively control woody plants with burning, fire must be applied regularly. Many rangelands occur in semi-arid environments in which forage-based livestock production is the primary agricultural activity and intermittent droughts are inevitable [205]). Therefore, accumulating sufficient fine fuel to carry fires in such environments requires the reduction in livestock numbers compared to areas where fire is not used. Hence, sustainable utilisation of semi-arid rangelands depends on complex management of animal species, stocking rates, and the vegetation composition,

The integration of bio-control agents and herbicides in a scientifically sound and rigorous management plan is the first step in a long-term approach to weed management. Such management plans should aim to maximise the benefits of all the respective control options and thereby ensure the infestation is contained and the density reduced to acceptable thresh‐ olds. Biological control is used as an important, long-term management solution to numerous weeds worldwide. When carefully integrated into management plans the combination of bio-

be controlled with the use of herbivores (goats) and herbicides could be useful.

encroachment by other noxious weed species [151].

294 Herbicides - Current Research and Case Studies in Use

structure, phenology and quality [129].

In this chapter, integration in the control of invasive species is not limited to control methods themselves in isolation but in all the processes relating to bush encroachment management. Primarily, it is important as the initial stage of integration to identify and characterise invasion/ encroachment of species. This should include establishment of their origin, mode of establish‐ ment and spread (seeds, cuttings etc), their phenological and morphological characteristics and assessing their favourable growth conditions. It is further important to determine the degree of invasion/encroachment, which will help setting economic and ecological thresholds of invasion. The analysis of the ecological and economic impact of invasion/encroachment in the environment should be carried out prior to any intervention. That will help in determining whether there is a need for intervention and magnitude of such intervention. The need for intervention should be assessed against the set thresholds for invasion. Setting objectives for invasion/encroachment management is very fundamental because the objectives will be used as the yardstick for the control.

A number of factors will guide selection of the approach to control bush encroachment. These factors include species to be controlled, the stage of invasion and landscape of an area. The approach to be selected would be chemical, mechanical and biological depending on the approach suited to the species to be controlled, the major landscape on which the invasion has occurred and the stage of invasion. The method that is ecologically and economically sound and practical should be selected. Integrated bush encroachment approaches may be practiced in combinations that could either be used simultaneously, alternatively or sequentially. In simultaneous integration of bush control methods, more than one method that could comple‐ ment each other under the prospects of chemical, biological or physical methods used together. Some methods cannot be used simultaneously because of the danger that they can cause on other organisms and environment. For example, the methods that can be integrated simulta‐ neously could be manual clearance and use of goats as browsers. The alternative integration could be executed through turns, thus, one method first and then the other. The alternate integration can be practiced in rotation if planned properly, for example, use fire with a given period in between goat treatment. Thus, burning can be applied every three years while goat use is continued. Sequential integration is executed in succession of methods where one method can be used to prepare for the next method in a sequence. In this integration there should be short-term objectives relating to each method and long-term objectives, which are based on the integrated approach. Thus, mechanical control in the form of fire or physical cutting can be used to reduce plant height to facilitate the use by goats as the maintenance stage of control. Where there is high density of bush, which impairs the movement of animals, or where the bush is above the browse line of goats or where the bush is unacceptable to browsing animals yet the fuel load is poor, mechanical cutting would be useful. This would reduce the bush density, which will open up for goats to be able to browse, that will further open up for grass to grow then fire can be used as a follow up. Where the bush has higher density but there is sufficient fuel load, fire will be the most applicable method. Fire will clear up the bush faster and relatively cost effectively, therefore, where there is enough fuel load fire is recommended as the first on the integration followed by use of goats. Biological control would always be the last in the sequence and it is the approach that helps in achieving longterm bush encroachment control objectives. The use of invertebrates (Weevils) could be integrated with the use of herbivore (goats) since the weevils take care of the seeds and the goats can take of the foliage to maintain the stands.

evaluate performance; however, performance measures should specifically address manage‐ ment goals and objectives and should be quantifiable, expressing status and trends of specific

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297

Bush encroachment and invasion could be attributed to a number of factors, which by their nature vary with species and locality. These factors cannot easily be ranked according to the strength of causation and/or according to the intensity of their effect on rangeland ecosystems. Factors that are blamed for bush encroachment include improper grazing practices, suppres‐ sion of fire, drought, rainfall intensity and distribution and climate change. The temporal and spatial distribution of bush encroachment follows a sigmoid distribution curve. Although some invasive species are abundant, they are localised in certain areas whilst, on the other hand, certain species are widely distributed but low in copiousness. There are three major methodological guidelines; these fall under chemical, mechanical/physical and biological and depend on a number of factors within economic and ecological impressions. Bush encroach‐ ment occurrences are generally caused by different factors, at different landscapes, by different plant species and with different effects. Therefore, the invasion control methods should consider this variation for success in treatments. Thus, there are areas and invasion situations where a single method can be used; however, a combination of different methods could be

Integrated plant invasion management should have four major stages of execution; these are comprised of diagnostic, preventative, control and management. The diagnostic stage should include identification and characterisation of invasion, determination of the degree of invasion, analysis of the ecological and economic impact of invasion, determination of the need for intervention, and setting objectives for intervention. The control stage should include selection of invasion control approach or combinations. Management stage includes post-treatment management, monitoring, evaluation, and ecosystems' performance appraisal. Preventative stage is more practical on the areas that are not yet invaded; at this stage management of areas that are not yet encroached is central. Assessment and characterisation of vulnerable areas for invasion will be important in developing an encroachment prevention plan. It is also important to assess plant invasion predisposing factors; however, these may vary with species and localities. In the diagnostic stage, determination of the level of spread is very fundamental and will serve as the background for selection of the bush encroachment control and management methods. The stage of bush encroachment spread can be divided into four broad phases viz, entry phase, adaptation and establishment phase, an exponential growth phase and domi‐ nance phase. It is in the exponential growth stage of weeds spread that integrated control programmes find a logical relevance. Prevention, and early detection and eradication, are more appropriate for the first two stages, while options may be severely limited once weed popu‐ lations reach the final stage of total ecosystem domination. Although there is massive literature on the plant invasion and bush encroachment, there is still a significant need for further research in establishing fundamental characteristics of bush encroachment phenomenon in

resource values of concern, such as unique ecosystem type.

used in simultaneous or alternative or subsequence approaches.

**5. Conclusions and recommendations**

A post treatment management plan should be part of integration in bush encroachment control, thus, there should be a clear plan on what rangeland management system will be practiced that will ensure that the control objectives are achieved. Thus, some invasion control methods such as the use of fire can leave the soil bare and susceptible to soil erosion and, therefore, there should be a clear objective plan on what practices will be taken immediately after treatment. Furthermore, on the areas that are severely encroached and grass biomass and basal cover are affected, use of herbicides will also leave the soil bare and grazing can worsen the situation and lead to soil erosion. Therefore, as part of integration, exclusion of treated areas to minimise grazing should be considered. This exclusion could be coupled with introduction of plant propagules, thus, revegetation through seeds or seedlings of the grass on the bare patches.

There is a need for periodic monitoring and evaluation as part of integration of the encroach‐ ment control. This will help in determining whether the treatment is achieving expected results within the given timeframes. That will help in realising if there is a need for the adjustment of the plan. Effective bush control monitoring and evaluation should be done according to the pre set objectives; it will help in the establishment of whether the objectives are achieved. Performance measures, monitoring, and adaptive management are necessary. Using these methods, status and trends can be tracked, analysis and accountability facilitated, and decisions adapted so that the intended balance among social, economic, and ecological concerns is achieved. Ecosystems' performance appraisal will be important at the end of the integration, this should be a pronouncement of whether the target ecosystem has been reached and should be coupled with sustainability management programme that will eliminate factors that could have lead to encroachment. Ecosystem performance measures can provide a quantitative basis for evaluating how well actions under the integrated bush control approach are meeting stated objectives. Performance measures allow for continuous learning, which broadens understanding about how ecosystems function. There are many approaches to evaluate performance; however, performance measures should specifically address manage‐ ment goals and objectives and should be quantifiable, expressing status and trends of specific resource values of concern, such as unique ecosystem type.

### **5. Conclusions and recommendations**

method can be used to prepare for the next method in a sequence. In this integration there should be short-term objectives relating to each method and long-term objectives, which are based on the integrated approach. Thus, mechanical control in the form of fire or physical cutting can be used to reduce plant height to facilitate the use by goats as the maintenance stage of control. Where there is high density of bush, which impairs the movement of animals, or where the bush is above the browse line of goats or where the bush is unacceptable to browsing animals yet the fuel load is poor, mechanical cutting would be useful. This would reduce the bush density, which will open up for goats to be able to browse, that will further open up for grass to grow then fire can be used as a follow up. Where the bush has higher density but there is sufficient fuel load, fire will be the most applicable method. Fire will clear up the bush faster and relatively cost effectively, therefore, where there is enough fuel load fire is recommended as the first on the integration followed by use of goats. Biological control would always be the last in the sequence and it is the approach that helps in achieving longterm bush encroachment control objectives. The use of invertebrates (Weevils) could be integrated with the use of herbivore (goats) since the weevils take care of the seeds and the

A post treatment management plan should be part of integration in bush encroachment control, thus, there should be a clear plan on what rangeland management system will be practiced that will ensure that the control objectives are achieved. Thus, some invasion control methods such as the use of fire can leave the soil bare and susceptible to soil erosion and, therefore, there should be a clear objective plan on what practices will be taken immediately after treatment. Furthermore, on the areas that are severely encroached and grass biomass and basal cover are affected, use of herbicides will also leave the soil bare and grazing can worsen the situation and lead to soil erosion. Therefore, as part of integration, exclusion of treated areas to minimise grazing should be considered. This exclusion could be coupled with introduction of plant propagules, thus, revegetation through seeds or seedlings of the grass

There is a need for periodic monitoring and evaluation as part of integration of the encroach‐ ment control. This will help in determining whether the treatment is achieving expected results within the given timeframes. That will help in realising if there is a need for the adjustment of the plan. Effective bush control monitoring and evaluation should be done according to the pre set objectives; it will help in the establishment of whether the objectives are achieved. Performance measures, monitoring, and adaptive management are necessary. Using these methods, status and trends can be tracked, analysis and accountability facilitated, and decisions adapted so that the intended balance among social, economic, and ecological concerns is achieved. Ecosystems' performance appraisal will be important at the end of the integration, this should be a pronouncement of whether the target ecosystem has been reached and should be coupled with sustainability management programme that will eliminate factors that could have lead to encroachment. Ecosystem performance measures can provide a quantitative basis for evaluating how well actions under the integrated bush control approach are meeting stated objectives. Performance measures allow for continuous learning, which broadens understanding about how ecosystems function. There are many approaches to

goats can take of the foliage to maintain the stands.

296 Herbicides - Current Research and Case Studies in Use

on the bare patches.

Bush encroachment and invasion could be attributed to a number of factors, which by their nature vary with species and locality. These factors cannot easily be ranked according to the strength of causation and/or according to the intensity of their effect on rangeland ecosystems. Factors that are blamed for bush encroachment include improper grazing practices, suppres‐ sion of fire, drought, rainfall intensity and distribution and climate change. The temporal and spatial distribution of bush encroachment follows a sigmoid distribution curve. Although some invasive species are abundant, they are localised in certain areas whilst, on the other hand, certain species are widely distributed but low in copiousness. There are three major methodological guidelines; these fall under chemical, mechanical/physical and biological and depend on a number of factors within economic and ecological impressions. Bush encroach‐ ment occurrences are generally caused by different factors, at different landscapes, by different plant species and with different effects. Therefore, the invasion control methods should consider this variation for success in treatments. Thus, there are areas and invasion situations where a single method can be used; however, a combination of different methods could be used in simultaneous or alternative or subsequence approaches.

Integrated plant invasion management should have four major stages of execution; these are comprised of diagnostic, preventative, control and management. The diagnostic stage should include identification and characterisation of invasion, determination of the degree of invasion, analysis of the ecological and economic impact of invasion, determination of the need for intervention, and setting objectives for intervention. The control stage should include selection of invasion control approach or combinations. Management stage includes post-treatment management, monitoring, evaluation, and ecosystems' performance appraisal. Preventative stage is more practical on the areas that are not yet invaded; at this stage management of areas that are not yet encroached is central. Assessment and characterisation of vulnerable areas for invasion will be important in developing an encroachment prevention plan. It is also important to assess plant invasion predisposing factors; however, these may vary with species and localities. In the diagnostic stage, determination of the level of spread is very fundamental and will serve as the background for selection of the bush encroachment control and management methods. The stage of bush encroachment spread can be divided into four broad phases viz, entry phase, adaptation and establishment phase, an exponential growth phase and domi‐ nance phase. It is in the exponential growth stage of weeds spread that integrated control programmes find a logical relevance. Prevention, and early detection and eradication, are more appropriate for the first two stages, while options may be severely limited once weed popu‐ lations reach the final stage of total ecosystem domination. Although there is massive literature on the plant invasion and bush encroachment, there is still a significant need for further research in establishing fundamental characteristics of bush encroachment phenomenon in rangelands. This will lead in systematic characterisation of bush encroachment and subse‐ quently that will lead to development of more practical and radical yet scientific bush encroachment control and management practices in rangelands.

[9] Van Auken, O. W. (2009). Causes and consequences of woody plant encroachment into western North American grasslands. Journal of Environental Management, , 90,

Integrated Plant Invasion and Bush Encroachment Management on Southern African Rangelands

http://dx.doi.org/10.5772/56182

299

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### **References**


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rangelands. This will lead in systematic characterisation of bush encroachment and subse‐ quently that will lead to development of more practical and radical yet scientific bush

and B. Moyo1

encroachment control and management practices in rangelands.

\*Address all correspondence to: lesolistar@gmail.com

, T. B. Solomon2

1 Fort Cox College of Agriculture and Forestry, King Williams Town, South Africa

2 Department of Livestock and Pasture Science, University of Fort Hare, Alice, South Africa

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**Chapter 12**

**New Natural Herbicide Candidate for**

*Sicyon angulatus* **Control**

Jung-Sup Choi and In-Taek Hwang

http://dx.doi.org/10.5772/54964

**1. Introduction**

Additional information is available at the end of the chapter

Most synthetic herbicides are used for controlling troublesome weed species in modern agriculture all over the world. However, consecutive use of the same herbicide brings about resistant weed problems and many countries are restricting repeated treatment in agricultural lands [1]. For these and environmental reasons, new herbicide discovery and subsequent registration is very challenging. Recently, evaluating natural products of ani‐ mals, plants, microorganisms and minerals for developing environmental friendly herbi‐ cides has increased [2]. Several compounds have been developed or in development as natural herbicides such as bialaphos [3], methoxyhygromycin (MHM) [4], and pelargonic acid [5]. Essential oils such as clove oil and cinnamon oil also contain allelochemicals that control a broad spectrum of weeds and can be used as natural herbicide source [6,7]. Plumbagin isolated from *Drosophyllum lusitanicum* and *Plumbago auriculata* inhibited the seed germination of lettuce and wheat [8,9]. Several classes of natural compounds in‐ cluding triketones, benzoquinones, naphthoquinones and anthraquinones have been re‐ ported as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and hence the novel classes of HPPD inhibitors could be developed based on their structural backbones [10].

Agricultural research for herbicide discovery with new target site is increasing due to the demand from farmers and multinational companies. Even so, new mode of action have not been succesfully introduced in the past 10 years [2,3]. We have recently reported : 7-keto-8 aminopelargonic acid synthase (EC 2.3.1.47, KAPAS, also known as 8-amino-7-oxononanoate synthase, AONS) and have suggested the potential KAPAS inhibitor triphenyltin [11]. KAPAS is a pyridoxal 5'-hophate dependent enzyme which catalyzes the decarboxylative condensa‐ tion of L-alanine with pimeloyl-CoA in a stereospecific manner to form7-keto-8-aminopelar‐

> © 2013 Choi and Hwang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 Choi and Hwang; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **Chapter 12**

## **New Natural Herbicide Candidate for** *Sicyon angulatus* **Control**

Jung-Sup Choi and In-Taek Hwang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54964

### **1. Introduction**

Most synthetic herbicides are used for controlling troublesome weed species in modern agriculture all over the world. However, consecutive use of the same herbicide brings about resistant weed problems and many countries are restricting repeated treatment in agricultural lands [1]. For these and environmental reasons, new herbicide discovery and subsequent registration is very challenging. Recently, evaluating natural products of ani‐ mals, plants, microorganisms and minerals for developing environmental friendly herbi‐ cides has increased [2]. Several compounds have been developed or in development as natural herbicides such as bialaphos [3], methoxyhygromycin (MHM) [4], and pelargonic acid [5]. Essential oils such as clove oil and cinnamon oil also contain allelochemicals that control a broad spectrum of weeds and can be used as natural herbicide source [6,7]. Plumbagin isolated from *Drosophyllum lusitanicum* and *Plumbago auriculata* inhibited the seed germination of lettuce and wheat [8,9]. Several classes of natural compounds in‐ cluding triketones, benzoquinones, naphthoquinones and anthraquinones have been re‐ ported as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and hence the novel classes of HPPD inhibitors could be developed based on their structural backbones [10].

Agricultural research for herbicide discovery with new target site is increasing due to the demand from farmers and multinational companies. Even so, new mode of action have not been succesfully introduced in the past 10 years [2,3]. We have recently reported : 7-keto-8 aminopelargonic acid synthase (EC 2.3.1.47, KAPAS, also known as 8-amino-7-oxononanoate synthase, AONS) and have suggested the potential KAPAS inhibitor triphenyltin [11]. KAPAS is a pyridoxal 5'-hophate dependent enzyme which catalyzes the decarboxylative condensa‐ tion of L-alanine with pimeloyl-CoA in a stereospecific manner to form7-keto-8-aminopelar‐

© 2013 Choi and Hwang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Choi and Hwang; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gonic acid, Coenzyme A, and carbon dioxide in the first committed step of biotin biosynthesis. Perhaps the most important role of biotin is in the carboxylation of acetyl-CoA to give malonyl-CoA, which is the first step in fatty-acid biosynthesis. Since fatty-acid synthesis is essential for the growth and development of most organisims, biotin is thus an essential nutrient for plants and animals. Plants, microorganisms, and some fungi biosynthesize their own biotin, while other organisms require trace amounts of the vitamin in their diet. Therefore, inhibition of the enzymes involved in the biotin biosynthesis pathway can cause irreparable damage to plants but be non-toxic to non-plant organisims, and for this reason, such enzymes can be useful targets for the rational design of inhibitors in the hopes of finding new herbicides [12,13].

**2. Development for** *Sicyon angulatus* **control**

available plumbagin was estimated over 90% by HPLC.

**2.2. Plumbagin as a KAPAS inhibitor**

controlled at 30o

1 mM NAD+

h at 4o

The specimens of *P. auriculata* grown in the greenhouse were collected, and the air-dried root (180 g) was soaked in 2 L of acetone at room temperature for 7 days. The extract was filtered and evaporated to dryness under negative pressure. The concentrated extract (1.5 g) was suspended in 100 ml of water and re-extracted with an equal volume of dichloromethane, which afforded 1.2 g of dichloromethane soluble fraction. The dichloromethane soluble fraction was subjected to silica gel column chromatography eluted with a mixture of hexane and ethyl acetate (20:1) to give 120 mg of plumbagin as a dark yellow crystal. The spectral data of isolated plumbagin (purity > 99%), such as UV, MS and 1H NMR and 13C-NMR were well accorded with the result of Bhattacharyya and Carvalho [24]. For field trial, plumbagin (5 hydroxy-2-methyl-1,4-naphthoquinone) was purchased from Sigma–Aldrich, which was originally isolated from Plumbago indica (Plumbaginaceae). The purity of commercially

New Natural Herbicide Candidate for *Sicyon angulatus* Control

http://dx.doi.org/10.5772/54964

317

The full-length of AtKAPAS cDNA was amplified and isolated from *Arabidopsis thaliana* cDNA and cloned into MBP fusion vector to generate the *Escherichia coli* expression construct pEMBPek-KAPAS [11]. SDS–PAGE analysis revealed that *E. coli* transformed with MBP fusion vector showed the expression of a very strongly induced fusion protein of ca. 98.2 kDa, which consisted of the AtKAPAS protein of 51.3 kDa and the maltose binding peptide MBP affinity tag of 46.9 kDa [11,32]. Pimeloyl-CoA was synthesized according to the method described previously [25]. KAPAS activity was determined according to the method described previ‐ ously [12] using a linked assay by monitoring the increase in absorption of NADH at 340 nm using a Microplate Spectrophotometer (Benchmark Plus, Bio-rad, USA), thermostatically

significant portion of these proteins was soluble, and their affinity-purified preparations contained a single major polypeptide. The lysates from IPTG-induced *E. coli* containing pEMBPek-KAPAS as well as from *E. coli* harboring control vector MBP fusion vector were loaded onto maltose affinity column (1.1 cm x 30 cm, Millipore, USA). The AtKAPAS protein bound to MBP resin was eluted with 10 mM maltose solution. A typical assay contained 20 mM potassium phosphate (pH 7.5), 1 mM α-ketoglutarate, 0.25 mM thiamine pyrophosphate,

(3 mg protein/ml) in a total volume of 200 μL. L-Alanine and pimeloyl-Co A were added to give the desirable final concentrations. Prior to analysis, enzyme samples were dialyzed for 2

C against 20 mM potassium phosphate (pH 7.5) containing 100 μM PLP. The KAPAS concentration in all analyses was 10 μM in 20 mM potassium phosphate (pH 7.5). 96-well microplates containing each 528 natural compounds prepared from various medicinal plants and exotic herbs were evaluated on KAPAS inhibition assay at the concentration of 1 mM. Through the consecutive experiment at lower concentration against samples showing 90% inhibition of KAPAS activity, plumbagin were selected as the most effective KAPAS inhibitor.

C. At KAPAS protein was expressed in *E. coli* at a very high level, and a

, 3 mM MgCl2, 0.1 unit of α-ketoglutarate dehydrogenase, and 2–10 μg of KAPAS

**2.1. Plumbagin preparation**

Also, we attempted to search for KAPAS inhibitors from plant-derived natural com‐ pounds. Several naturally occurring quinones including chrysophanic acid, tanshinones, 5,8-dihydroxy-1,4-naphthoquinone, and plumbagin was selected as potent inhibitors against KAPAS. We evaluated the plumbagin showing most effective KAPAS inhibition, as a natural herbicide under greenhouse and field tests. Field tests were focused on the annual noxious weed species of *Sicyos angulatus* (burcucumber or star-cucumber) which have migrated from eastern North America and have been designated as one of the eco‐ logical disturbance plants listed by the Ministry of Environment in Korea. The alien plant *S. angulatus* was first observed in 1989 and rapidly emerged in the marginal of ag‐ ricultural fields close to riparian zone where it has been rapidly spreading along rivers in Korea over the past two decades [14,15]. Invasion into the natural ecosystems by exot‐ ic species is a major global threat to biodiversity. *S. angulatus* was also listed in Federal and State Noxious Weeds, USA and its geographical distribution was published in the OEPP/EPPO Bulletin [16]. It is adapted to wet habitats: deciduous swamps, woodland floodplains, and river floodplains. It also colonizes open habitats along fencerows, road‐ sides, and woodland borders. *S. angulatus* is found in every state east of the Rocky Mountains and also found in Canada's eastern provinces, Mexico, the Caribbean, and Eastern Asia. It was first introduced to Europe as an ornamental plant, but has since es‐ caped cultivation and become a weedy invasive species. Asaeda et al. [17] reported the most dominant liana species in the floodplain is *S. angulatus* and it was first sighted in Japan in 1952. Ceschin et al. [18] reported exotic species of *S. angulatus* as a new arrival alien in the Tiber River in Rome. Many reports of its invasiveness have been published in the United Kingdom [19], Norway [20], Japan [21], Korea [14], and Spain [22] etc.

In this chapter, we briefly describe the KAPAS inhibitory activity of plumbagin, which showed the most potent inhibition during the preliminary survey of many natural prod‐ ucts. Also the herbicidal activity of plumbagin was evaluated under greenhouse condi‐ tions and field trials. Physiological responses caused by the plumbagin treatment with respect to cellular leakage, chlorophyll loss and the rescue effect with biotin supplement through tissue section experiments or seed germination are reported. Plumbagin is under examination as a LOHAS (Lifestyles of Health and Sustainability) [23] herbicide against an invasive alien vine plant species.

### **2. Development for** *Sicyon angulatus* **control**

#### **2.1. Plumbagin preparation**

gonic acid, Coenzyme A, and carbon dioxide in the first committed step of biotin biosynthesis. Perhaps the most important role of biotin is in the carboxylation of acetyl-CoA to give malonyl-CoA, which is the first step in fatty-acid biosynthesis. Since fatty-acid synthesis is essential for the growth and development of most organisims, biotin is thus an essential nutrient for plants and animals. Plants, microorganisms, and some fungi biosynthesize their own biotin, while other organisms require trace amounts of the vitamin in their diet. Therefore, inhibition of the enzymes involved in the biotin biosynthesis pathway can cause irreparable damage to plants but be non-toxic to non-plant organisims, and for this reason, such enzymes can be useful targets for the rational design of inhibitors in the hopes of finding new herbicides [12,13].

Also, we attempted to search for KAPAS inhibitors from plant-derived natural com‐ pounds. Several naturally occurring quinones including chrysophanic acid, tanshinones, 5,8-dihydroxy-1,4-naphthoquinone, and plumbagin was selected as potent inhibitors against KAPAS. We evaluated the plumbagin showing most effective KAPAS inhibition, as a natural herbicide under greenhouse and field tests. Field tests were focused on the annual noxious weed species of *Sicyos angulatus* (burcucumber or star-cucumber) which have migrated from eastern North America and have been designated as one of the eco‐ logical disturbance plants listed by the Ministry of Environment in Korea. The alien plant *S. angulatus* was first observed in 1989 and rapidly emerged in the marginal of ag‐ ricultural fields close to riparian zone where it has been rapidly spreading along rivers in Korea over the past two decades [14,15]. Invasion into the natural ecosystems by exot‐ ic species is a major global threat to biodiversity. *S. angulatus* was also listed in Federal and State Noxious Weeds, USA and its geographical distribution was published in the OEPP/EPPO Bulletin [16]. It is adapted to wet habitats: deciduous swamps, woodland floodplains, and river floodplains. It also colonizes open habitats along fencerows, road‐ sides, and woodland borders. *S. angulatus* is found in every state east of the Rocky Mountains and also found in Canada's eastern provinces, Mexico, the Caribbean, and Eastern Asia. It was first introduced to Europe as an ornamental plant, but has since es‐ caped cultivation and become a weedy invasive species. Asaeda et al. [17] reported the most dominant liana species in the floodplain is *S. angulatus* and it was first sighted in Japan in 1952. Ceschin et al. [18] reported exotic species of *S. angulatus* as a new arrival alien in the Tiber River in Rome. Many reports of its invasiveness have been published in the United Kingdom [19], Norway [20], Japan [21], Korea [14], and Spain [22] etc.

In this chapter, we briefly describe the KAPAS inhibitory activity of plumbagin, which showed the most potent inhibition during the preliminary survey of many natural prod‐ ucts. Also the herbicidal activity of plumbagin was evaluated under greenhouse condi‐ tions and field trials. Physiological responses caused by the plumbagin treatment with respect to cellular leakage, chlorophyll loss and the rescue effect with biotin supplement through tissue section experiments or seed germination are reported. Plumbagin is under examination as a LOHAS (Lifestyles of Health and Sustainability) [23] herbicide against

an invasive alien vine plant species.

316 Herbicides - Current Research and Case Studies in Use

The specimens of *P. auriculata* grown in the greenhouse were collected, and the air-dried root (180 g) was soaked in 2 L of acetone at room temperature for 7 days. The extract was filtered and evaporated to dryness under negative pressure. The concentrated extract (1.5 g) was suspended in 100 ml of water and re-extracted with an equal volume of dichloromethane, which afforded 1.2 g of dichloromethane soluble fraction. The dichloromethane soluble fraction was subjected to silica gel column chromatography eluted with a mixture of hexane and ethyl acetate (20:1) to give 120 mg of plumbagin as a dark yellow crystal. The spectral data of isolated plumbagin (purity > 99%), such as UV, MS and 1H NMR and 13C-NMR were well accorded with the result of Bhattacharyya and Carvalho [24]. For field trial, plumbagin (5 hydroxy-2-methyl-1,4-naphthoquinone) was purchased from Sigma–Aldrich, which was originally isolated from Plumbago indica (Plumbaginaceae). The purity of commercially available plumbagin was estimated over 90% by HPLC.

#### **2.2. Plumbagin as a KAPAS inhibitor**

The full-length of AtKAPAS cDNA was amplified and isolated from *Arabidopsis thaliana* cDNA and cloned into MBP fusion vector to generate the *Escherichia coli* expression construct pEMBPek-KAPAS [11]. SDS–PAGE analysis revealed that *E. coli* transformed with MBP fusion vector showed the expression of a very strongly induced fusion protein of ca. 98.2 kDa, which consisted of the AtKAPAS protein of 51.3 kDa and the maltose binding peptide MBP affinity tag of 46.9 kDa [11,32]. Pimeloyl-CoA was synthesized according to the method described previously [25]. KAPAS activity was determined according to the method described previ‐ ously [12] using a linked assay by monitoring the increase in absorption of NADH at 340 nm using a Microplate Spectrophotometer (Benchmark Plus, Bio-rad, USA), thermostatically controlled at 30o C. At KAPAS protein was expressed in *E. coli* at a very high level, and a significant portion of these proteins was soluble, and their affinity-purified preparations contained a single major polypeptide. The lysates from IPTG-induced *E. coli* containing pEMBPek-KAPAS as well as from *E. coli* harboring control vector MBP fusion vector were loaded onto maltose affinity column (1.1 cm x 30 cm, Millipore, USA). The AtKAPAS protein bound to MBP resin was eluted with 10 mM maltose solution. A typical assay contained 20 mM potassium phosphate (pH 7.5), 1 mM α-ketoglutarate, 0.25 mM thiamine pyrophosphate, 1 mM NAD+ , 3 mM MgCl2, 0.1 unit of α-ketoglutarate dehydrogenase, and 2–10 μg of KAPAS (3 mg protein/ml) in a total volume of 200 μL. L-Alanine and pimeloyl-Co A were added to give the desirable final concentrations. Prior to analysis, enzyme samples were dialyzed for 2 h at 4o C against 20 mM potassium phosphate (pH 7.5) containing 100 μM PLP. The KAPAS concentration in all analyses was 10 μM in 20 mM potassium phosphate (pH 7.5). 96-well microplates containing each 528 natural compounds prepared from various medicinal plants and exotic herbs were evaluated on KAPAS inhibition assay at the concentration of 1 mM. Through the consecutive experiment at lower concentration against samples showing 90% inhibition of KAPAS activity, plumbagin were selected as the most effective KAPAS inhibitor. IC50 value of KAPAS inhibition by plumbagin was calculated from the regression curve prepared with the extensive assay performed with the plumbagin ranged from 0.1 to 250 μM with five replications. A reference was prepared with all components except plumbagin.

**2.3. Herbicidal activity of plumbagin**

Herbicidal activity and spectrum of plumbagin were investigated against eight weed species, consisting of three grass species of *Sorghum bicolor* (sorghum), *Echinochloa crus-galli* (barnyard grass), *Digitaria sanguinalis* (large crabgrass) and five broad leaf species of *Solanum nigrum* (black nightshade), *Aeschynomene indica* (Indian joint vetch), *Abutilon avicennae* (velvetleaf), *Xanthium strumarium* (common cocklebur), *Calystegia japonica* (Japanese bindweed). Seeds of weeds for foliar application were germinated in a commercial greenhouse substrate (Boo-Nong Soil, Seoul, Korea) and watered with tap water. About five plants were grown at 30/20 ± 3o

day/night temperature with an about 14 h photoperiod for 12 days under greenhouse. Foliar ap‐ plication was conducted at 12 days after sowing, the test solution was sprayed into the test pot grown with 10 ~ 15 seedlings of sorghum, barnyard grass, large crabgrass, black nightshade, In‐ dian joint vetch and velvetleaf, and two seedling of common cocklebur and Japanese bindweed. Various concentrations of the purified plumbagin from *P. auriculata* prepared with 50% acetone solution containing 0.1% Tween-20 were sprayed onto plants with a laboratory spray gun deliv‐ ering spray volume of 5 ml per pot. The control treatment recieved the same volume of spray without plumbagin. After treatment, the plants were placed in a vented cabinet to dry and re‐ turned to the same greenhouse without replication. At 5 days after treatments, visual injury of plants assessed on a scale from 0 (no injury) to 100 (complete death). A field trial was performed against 10 ~ 15 leaf-stage and 2 ~ 3 m vine length of natural *S. angulatus* habitats around riparian zones in Nam-Han River. Foliar applications were conducted with 1,000 and 2,000 μg/mL of plumbagin in 50% acetone solution containing 0.1% Tween-20 using a laboratory sprayer deliv‐

out plumbagin. The field trial was performed from 22th September to 6th October, 2011, and the

Under greenhouse conditions, all eight weed species were completely controlled by the fo‐ liar application of 1,000 and 2,000 μg/mL plumbagin, while 500 μg/mL applications also showed 100% herbicidal efficacy against seven weed species with the exception of *A. avicen‐ nae* (Fig. 2). 250 μg/mL applications against eight weeds showed 60 ~ 100% control (Table 1), and especially a concentration as low as 32 μg/mL had a herbicidal efficacy of 70% on *D. sanguinalis* (data not shown). With a plumbagin treatment of eight weed species, the main herbicidal symptoms were desiccation or extensive necrosis within 2 h. The difference of symptoms caused by the plumbagin between grass species and broad leaf species was insig‐ nificant after foliar application. Field test results revealed that the natural compound plum‐ bagin controlled alien weed *S. angulatus* completely at 2,000 μg/mL under foliar application. Visual symptoms of plant injury after plumbagin foliar application against natural *S. angula‐ tus* were desiccation or burn down within 2 h after treatment. Control values were evaluated as 95–100% by a visual rating scale of 0–100 at 5, 8, and 14 days after treatment with 1,000 or

and 14 days after treatments. Test plots were situated directly adjacent to each other.

2,000 μg/mL. The residual activity lasted for 2 weeks without any regrowth.

with a control treatment of the same preparation solution with‐

plot size. The control value was evaluated visually at 5, 8,

New Natural Herbicide Candidate for *Sicyon angulatus* Control

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C,

319

*2.3.1. Materials and methods*

ering spray volume of 300 ml/m2

*2.3.2. Results*

trial contained three replicates of 1 m2

Enzyme activity was tested with the partially purified AtKAPAS protein extracted from transgenic *E. coli*. AtKAPAS protein was expressed *in E. coli* at a very high level, and a significant portion of these proteins was soluble, and their affinity-purified preparations contained a single major polypeptide. The inhibitory effect of 528 plant-derived natural compounds collected in Korea Chemical Bank, KRICT on KAPAS was evaluated using the partially purified AtKAPAS protein, *in vitro*. Less than 2% of tested compounds exhibited significant inhibitory effect on KAPAS at the concentration lower than 20 μM. Interestingly, several naturally occurring quinones including chrysophanic acid, tanshinones, 5,8-dihy‐ droxy-1,4-naphthoquinone, and plumbagin were observed to give a potent inhibitory effect on KAPAS. Plumbagin, a natural naphthoquinone demonstrated the most effective inhibitory effect on KAPAS with an IC50 of 2.1 μM (Fig. 1).

**Figure 1.** KAPAS inhibition by plumbagin *in vitro* assay. Vertical bars represent standard deviation. In some cases the vertical bar is obscured by the datum symbol.

#### **2.3. Herbicidal activity of plumbagin**

#### *2.3.1. Materials and methods*

IC50 value of KAPAS inhibition by plumbagin was calculated from the regression curve prepared with the extensive assay performed with the plumbagin ranged from 0.1 to 250 μM with five replications. A reference was prepared with all components except plumbagin.

Enzyme activity was tested with the partially purified AtKAPAS protein extracted from transgenic *E. coli*. AtKAPAS protein was expressed *in E. coli* at a very high level, and a significant portion of these proteins was soluble, and their affinity-purified preparations contained a single major polypeptide. The inhibitory effect of 528 plant-derived natural compounds collected in Korea Chemical Bank, KRICT on KAPAS was evaluated using the partially purified AtKAPAS protein, *in vitro*. Less than 2% of tested compounds exhibited significant inhibitory effect on KAPAS at the concentration lower than 20 μM. Interestingly, several naturally occurring quinones including chrysophanic acid, tanshinones, 5,8-dihy‐ droxy-1,4-naphthoquinone, and plumbagin were observed to give a potent inhibitory effect on KAPAS. Plumbagin, a natural naphthoquinone demonstrated the most effective inhibitory

**Figure 1.** KAPAS inhibition by plumbagin *in vitro* assay. Vertical bars represent standard deviation. In some cases the

effect on KAPAS with an IC50 of 2.1 μM (Fig. 1).

318 Herbicides - Current Research and Case Studies in Use

vertical bar is obscured by the datum symbol.

Herbicidal activity and spectrum of plumbagin were investigated against eight weed species, consisting of three grass species of *Sorghum bicolor* (sorghum), *Echinochloa crus-galli* (barnyard grass), *Digitaria sanguinalis* (large crabgrass) and five broad leaf species of *Solanum nigrum* (black nightshade), *Aeschynomene indica* (Indian joint vetch), *Abutilon avicennae* (velvetleaf), *Xanthium strumarium* (common cocklebur), *Calystegia japonica* (Japanese bindweed). Seeds of weeds for foliar application were germinated in a commercial greenhouse substrate (Boo-Nong Soil, Seoul, Korea) and watered with tap water. About five plants were grown at 30/20 ± 3o C, day/night temperature with an about 14 h photoperiod for 12 days under greenhouse. Foliar ap‐ plication was conducted at 12 days after sowing, the test solution was sprayed into the test pot grown with 10 ~ 15 seedlings of sorghum, barnyard grass, large crabgrass, black nightshade, In‐ dian joint vetch and velvetleaf, and two seedling of common cocklebur and Japanese bindweed. Various concentrations of the purified plumbagin from *P. auriculata* prepared with 50% acetone solution containing 0.1% Tween-20 were sprayed onto plants with a laboratory spray gun deliv‐ ering spray volume of 5 ml per pot. The control treatment recieved the same volume of spray without plumbagin. After treatment, the plants were placed in a vented cabinet to dry and re‐ turned to the same greenhouse without replication. At 5 days after treatments, visual injury of plants assessed on a scale from 0 (no injury) to 100 (complete death). A field trial was performed against 10 ~ 15 leaf-stage and 2 ~ 3 m vine length of natural *S. angulatus* habitats around riparian zones in Nam-Han River. Foliar applications were conducted with 1,000 and 2,000 μg/mL of plumbagin in 50% acetone solution containing 0.1% Tween-20 using a laboratory sprayer deliv‐ ering spray volume of 300 ml/m2 with a control treatment of the same preparation solution with‐ out plumbagin. The field trial was performed from 22th September to 6th October, 2011, and the trial contained three replicates of 1 m2 plot size. The control value was evaluated visually at 5, 8, and 14 days after treatments. Test plots were situated directly adjacent to each other.

#### *2.3.2. Results*

Under greenhouse conditions, all eight weed species were completely controlled by the fo‐ liar application of 1,000 and 2,000 μg/mL plumbagin, while 500 μg/mL applications also showed 100% herbicidal efficacy against seven weed species with the exception of *A. avicen‐ nae* (Fig. 2). 250 μg/mL applications against eight weeds showed 60 ~ 100% control (Table 1), and especially a concentration as low as 32 μg/mL had a herbicidal efficacy of 70% on *D. sanguinalis* (data not shown). With a plumbagin treatment of eight weed species, the main herbicidal symptoms were desiccation or extensive necrosis within 2 h. The difference of symptoms caused by the plumbagin between grass species and broad leaf species was insig‐ nificant after foliar application. Field test results revealed that the natural compound plum‐ bagin controlled alien weed *S. angulatus* completely at 2,000 μg/mL under foliar application. Visual symptoms of plant injury after plumbagin foliar application against natural *S. angula‐ tus* were desiccation or burn down within 2 h after treatment. Control values were evaluated as 95–100% by a visual rating scale of 0–100 at 5, 8, and 14 days after treatment with 1,000 or 2,000 μg/mL. The residual activity lasted for 2 weeks without any regrowth.

**2.4. Reversal study** 

**2.4.1. Materials and methods** 

for each measurement were triplicates.

**2.4.2. Results** 


1)Herbicida1 activity was determined 7 days after treatment by visual injury. SORBI, Sorghum bicolor (sorghum); ECHCG, Echinochloa crus-galli (barnyard grass); DIGSA, Digitaria sanguinalis (large crabgrass); SOLNI, Solanum nigrum (black night‐ shade); AESIN, Aeschynomene indica (Indian joint vetch); ABUTH,Abutilon avicennae (velvetleai); XANSI,Xanthium struma‐ rium (common cocklebur); CAGEH, Calystegia japonica (Japanese bindweed). ' 2,000 pg/ml. can change to 4 kg/ha.

**Table 1.** Herbicidal efficacy of plumbagin post-emergence foliar application against several weeds in a greenhouse condition Table 1. Herbicidal efficacy of plumbagin post-emergence foliar application against several weeds in a greenhouse condition

1)Herbicida1 activity was determined 7 days after treatment by visual injury. SORBI, Sorghum bicolor (sorghum); ECHCG, Echinochloa crus-galli (barnyard grass); DIGSA, Digitaria sanguinalis (large crabgrass); SOLNI, Solanum nigrum (black nightshade); AESIN, Aeschynomene indica (Indian joint vetch); ABUTH,Abutilon avicennae (velvetleai); XANSI,Xanthium

**2.4. Reversal study**

*2.4.1. Materials and methods*

growth chamber at 25o

*2.4.2. Results*

All treatments for each measurement were triplicates.

*ana* seeds caused by the treatment of plumbagin.

1)Germination rate of *A. thaliana* seed at 7 days after application.

**Table 2.** Reversal effect of *Arabidopsis thaliana* seed germination with biotin supplement

Seeds of *A. thaliana* were germinated on a 55 mm Polystyrene Petri-dish lined with one-layer filter paper (Advantec No. 2). One milliliter of each plumbagin solution dis‐ solved in absolute acetone with various concentrations of 0, 25, 50 and 100 μM was dropped evenly onto the filter paper and placed in a vented cabinet to dry. After complete drying, 1 ml of distilled water with or without supplement of 0, 0.25, 0.5 and 1 mM biotin (Sigma, USA) was added, and 30 seeds were placed onto the filter paper in Petri-dish. Each Petri-dish was sealed with laboratory film and incubated in a

calculated with the number of germinated *A. thaliana* seeds at 7 days after application.

The inhibited germination of *A. thaliana* seeds treated with plumbagin was significantly rescued in a dose dependent manner by biotin supplement. Germination rate of *A. thaliana* seeds at plumbagin levels of 25, 50, and 100 μM was 33.3%, 23.3%, and 16.7%, respectively. However, the inhibited germination by plumbagin was negated up to 93.3%, 86.7%, and 83.3% with the supplement of 1 mM biotin, and also it was negated up to 66.7%, 63.3%, and 60.0% with the supplement of 0.5 mM biotin, respectively (Ta‐ ble 2, Fig. 3). Biotin supplement apparently rescued the inhibited germination *A. thali‐*

C, 14/10 h (Light/Dark). Germination inhibition percentages were

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Figure 2. Herbicidal symptoms of post-emergence foliar application of plumbagin (g/mL). (A) Pot test in a greenhouse condition against 8 weed

Seeds of *A. thaliana* were germinated on a 55 mm Polystyrene Petri-dish lined with one-layer filter paper (Advantec No. 2). One milliliter of each plumbagin solution dissolved in absolute acetone with various concentrations of 0, 25, 50 and 100 lM was dropped evenly onto the filter paper and placed in a vented cabinet to dry. After complete drying, 1 ml of distilled water with or without supplement of 0, 0.25, 0.5 and 1 mM biotin (Sigma, USA) was added, and 30 seeds were placed onto the filter paper in Petri-dish. Each Petri-dish was sealed with laboratory film and incubated in a growth chamber at 25oC, 14/10 h (Light/Dark). Germination inhibition percentages were calculated with the number of germinated *A. thaliana* seeds at 7 days after application. All treatments

The inhibited germination of *A. thaliana* seeds treated with plumbagin was significantly rescued in a dose dependent manner by biotin supplement. Germination rate of *A. thaliana* seeds at plumbagin levels of 25, 50, and 100 M was 33.3%, 23.3%, and 16.7%, respectively. However, the inhibited germination by plumbagin was negated up to 93.3%, 86.7%, and 83.3% with the supplement of 1 mM biotin, and also it was negated up to 66.7%, 63.3%, and 60.0% with the supplement of 0.5 mM biotin, respectively (Table 2, Fig 3). Biotin supplement apparently rescued the inhibited germination *A. thaliana* seeds caused by the treatment of plumbagin.

strumarium (common cocklebur); CAGEH, Calystegia jqponica (Japanese bindweed). ' 2,000 pg/ml. can change to 4 kg/ha.

species. (B) Field trials for *Sicyos angulatus* control. \* 2,000 g/mL can change to 4 kg/ha. **Figure 2.** Herbicidal symptoms of post-emergence foliar application of plumbagin (μg/mL). (A) Pot test in a greenhouse condition against 8 weed species. (B) Field trials for *Sicyos angulatus* control. \* 2,000 μg/mL can change to 4 kg/ha.

#### **2.4. Reversal study**

#### *2.4.1. Materials and methods*

Seeds of *A. thaliana* were germinated on a 55 mm Polystyrene Petri-dish lined with one-layer filter paper (Advantec No. 2). One milliliter of each plumbagin solution dis‐ solved in absolute acetone with various concentrations of 0, 25, 50 and 100 μM was dropped evenly onto the filter paper and placed in a vented cabinet to dry. After complete drying, 1 ml of distilled water with or without supplement of 0, 0.25, 0.5 and 1 mM biotin (Sigma, USA) was added, and 30 seeds were placed onto the filter paper in Petri-dish. Each Petri-dish was sealed with laboratory film and incubated in a growth chamber at 25o C, 14/10 h (Light/Dark). Germination inhibition percentages were calculated with the number of germinated *A. thaliana* seeds at 7 days after application. All treatments for each measurement were triplicates.

#### 1)Herbicida1 activity was determined 7 days after treatment by visual injury. SORBI, Sorghum bicolor (sorghum); ECHCG, Echinochloa crus-galli (barnyard grass); DIGSA, Digitaria sanguinalis (large crabgrass); SOLNI, Solanum nigrum (black *2.4.2. Results*

1)Herbicida1 activity was determined 7 days after treatment by visual injury. SORBI, Sorghum bicolor (sorghum); ECHCG, Echinochloa crus-galli (barnyard grass); DIGSA, Digitaria sanguinalis (large crabgrass); SOLNI, Solanum nigrum (black night‐ shade); AESIN, Aeschynomene indica (Indian joint vetch); ABUTH,Abutilon avicennae (velvetleai); XANSI,Xanthium struma‐ rium (common cocklebur); CAGEH, Calystegia japonica (Japanese bindweed). ' 2,000 pg/ml. can change to 4 kg/ha.

**Table 1.** Herbicidal efficacy of plumbagin post-emergence foliar application against several weeds in a greenhouse

species. (B) Field trials for *Sicyos angulatus* control. \* 2,000 g/mL can change to 4 kg/ha.

**Figure 2.** Herbicidal symptoms of post-emergence foliar application of plumbagin (μg/mL). (A) Pot test in a greenhouse condition against 8 weed species. (B) Field trials for *Sicyos angulatus* control. \* 2,000 μg/mL can change to 4 kg/ha.

**2.4. Reversal study** 

**2.4.1. Materials and methods** 

for each measurement were triplicates.

**2.4.2. Results** 

Table 1. Herbicidal efficacy of plumbagin post-emergence foliar application against several weeds in a greenhouse condition

Figure 2. Herbicidal symptoms of post-emergence foliar application of plumbagin (g/mL). (A) Pot test in a greenhouse condition against 8 weed

Seeds of *A. thaliana* were germinated on a 55 mm Polystyrene Petri-dish lined with one-layer filter paper (Advantec No. 2). One milliliter of each plumbagin solution dissolved in absolute acetone with various concentrations of 0, 25, 50 and 100 lM was dropped evenly onto the filter paper and placed in a vented cabinet to dry. After complete drying, 1 ml of distilled water with or without supplement of 0, 0.25, 0.5 and 1 mM biotin (Sigma, USA) was added, and 30 seeds were placed onto the filter paper in Petri-dish. Each Petri-dish was sealed with laboratory film and incubated in a growth chamber at 25oC, 14/10 h (Light/Dark). Germination inhibition percentages were calculated with the number of germinated *A. thaliana* seeds at 7 days after application. All treatments

The inhibited germination of *A. thaliana* seeds treated with plumbagin was significantly rescued in a dose dependent manner by biotin supplement. Germination rate of *A. thaliana* seeds at plumbagin levels of 25, 50, and 100 M was 33.3%, 23.3%, and 16.7%, respectively. However, the inhibited germination by plumbagin was negated up to 93.3%, 86.7%, and 83.3% with the supplement of 1 mM biotin, and also it was negated up to 66.7%, 63.3%, and 60.0% with the supplement of 0.5 mM biotin, respectively (Table 2, Fig 3). Biotin supplement apparently rescued the inhibited germination *A. thaliana* seeds caused by the treatment of plumbagin.

condition

320 Herbicides - Current Research and Case Studies in Use

nightshade); AESIN, Aeschynomene indica (Indian joint vetch); ABUTH,Abutilon avicennae (velvetleai); XANSI,Xanthium strumarium (common cocklebur); CAGEH, Calystegia jqponica (Japanese bindweed). ' 2,000 pg/ml. can change to 4 kg/ha. The inhibited germination of *A. thaliana* seeds treated with plumbagin was significantly rescued in a dose dependent manner by biotin supplement. Germination rate of *A. thaliana* seeds at plumbagin levels of 25, 50, and 100 μM was 33.3%, 23.3%, and 16.7%, respectively. However, the inhibited germination by plumbagin was negated up to 93.3%, 86.7%, and 83.3% with the supplement of 1 mM biotin, and also it was negated up to 66.7%, 63.3%, and 60.0% with the supplement of 0.5 mM biotin, respectively (Ta‐ ble 2, Fig. 3). Biotin supplement apparently rescued the inhibited germination *A. thali‐ ana* seeds caused by the treatment of plumbagin.


1)Germination rate of *A. thaliana* seed at 7 days after application.

using the partially purified AtKAPAS protein, *in vitro*. Less than 2% of 528 compounds exhibited inhibitory effect under a concentration of 20 μM. Interestingly, several naturally occurring quinone compounds including chrysophanic acid, tanshinones, 5,8-dihydroxy-1,4 naphthoquinone, and plumbagin were observed to give a potent inhibitory effect on KAPAS. Plumbagin, a natural naphthoquinone demonstrated the most effective inhibition on KAPAS in a concentration-dependent manner, and the IC50 was calculated as 2.1 μM. Webster et al. [12] reported that biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions. Abell [28] and Pillmoor et al. [29] suggested that if an enzyme is a potential target, a 60–80% inhibition of its activity leads to a severe growth. However, this requires the confirmation of potential target. For the purpose of target validation, a rescue study was carried out. Plumbagin inhibited germination of *A. thaliana* seeds but this effect was rescued by a biotin supplement. From this point of view, our results suggest that strong inhibition of KAPAS by plumbagin leads to restriction on the biotin biosynthesis in plants, ultimately the stems or leaves of plant treated with plumbagin die. Hwang et al. [11] argued that knowledge of biochemical pathways in plants is incomplete, and the next major herbicide target may lie in an unexpected area of plant metabolism; knowledge in detail how plants actually die as a result of inhibition of some known targets is still ambiguous. Also, we should note that the complete inhibition of enzyme activity at some known targets is not necessary for plant death [30]. However, it can be predicted that the herbicidal activity is somewhat connected between the reduced level of target enzyme activity and plant death. The enzyme inhibition results and rescue effect by biotin strongly suggested that the herbicidal activity by foliar treatment was due to the inhibition of KAPAS caused by the plumbagin. The natural chemical plumbagin has been shown by our research to effectively control eight weed species of *S. bicolor*, *E. crusgalli*, *D. sanguinalis*, *S. nigrum*, *A. indica*, *A. avicennae*, *X. strumarium*, *C. japonica* under nonreplicated greenhouse conditions. Also, the foliar application of the natural compound plumbagin at 2,000 Mg/mL has completely controlled 10 ~ 15 leaf-stage and 2 ~ 3 m vine length natural *S. Angulatus*, with sustantial residual activity under field conditions. The residual activity lasted for 2 weeks because regrowth was not observed until then. Visual symptoms of browning and necrosis of leaf tissue after plumbagin foliar applications appear to be intro‐ duced by cellular leakage rather than the inhibition of photosynthesis since cellular leakage occurred under light and dark conditions without chlorophyll loss. It seems closely related to the membrane lipid peroxidation as a result of the biotinyl carboxylase and transcarboxylase inhibition attributable to the biotin deficiency by KAPAS inhibition. Biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions in plant leaf, and KAPAS inhibition resulted in biotin depletion. As reviewed by Delye et al. [31] and Hwang et al. [32], these pathways in plant have been well established by acetyl-CoA carboxylase (ACCase) inhibiting herbicides, like as aryloxyphenoxypropionates and cyclohexanediones. ACCase is involved in the first step of lipid synthesis. The target site of acetyl-CoA carboxylase is a biotindependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA. The inhibition of KAPAS by plumbagin might result in the deficiency of substrate biotin to the biotinyl carboxylase in plants. However, the mechanism of action should be studied for better understanding of whole plant-compound interactions confirmative for

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this speculation.

**Figure 3.** Proposed target site of plumbagin on KAPAS and biotin synthesis pathway in plant.

#### **5. Summary**

A new herbicide developed the lifestyle of health and sustainability (LOHAS) initiative is required to satisfy environmental and regulatory pressures. LOHAS describes an estimated \$290 billion US marketplace for goods and services focused on health, the environment, social justice, personal development and sustainable living. Approximately 13–19% of the adults in the U.S. are currently considered LOHAS consumers. This is based on surveys of the U.S. adult population estimated at 215 million [23]. Also world-wide consumers demand these types of compounds as potential natural-product based herbicides. In this chapter, we attempted to develop a new herbicide from natural compounds having the new target KAPAS, and we applied this to annual noxious weed species of *S. angulatus* (burcucumber or star-cucumber). Our laboratory has performed molecular genetics dissection using anti-sense approach to identify new target AtKAPAS on the pathway of biotin biosynthesis and to characterize the phenotypic consequences of loss-of-function mutations [11]. The 528 plant-derived natural compounds stored in KRICT Chemical Bank were assessed on the inhibitory effect on KAPAS using the partially purified AtKAPAS protein, *in vitro*. Less than 2% of 528 compounds exhibited inhibitory effect under a concentration of 20 μM. Interestingly, several naturally occurring quinone compounds including chrysophanic acid, tanshinones, 5,8-dihydroxy-1,4 naphthoquinone, and plumbagin were observed to give a potent inhibitory effect on KAPAS. Plumbagin, a natural naphthoquinone demonstrated the most effective inhibition on KAPAS in a concentration-dependent manner, and the IC50 was calculated as 2.1 μM. Webster et al. [12] reported that biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions. Abell [28] and Pillmoor et al. [29] suggested that if an enzyme is a potential target, a 60–80% inhibition of its activity leads to a severe growth. However, this requires the confirmation of potential target. For the purpose of target validation, a rescue study was carried out. Plumbagin inhibited germination of *A. thaliana* seeds but this effect was rescued by a biotin supplement. From this point of view, our results suggest that strong inhibition of KAPAS by plumbagin leads to restriction on the biotin biosynthesis in plants, ultimately the stems or leaves of plant treated with plumbagin die. Hwang et al. [11] argued that knowledge of biochemical pathways in plants is incomplete, and the next major herbicide target may lie in an unexpected area of plant metabolism; knowledge in detail how plants actually die as a result of inhibition of some known targets is still ambiguous. Also, we should note that the complete inhibition of enzyme activity at some known targets is not necessary for plant death [30]. However, it can be predicted that the herbicidal activity is somewhat connected between the reduced level of target enzyme activity and plant death. The enzyme inhibition results and rescue effect by biotin strongly suggested that the herbicidal activity by foliar treatment was due to the inhibition of KAPAS caused by the plumbagin. The natural chemical plumbagin has been shown by our research to effectively control eight weed species of *S. bicolor*, *E. crusgalli*, *D. sanguinalis*, *S. nigrum*, *A. indica*, *A. avicennae*, *X. strumarium*, *C. japonica* under nonreplicated greenhouse conditions. Also, the foliar application of the natural compound plumbagin at 2,000 Mg/mL has completely controlled 10 ~ 15 leaf-stage and 2 ~ 3 m vine length natural *S. Angulatus*, with sustantial residual activity under field conditions. The residual activity lasted for 2 weeks because regrowth was not observed until then. Visual symptoms of browning and necrosis of leaf tissue after plumbagin foliar applications appear to be intro‐ duced by cellular leakage rather than the inhibition of photosynthesis since cellular leakage occurred under light and dark conditions without chlorophyll loss. It seems closely related to the membrane lipid peroxidation as a result of the biotinyl carboxylase and transcarboxylase inhibition attributable to the biotin deficiency by KAPAS inhibition. Biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions in plant leaf, and KAPAS inhibition resulted in biotin depletion. As reviewed by Delye et al. [31] and Hwang et al. [32], these pathways in plant have been well established by acetyl-CoA carboxylase (ACCase) inhibiting herbicides, like as aryloxyphenoxypropionates and cyclohexanediones. ACCase is involved in the first step of lipid synthesis. The target site of acetyl-CoA carboxylase is a biotindependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA. The inhibition of KAPAS by plumbagin might result in the deficiency of substrate biotin to the biotinyl carboxylase in plants. However, the mechanism of action should be studied for better understanding of whole plant-compound interactions confirmative for this speculation.

**Figure 3.** Proposed target site of plumbagin on KAPAS and biotin synthesis pathway in plant.

A new herbicide developed the lifestyle of health and sustainability (LOHAS) initiative is required to satisfy environmental and regulatory pressures. LOHAS describes an estimated \$290 billion US marketplace for goods and services focused on health, the environment, social justice, personal development and sustainable living. Approximately 13–19% of the adults in the U.S. are currently considered LOHAS consumers. This is based on surveys of the U.S. adult population estimated at 215 million [23]. Also world-wide consumers demand these types of compounds as potential natural-product based herbicides. In this chapter, we attempted to develop a new herbicide from natural compounds having the new target KAPAS, and we applied this to annual noxious weed species of *S. angulatus* (burcucumber or star-cucumber). Our laboratory has performed molecular genetics dissection using anti-sense approach to identify new target AtKAPAS on the pathway of biotin biosynthesis and to characterize the phenotypic consequences of loss-of-function mutations [11]. The 528 plant-derived natural compounds stored in KRICT Chemical Bank were assessed on the inhibitory effect on KAPAS

**5. Summary**

322 Herbicides - Current Research and Case Studies in Use

In a competing mechanism, proton abstraction is involved with the attack of acetyl-CoA. When the biotin is deficient, the product, malonyl-CoA is not produced. Malonyl-CoA is a building block for new fatty acids and can inhibit the transfer of the fatty acyl group from acyl-CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria. *S. angulatus* have been designated as one of the ecological disturbance plants by the Ministry of Environment in Korea. *S. angulatus* has spread across the marginal of agricultural field close to riparian zones along the rivers in Korea within the 15 years since its first appearance in 1989 (An Dong), covering more than 110 ha in 2005 [14,15]. The social and agricultural impact, risk assessment, invasion plants identification, and control management methods for alien vine plant such as *Humulus japonica* and *S. angulatus* have become a great problem in Korea. In conclusion, our results show that the herbicidal effect of plumbagin, a naturally occurring naphthoquionone, is closely associated with its inhibitory effect on KAPAS, a new target site of herbicide. Plumbagin and related 1,4-naphthoquinone compounds could be employed as a good chemical lead for an *S. angulatus* herbicide with a new mode of action (Fig. 4). the biotinyl carboxylase in plants. However, the mechanism of action should be studied for better understanding of whole plantcompound interactions confirmative for this speculation. In a competing mechanism, proton abstraction is involved with the attack of acetyl-CoA. When the biotin is deficient, the product, malonyl-CoA is not produced. Malonyl-CoA is a building block for new fatty acids and can inhibit the transfer of the fatty acyl group from acyl-CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria. *S. angulatus* have been designated as one of the ecological disturbance plants by the Ministry of Environment in Korea. *S. angulatus* has spread across the marginal of agricultural field close to riparian zones along the rivers in Korea within the 15 years since its first appearance in 1989 (An Dong), covering more than 110 ha in 2005 [14,15]. The social and agricultural impact, risk assessment, invasion plants identification, and control management methods for alien vine plant such as *Humulus japonica* and *S. angulatus* have become a great problem in Korea. In conclusion, our results show that the herbicidal effect of plumbagin, a naturally occurring naphthoquionone, is closely associated with its inhibitory effect on KAPAS, a new target site of herbicide. Plumbagin and related 1,4-naphthoquinone compounds could be employed as a good chemical lead for an *S. angulatus* herbicide with a new mode of action (Fig. 4).

suggested that the herbicidal activity by foliar treatment was due to the inhibition of KAPAS caused by the plumbagin. The natural chemical plumbagin has been shown by our research to effectively control eight weed species of *S. bicolor*, *E. crus-galli*, *D. sanguinalis*, *S. nigrum*, *A. indica*, *A. avicennae*, *X. strumarium*, *C. japonica* under non-replicated greenhouse conditions. Also, the foliar application of the natural compound plumbagin at 2,000 gmL-1 has completely controlled 10 ~ 15 leaf-stage and 2 ~ 3 m vine length natural *S. Angulatus*, with sustantial residual activity under field conditions. The residual activity lasted for 2 weeks because regrowth was not observed until then. Visual symptoms of browning and necrosis of leaf tissue after plumbagin foliar applications appear to be introduced by cellular leakage rather than the inhibition of photosynthesis since cellular leakage occurred under light and dark conditions without chlorophyll loss. It seems closely related to the membrane lipid peroxidation as a result of the biotinyl carboxylase and transcarboxylase inhibition attributable to the biotin deficiency by KAPAS inhibition. Biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions in plant leaf, and KAPAS inhibition resulted in biotin depletion. As reviewed by Delye et al. [31] and Hwang et al. [32], these pathways in plant have been well established by acetyl-CoA carboxylase

lipid synthesis. The target site of Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA. The inhibition of KAPAS by plumbagin might result in the deficiency of substrate biotin to

**Acknowledgements**

**Author details**

Jung-Sup Choi1

**References**

This work was supported by the R&D Program of MKE/KEIT [10035386, Biochemical Crop

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1 Korea Research Institute of Chemical Technology, Yusung, Daejon, Republic of Korea

2 Department of Green Chemistry and Environmental Biotechnology, University of Science

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[4] H.B. Lee, C.J. Kim, J.S. Kim, K.S. Hong, K.Y. Cho, A bleaching herbicidal activity of methoxyhygromycin (MHM) produced by an actinomycetes strain Streptomyces sp

[5] M. Fukuda, Y. Tsujino, T. Fujimori, K. Wakabayashi, P. Böger, Phytotoxicity activity of middle-chain fatty acids I: effect on cell constituents, Pest Biochem. Physiol.

[7] L.D. Bainard, M.B. Isman, Phyto-toxicity of clove oil and its primary constituent eu‐ genol and the role of leaf epicuticular wax in the susceptibility to these essential oils,

[8] S. Goncalves, M. Ferraz, A. Romano, Phytotoxic properties of *Drosophyllum lusitani‐ cum* leaf extracts and its main compound plumbagin, Sci. Hortic. 2009;122:96–101.

[6] T. Tworkoski, Herbicide effects of essential oils, Weed Sci. 2002;50:425–431.

vol. 35, Society for Chemical Industries, Cambridge, UK, 1996, pp. 82-113.

Protecting Agents for LOHAS] and by the KRICT's own project [KK-1104-B0].

and In-Taek Hwang1,2

we learned?, Weed Sci 2002;50:700–712.

8E-12, Lett. Appl. Microbiol. 2003;36:387–391.

2004;80:143–150.

Weed Sci. 2006;54:833–837.

& Technology, Gajungro, Yuseong-gu, Daejon, Republic of Korea

This work was supported by the R&D Program of MKE/KEIT [10035386, Biochemical Crop Protecting Agents for LOHAS] and by

Figure 4. Proposed target site of plumbagin and herbicidal activity **Figure 4.** Proposed target site of plumbagin and herbicidal activity

the KRICT's own project [KK-1104-B0].

**Acknowledgements** 

**References** 

#### of acetyl-CoA to produce malonyl-CoA. The inhibition of KAPAS by plumbagin might result in the deficiency of substrate biotin to the biotinyl carboxylase in plants. However, the mechanism of action should be studied for better understanding of whole plant-**Acknowledgements**

In a competing mechanism, proton abstraction is involved with the attack of acetyl-CoA. When the biotin is deficient, the product, malonyl-CoA is not produced. Malonyl-CoA is a building block for new fatty acids and can inhibit the transfer of the fatty acyl group from acyl-CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria. *S. angulatus* have been designated as one of the ecological disturbance plants by the Ministry of Environment in Korea. *S. angulatus* has spread across the marginal of agricultural field close to riparian zones along the rivers in Korea within the 15 years since its first appearance in 1989 (An Dong), covering more than 110 ha in 2005 [14,15]. The social and agricultural impact, risk assessment, invasion plants identification, and control management methods for alien vine plant such as *Humulus japonica* and *S. angulatus* have become a great problem in Korea. In conclusion, our results show that the herbicidal effect of plumbagin, a naturally occurring naphthoquionone, is closely associated with its inhibitory effect on KAPAS, a new target site of herbicide. Plumbagin and related 1,4-naphthoquinone compounds could be employed as a good chemical lead for an *S. angulatus* herbicide with a new mode of

compound interactions confirmative for this speculation.

Figure 4. Proposed target site of plumbagin and herbicidal activity

**Acknowledgements** 

**Figure 4.** Proposed target site of plumbagin and herbicidal activity

**References** 

the KRICT's own project [KK-1104-B0].

with a new mode of action (Fig. 4).

324 Herbicides - Current Research and Case Studies in Use

suggested that the herbicidal activity by foliar treatment was due to the inhibition of KAPAS caused by the plumbagin. The natural chemical plumbagin has been shown by our research to effectively control eight weed species of *S. bicolor*, *E. crus-galli*, *D. sanguinalis*, *S. nigrum*, *A. indica*, *A. avicennae*, *X. strumarium*, *C. japonica* under non-replicated greenhouse conditions. Also, the foliar application of the natural compound plumbagin at 2,000 gmL-1 has completely controlled 10 ~ 15 leaf-stage and 2 ~ 3 m vine length natural *S. Angulatus*, with sustantial residual activity under field conditions. The residual activity lasted for 2 weeks because regrowth was not observed until then. Visual symptoms of browning and necrosis of leaf tissue after plumbagin foliar applications appear to be introduced by cellular leakage rather than the inhibition of photosynthesis since cellular leakage occurred under light and dark conditions without chlorophyll loss. It seems closely related to the membrane lipid peroxidation as a result of the biotinyl carboxylase and transcarboxylase inhibition attributable to the biotin deficiency by KAPAS inhibition. Biotin is an essential enzyme cofactor for carboxylase and transcarboxylase reactions in plant leaf, and KAPAS inhibition resulted in biotin depletion. As reviewed by Delye et al. [31] and Hwang et al. [32], these pathways in plant have been well established by acetyl-CoA carboxylase (ACCase) inhibiting herbicides, like as aryloxyphenoxypropionates and cyclohexanediones. ACCase is involved in the first step of lipid synthesis. The target site of Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the irreversible carboxylation

malonyl-CoA is not produced. Malonyl-CoA is a building block for new fatty acids and can inhibit the transfer of the fatty acyl

Korea. *S. angulatus* has spread across the marginal of agricultural field close to riparian zones along the rivers in Korea within the

This work was supported by the R&D Program of MKE/KEIT [10035386, Biochemical Crop Protecting Agents for LOHAS] and by

action (Fig. 4).

In a competing mechanism, proton abstraction is involved with the attack of acetyl-CoA. When the biotin is deficient, the product, This work was supported by the R&D Program of MKE/KEIT [10035386, Biochemical Crop Protecting Agents for LOHAS] and by the KRICT's own project [KK-1104-B0].

#### group from acyl-CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria. *S. angulatus* have been designated as one of the ecological disturbance plants by the Ministry of Environment in **Author details**

15 years since its first appearance in 1989 (An Dong), covering more than 110 ha in 2005 [14,15]. The social and agricultural impact, Jung-Sup Choi1 and In-Taek Hwang1,2

risk assessment, invasion plants identification, and control management methods for alien vine plant such as *Humulus japonica* and *S. angulatus* have become a great problem in Korea. In conclusion, our results show that the herbicidal effect of plumbagin, a 1 Korea Research Institute of Chemical Technology, Yusung, Daejon, Republic of Korea

naturally occurring naphthoquionone, is closely associated with its inhibitory effect on KAPAS, a new target site of herbicide. Plumbagin and related 1,4-naphthoquinone compounds could be employed as a good chemical lead for an *S. angulatus* herbicide 2 Department of Green Chemistry and Environmental Biotechnology, University of Science & Technology, Gajungro, Yuseong-gu, Daejon, Republic of Korea

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[24] J. Bhattacharyya, V.R. De Carvalho, Epi-isoshinanolone from *Plumbago scandens*, Phy‐

New Natural Herbicide Candidate for *Sicyon angulatus* Control

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[25] O. Ploux, A. Marquet, The 8-amino-7-oxopelargonate synthase from *Bacillus sphaeri‐ cus*. Purification and preliminary characterization of the cloned enzyme overpro‐

[26] W.H. Kenyon, S.O. Duke, K.C. Vaughn, Sequence of effects of acifluorfen on physio‐ logical and ultrastructural parameters in cucumber cotyledon discs, Pest Biochem.

[27] J.D. Hiscox, G.F. Israelstam, A method for the extraction of chlorophyll from leaf tis‐

[28] L.M. Abell, Biochemica lapproaches to herbicide discovery: advances in enzyme tar‐

[29] J.B. Pillmoor, S.D. Lindell, G.G. Briggs, K. Wright, The influences of molecular mech‐ anisms of action on herbicide design, in: N.N. Ragsdale, P.C. Kearney, J.R. Plimmer (Eds.), Processing of the Eighth of the English International Congress of Pesticide

[30] C. Alban, D. Job, R. Douce, Biotin metabolism in plants, Annu. Rev. Plant Physiol.

[31] C. Délye, A. Matéjicek, J. Gasquez, PCR-based detection of resistance to acetyl-CoA carboxylase-inhibiting herbicides in black-grass (*Alopecurus myosuroides* Huds) and

[32] I.T. Hwang, D.H. Lee, And N-J Park, 7-Keto-8-aminopelargonic acid synthase as a potential herbicide target, in: S. Soloneski, M. Larramendy (Eds.), Herbicides, IN‐

Chemistry, America Chemical Society, Washington, DC, 1995, pp. 292–303.

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get identification and inhibitor design, Weed Sci. 1996;44:734–742.

ryegrass (*Lolium rigidum* Gaud), Pest Manag. Sci. 2002;58:474–478.

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[9] J.J.M. Meyer, F. Van der Kooy, A. Joubert, Identification of plumbagin epoxide as a germination inhibitory compound through a rapid bioassay on TLC, S. Afr. J. Bot.

[10] G. Meazza, B.E. Scheffler, M.R. Tellez, A.M. Rimando, J.G. Romagni, S.O. Duke, D. Nanayakkara, I.K. Khan, E.A. Abourashed, F.E. Dayan, The inhibitory activity of nat‐ ural products on plant p-hydroxyphenylpyruvate dioxygenase, Phytochemistry

[11] I.T. Hwang, J.S. Choi, H.Y. Song, S.J. Cho, H.K. Lim, N.J. Park, D.H. Lee, Validation of 7-keto-8-aminopelargonic acid synthase as a potential herbicide target with lead

[12] S.P. Webster, D. Alexeev, D.J. Campopiano, R.M. Watt, M. Alexeeva, L. Sawyer, R.L. Baxter, Mechanism of 8-Amino-7-oxononanoate synthase: spectroscopic, kinetic, and

[13] A. Nudelman, D. Marcovici-Mizrahi, A. Nudelman, D. Flintd, V. Wittenbache, Inhib‐ itors of biotin biosynthesis as potential herbicides, Tetrahedron 2004;60:1731–1748. [14] J.H. Kil, H.Y. Kong, K.S. Koh & J.M. Kim, Management of Sicyos angulata spread in Korea. In: Neobiota, From Ecology to Conservation, 4th European Conference on Bio‐

[15] J.H. Kang, B.S. Jeon, S.W. Lee, Z.R. Choe, S.I. Shim, Enhancement of seed germina‐ tion by aging, cold stratification, and light quality during desiccation in bur cucum‐

[16] European and Mediterranean Plant Protection Organization, EPPO data sheet on In‐ vasive Alien Plants Sicyos angulatus, OEPP/EPPO Bulletin 2010;40:401–406.

[17] T. Asaeda, MD.H. Rashid, S. Kotagiri, T. Uchida, The role of soil characteristics in the succession of two herbaceous lianas in a modified river flood plain, River Res. Appl.

[18] S. Ceschin, G. Salerno, S. Bisceglie, A. Kumbaric, Temporal floristic variations as in‐ dicator of environmental changes in the Tiber River in Rome, Aquat. Ecol.

[19] C.G. Hanson and J.L. Mason, Bird seed aliens in Britain. Watsonia 1985;15:237–252.

[21] S. Kurokawa, Invasion of exotic weed seeds into Japan, mixed in imported feed

[22] J.F. Larché, Sicyos angulatus, nouvelle adventice du maïs dans le Sud-Ouest de la

[20] T. Ouren, Soybean adventitious weeds in Norway, Blyttia 1987;45:175–185.

compound triphenyltin acetate, Pest Biochem. Physiol. 2010;97:24–31.

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<http://www.watsonia.org.uk/html/watsonia\_15.html>.

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2007;73:654–656.

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2011;27:591–601.

2010;44:93–100.


**Chapter 13**

**Herbicides in Aquatic Systems**

Additional information is available at the end of the chapter

Water plays a crucial role in maintaining the health of our global ecosystem. We rely on this valuable resource to provide drinking water, irrigation, and recreation; in addition, appropri‐ ate management of our waters is critical for flood control efforts. A diversity of native aquatic plants constitutes an integral part of the aquatic environment. These mixed populations of hydrophytes provide structure, habitat and food for fish, waterfowl, and other wildlife and act as nutrient sinks by removing phosphorus, nitrogen, and other elements from the water column. Many regions of the world – but especially those with mild climates – provide an ideal habitat for many organisms, including aquatic plants. Non-native aquatic plants are frequently introduced to aquatic systems through a number of pathways, including transport by animals, currents, or wind, but the majority of problematic plants are brought in as a result of anthro‐ pogenic activities. Human introduction of non-native aquatic plants may be accidental (e.g.,

Many of the worst aquatic weed problems in the United States are the result of intentional introduction. For example, waterhyacinth [*Eichhornia crassipes* (Mart.) Solms] (Fig. 1) was reportedly introduced to the United States at the Southern States Cotton Expo in New Orleans in 1884. Visitors to the Expo were given waterhyacinth plants as souvenirs and many of these plants found their way into the waters of Louisiana, Texas, and Florida [1]. Local legend states that a Florida resident was entranced by the beautiful, showy flowers of this Amazonian native and brought plants back to his water garden near the St. Johns River. The plants grew abundantly and the backyard water gardener decided to share his "bounty of beauties" with

> © 2013 Gettys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Gettys et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

via ballast water or as contaminants in desirable flora) or intentional.

Lyn A. Gettys, William T. Haller and

Gregory E. MacDonald

http://dx.doi.org/10.5772/56015

**1. Introduction**

**2. Aquatic weeds**

### **Chapter 13**

## **Herbicides in Aquatic Systems**

Lyn A. Gettys, William T. Haller and Gregory E. MacDonald

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56015

### **1. Introduction**

Water plays a crucial role in maintaining the health of our global ecosystem. We rely on this valuable resource to provide drinking water, irrigation, and recreation; in addition, appropri‐ ate management of our waters is critical for flood control efforts. A diversity of native aquatic plants constitutes an integral part of the aquatic environment. These mixed populations of hydrophytes provide structure, habitat and food for fish, waterfowl, and other wildlife and act as nutrient sinks by removing phosphorus, nitrogen, and other elements from the water column. Many regions of the world – but especially those with mild climates – provide an ideal habitat for many organisms, including aquatic plants. Non-native aquatic plants are frequently introduced to aquatic systems through a number of pathways, including transport by animals, currents, or wind, but the majority of problematic plants are brought in as a result of anthro‐ pogenic activities. Human introduction of non-native aquatic plants may be accidental (e.g., via ballast water or as contaminants in desirable flora) or intentional.

### **2. Aquatic weeds**

Many of the worst aquatic weed problems in the United States are the result of intentional introduction. For example, waterhyacinth [*Eichhornia crassipes* (Mart.) Solms] (Fig. 1) was reportedly introduced to the United States at the Southern States Cotton Expo in New Orleans in 1884. Visitors to the Expo were given waterhyacinth plants as souvenirs and many of these plants found their way into the waters of Louisiana, Texas, and Florida [1]. Local legend states that a Florida resident was entranced by the beautiful, showy flowers of this Amazonian native and brought plants back to his water garden near the St. Johns River. The plants grew abundantly and the backyard water gardener decided to share his "bounty of beauties" with

© 2013 Gettys et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Gettys et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

others by tossing his extra plants into the St. Johns River [2]. Within a decade, the St. Johns was so clogged with waterhyacinths that navigation had become impossible [1-3].

**Figure 2.** Mechanical harvesting of waterhyacinth. Photo courtesy UF/IFAS Center for Aquatic and Invasive Plants.

ecosystem services and anthropogenic uses of aquatic resources.

Floating weeds such as waterhyacinth are readily visible and many stakeholders understand the need to control these types of noxious species. Submersed invasive species, however, are often hidden from view and the problems associated with them are not readily apparent. Submersed weeds often go unnoticed until they form surface mats; by this point, plants have been growing unchecked, often for months, and the water column is filled with plant material. This is often the case with hydrilla [*Hydrilla verticillata*) (L.f.) Royle] (Fig. 3), a noxious invader with multiple centers of origin that has been called the world's worst weed [6]. Hydrilla was introduced to the United States intentionally via the aquarium industry [7], and historical accounts suggest that some aquarium plant dealers cultivated hydrilla in canals and waters near their nurseries to have a ready supply of plant material for their customers [8]. However, the species has undoubtedly been introduced to the country's waterways repeatedly, as hobbyists regularly dispose of extra aquarium plants by tossing them in the nearest body of water. Because hydrilla is able to root from extremely small fragments [9], other pathways of introduction include waterfowl, other fauna and recreational equipment such as boats and trailers. Hydrilla causes a host of problems in its regions of invasion and greatly reduces

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 331

Hydrilla can reportedly grow 1 inch (2.5 cm) per day [6], but most researchers agree that this is a gross underestimate of the plant's actual productivity [10]. This noxious weed wreaks havoc on the ecosystem by forming monocultures [11], which serve as poor habitat for resident fauna. Dense plant growth traps heat, raises the temperature of surface water and depletes dissolved oxygen, resulting in conditions that negatively impact fish survival [12, 13]. Hydrilla also obstructs water flow, which can have catastrophic consequences if resource managers need to quickly move water to prevent flooding during tropical storms, hurricanes, and other severe weather events. Recreational uses of hydrilla-infested waters are limited as well; boats motors quickly become clogged and strangled with weeds (Fig. 4), fishing lines are snagged within moments of being cast, and swimmers have reportedly drowned after becoming entangled in hydrilla [14]. Hydrilla is intensively managed in its regions of invasion. Popula‐ tions of this submersed weed are reduced by a number of means, including mechanical harvesting, hand-pulling, benthic barriers, and biological control organisms such as Asian or

**Figure 1.** Waterhyacinth. Photo courtesy Lyn Gettys.

The St. Johns constituted a major shipping passage through Florida; in order to mitigate this important resource and make it available to commercial concerns for the transport of goods, the US Army Corps of Engineers was authorized by the US Congress to use "any means necessary" to clear the system of these noxious weeds [1]. Attempts to control floating waterhyacinths utilized applications of a wide variety of substances, including arsenic, sulfuric acid, and other toxic chemicals [1]. Some of these substances effectively controlled waterhya‐ cinths, but proved toxic to cattle that grazed on treated plants [2]. Feeding deterrents such as rotten eggs and manure were added to chemical applications to discourage grazing, but were ultimately ineffective or too expensive to use under operational conditions [1]. After these disappointing results, resource managers were forced to resort to mechanical control – manually removing plants from the surface of the water and offloading them to shore – in their attempts to clear Florida's waterways (Fig. 2). This method proved expensive and ineffective, as plants grew faster than they could be harvested from the system, but was the only man‐ agement tool available until the discovery of synthetic herbicides in the 1940s [3]. Waterhya‐ cinth is now controlled in many regions via chemical means (e.g., application of herbicides), but this Brazilian native is still considered one of the world's worst weeds [4, 5] and is intensively managed in virtually all areas the species has managed to invade.

others by tossing his extra plants into the St. Johns River [2]. Within a decade, the St. Johns was

The St. Johns constituted a major shipping passage through Florida; in order to mitigate this important resource and make it available to commercial concerns for the transport of goods, the US Army Corps of Engineers was authorized by the US Congress to use "any means necessary" to clear the system of these noxious weeds [1]. Attempts to control floating waterhyacinths utilized applications of a wide variety of substances, including arsenic, sulfuric acid, and other toxic chemicals [1]. Some of these substances effectively controlled waterhya‐ cinths, but proved toxic to cattle that grazed on treated plants [2]. Feeding deterrents such as rotten eggs and manure were added to chemical applications to discourage grazing, but were ultimately ineffective or too expensive to use under operational conditions [1]. After these disappointing results, resource managers were forced to resort to mechanical control – manually removing plants from the surface of the water and offloading them to shore – in their attempts to clear Florida's waterways (Fig. 2). This method proved expensive and ineffective, as plants grew faster than they could be harvested from the system, but was the only man‐ agement tool available until the discovery of synthetic herbicides in the 1940s [3]. Waterhya‐ cinth is now controlled in many regions via chemical means (e.g., application of herbicides), but this Brazilian native is still considered one of the world's worst weeds [4, 5] and is

intensively managed in virtually all areas the species has managed to invade.

so clogged with waterhyacinths that navigation had become impossible [1-3].

**Figure 1.** Waterhyacinth. Photo courtesy Lyn Gettys.

330 Herbicides - Current Research and Case Studies in Use

**Figure 2.** Mechanical harvesting of waterhyacinth. Photo courtesy UF/IFAS Center for Aquatic and Invasive Plants.

Floating weeds such as waterhyacinth are readily visible and many stakeholders understand the need to control these types of noxious species. Submersed invasive species, however, are often hidden from view and the problems associated with them are not readily apparent. Submersed weeds often go unnoticed until they form surface mats; by this point, plants have been growing unchecked, often for months, and the water column is filled with plant material. This is often the case with hydrilla [*Hydrilla verticillata*) (L.f.) Royle] (Fig. 3), a noxious invader with multiple centers of origin that has been called the world's worst weed [6]. Hydrilla was introduced to the United States intentionally via the aquarium industry [7], and historical accounts suggest that some aquarium plant dealers cultivated hydrilla in canals and waters near their nurseries to have a ready supply of plant material for their customers [8]. However, the species has undoubtedly been introduced to the country's waterways repeatedly, as hobbyists regularly dispose of extra aquarium plants by tossing them in the nearest body of water. Because hydrilla is able to root from extremely small fragments [9], other pathways of introduction include waterfowl, other fauna and recreational equipment such as boats and trailers. Hydrilla causes a host of problems in its regions of invasion and greatly reduces ecosystem services and anthropogenic uses of aquatic resources.

Hydrilla can reportedly grow 1 inch (2.5 cm) per day [6], but most researchers agree that this is a gross underestimate of the plant's actual productivity [10]. This noxious weed wreaks havoc on the ecosystem by forming monocultures [11], which serve as poor habitat for resident fauna. Dense plant growth traps heat, raises the temperature of surface water and depletes dissolved oxygen, resulting in conditions that negatively impact fish survival [12, 13]. Hydrilla also obstructs water flow, which can have catastrophic consequences if resource managers need to quickly move water to prevent flooding during tropical storms, hurricanes, and other severe weather events. Recreational uses of hydrilla-infested waters are limited as well; boats motors quickly become clogged and strangled with weeds (Fig. 4), fishing lines are snagged within moments of being cast, and swimmers have reportedly drowned after becoming entangled in hydrilla [14]. Hydrilla is intensively managed in its regions of invasion. Popula‐ tions of this submersed weed are reduced by a number of means, including mechanical harvesting, hand-pulling, benthic barriers, and biological control organisms such as Asian or

Waterhyacinth and hydrilla quickly establish and become invasive in virtually all areas where they have been introduced, but these species are not the only aquatic plants that cause problems in natural systems, reservoirs, and canals through the world. For example, resource managers charged with protecting the waters of the Pacific Northwest and many other parts of the US struggle with invasions of Eurasian watermilfoil (*Myriophyllum spicatum* L.), flowering rush (*Butomus umbellatus* L.), and curlyleaf pondweed (*Potamogeton crispus* L.) (Fig. 5). It is thought that these species were initially introduced through the aquarium and nursery trade, but have since spread throughout the country's waters as a result of improper or inadequate cleaning of contaminated equipment that has been moved from infested sites to

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 333

**Figure 5.** Other common aquatic invaders in the US. Left: curlyleaf pondweed. Right: flowering rush (emerged) and

It is clear that aquatic weeds can severely reduce ecosystem functions and limit the use of infested waters for anthropogenic activities such as recreation and flood control. However, invasive aquatic plants can pose serious risks to human health as well. For example, a number of floating species provide ideal conditions for mosquito breeding activities. Even in fastflowing water, the stagnant water needed for mosquito reproduction is often present in the rosettes of floating weeds such as waterhyacinth and waterlettuce (*Pistia stratiotes* L.) [17-19]

A number of techniques can be employed to control or reduce populations of aquatic weeds. Clearly, the most effective way to avoid the problems associated with invasive plants is through exclusion, or preventing them from entering uninfested aquatic systems. Public education programs that emphasize proper disposal of cultivated introduced plants and animals can be helpful, but target audiences (i.e., pet and aquarium owners) often remain unaware of the ecological consequences associated with the release of these organisms into

Eurasian watermilfoil (submersed). Photos courtesy Lyn Gettys.

**3. Weed control methods in aquatic systems**

pristine waters.

(Fig. 6).

**Figure 3.** Hydrilla. Photo courtesy William Haller.

Chinese grass carp (*Ctenopharyngodon idella* Val.) [15, 16], but the vast majority of resource managers rely on chemical control to keep the growth of hydrilla in check.

**Figure 4.** Boat motor clogged with hydrilla. Photo courtesy UF/IFAS Center for Aquatic and Invasive Plants.

Waterhyacinth and hydrilla quickly establish and become invasive in virtually all areas where they have been introduced, but these species are not the only aquatic plants that cause problems in natural systems, reservoirs, and canals through the world. For example, resource managers charged with protecting the waters of the Pacific Northwest and many other parts of the US struggle with invasions of Eurasian watermilfoil (*Myriophyllum spicatum* L.), flowering rush (*Butomus umbellatus* L.), and curlyleaf pondweed (*Potamogeton crispus* L.) (Fig. 5). It is thought that these species were initially introduced through the aquarium and nursery trade, but have since spread throughout the country's waters as a result of improper or inadequate cleaning of contaminated equipment that has been moved from infested sites to pristine waters.

**Figure 5.** Other common aquatic invaders in the US. Left: curlyleaf pondweed. Right: flowering rush (emerged) and Eurasian watermilfoil (submersed). Photos courtesy Lyn Gettys.

It is clear that aquatic weeds can severely reduce ecosystem functions and limit the use of infested waters for anthropogenic activities such as recreation and flood control. However, invasive aquatic plants can pose serious risks to human health as well. For example, a number of floating species provide ideal conditions for mosquito breeding activities. Even in fastflowing water, the stagnant water needed for mosquito reproduction is often present in the rosettes of floating weeds such as waterhyacinth and waterlettuce (*Pistia stratiotes* L.) [17-19] (Fig. 6).

### **3. Weed control methods in aquatic systems**

Chinese grass carp (*Ctenopharyngodon idella* Val.) [15, 16], but the vast majority of resource

managers rely on chemical control to keep the growth of hydrilla in check.

**Figure 3.** Hydrilla. Photo courtesy William Haller.

332 Herbicides - Current Research and Case Studies in Use

**Figure 4.** Boat motor clogged with hydrilla. Photo courtesy UF/IFAS Center for Aquatic and Invasive Plants.

A number of techniques can be employed to control or reduce populations of aquatic weeds. Clearly, the most effective way to avoid the problems associated with invasive plants is through exclusion, or preventing them from entering uninfested aquatic systems. Public education programs that emphasize proper disposal of cultivated introduced plants and animals can be helpful, but target audiences (i.e., pet and aquarium owners) often remain unaware of the ecological consequences associated with the release of these organisms into

```
Figure 6. Waterlettuce. Photo courtesy Lyn Gettys.
```
public waters. Although this sort of intentional release is certainly a vector for the introduction of new invaders (see Section 2 of this chapter describing the introduction of waterhyacinth), accidental transfer of aquatic weeds frequently occurs when boats, trailers, and other equip‐ ment is moved from an invaded site to a pristine body of water (Fig. 7). The likelihood of introduction via this route can be reduced by requiring careful inspection of any object before movement from one body of water to another. This is especially important when boats and other equipment are being relocated from a body of water that is suspected of or known to harbor invasive species to one that is pristine. These inspections can identify seeds, vegetative fragments, larvae, veligers, and other propagules of invasive aquatic species and ensure their removal before launching at a new site, thus preventing the introduction of exotic organisms into an uninfested body of water. This method has been employed with some success in the northern US, where rigorous boat inspection programs have kept invasive aquatic plants and animals such as zebra and quagga mussels (*Dreissena polymorpha* and *D. rostriformis bugensis*, respectively) from spreading to new sites [20, 21].

stances. However, a number of factors must be taken into consideration before starting mechanical control efforts, regardless of whether volunteer labor or mechanical harvesters are employed. For example, it may be logistically difficult or prohibitively expensive to dispose of harvested plant material. Resource managers sometimes have access to a nearby "high and dry" site where collected weeds can be stockpiled and allowed to desiccate and decay, but harvested material must often be transported off-site for disposal. This process can add significantly to the cost of the project, especially if the weeds must be disposed of in a landfill that charges tipping fees. As much as 95% of the fresh weight of aquatic weeds is water; a single acre of hydrilla can weigh as much as 24,000 pounds (10,886 kg), but only 1,200 pounds (544 kg) of that weight is plant material and the remaining 22,800 pounds (10,342 kg) is water [22]. Also, removal of weeds by volunteers or mechanical harvesters typically causes frag‐ mentation of plant material and fails to capture root crowns, tubers, seeds, and other propa‐ gules in the sediment. Many aquatic weeds – including hydrilla, curlyleaf pondweed, and Eurasian watermilfoil – easily root from fragments and quickly regrow from sediment- borne propagules. As a result, initial observations at many sites that are managed using hand or mechanical removal of aquatic weeds may suggest that these methods have successfully addressed the problem, but control of the new invader is often ephemeral and weed popula‐ tions regenerate in as little as a few weeks. A third factor to consider when hand-pulling or using mechanical harvesters to remove aquatic weeds is water depth. Volunteers are unlikely to be able to remove plants growing in water that is deeper than 3 feet (1 m) without diving gear and most traditional mechanical harvesters can only remove plant material in the upper 5 feet (1.5 m) of the water column, although newer equipment can harvest weeds in the upper 10 feet (3 m) of water. These factors should be considered before launching a weed removal program, regardless of whether weeds are taken out of the system by hand or by utilizing mechanical harvesters, but there are additional challenges inherent to each method. For example, volunteers tasked with hand-pulling invaders must be adequately trained to ensure that they will be able to successfully identify the target weed, especially when the invader is

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 335

**Figure 7.** Aquatic weeds on a boat trailer. Photo courtesy Lyn Gettys.

When exclusion programs fail and an exotic plant species colonizes a new system, managers often attempt to manually remove the invader as a first line of defense. The methods employed for removal efforts vary and are often dependent on available resources. For example, handpulling of target weeds may be effective, especially if the infestation is small and localized, and may be cost-effective if a pool of engaged stakeholders and volunteers can be mobilized to accomplish the task. If the new invader has colonized a relatively large area or has estab‐ lished in water deeper than 1 meter, the use of specialized equipment such as mechanical harvesters (Fig. 8) may be employed. Mechanical removal of aquatic weeds is often viewed by the public as the most "environmentally friendly" control method, especially among clientele that dislike the use of pesticides, and the technique certainly has utility under some circum‐

**Figure 7.** Aquatic weeds on a boat trailer. Photo courtesy Lyn Gettys.

public waters. Although this sort of intentional release is certainly a vector for the introduction of new invaders (see Section 2 of this chapter describing the introduction of waterhyacinth), accidental transfer of aquatic weeds frequently occurs when boats, trailers, and other equip‐ ment is moved from an invaded site to a pristine body of water (Fig. 7). The likelihood of introduction via this route can be reduced by requiring careful inspection of any object before movement from one body of water to another. This is especially important when boats and other equipment are being relocated from a body of water that is suspected of or known to harbor invasive species to one that is pristine. These inspections can identify seeds, vegetative fragments, larvae, veligers, and other propagules of invasive aquatic species and ensure their removal before launching at a new site, thus preventing the introduction of exotic organisms into an uninfested body of water. This method has been employed with some success in the northern US, where rigorous boat inspection programs have kept invasive aquatic plants and animals such as zebra and quagga mussels (*Dreissena polymorpha* and *D. rostriformis bugensis*,

When exclusion programs fail and an exotic plant species colonizes a new system, managers often attempt to manually remove the invader as a first line of defense. The methods employed for removal efforts vary and are often dependent on available resources. For example, handpulling of target weeds may be effective, especially if the infestation is small and localized, and may be cost-effective if a pool of engaged stakeholders and volunteers can be mobilized to accomplish the task. If the new invader has colonized a relatively large area or has estab‐ lished in water deeper than 1 meter, the use of specialized equipment such as mechanical harvesters (Fig. 8) may be employed. Mechanical removal of aquatic weeds is often viewed by the public as the most "environmentally friendly" control method, especially among clientele that dislike the use of pesticides, and the technique certainly has utility under some circum‐

respectively) from spreading to new sites [20, 21].

**Figure 6.** Waterlettuce. Photo courtesy Lyn Gettys.

334 Herbicides - Current Research and Case Studies in Use

stances. However, a number of factors must be taken into consideration before starting mechanical control efforts, regardless of whether volunteer labor or mechanical harvesters are employed. For example, it may be logistically difficult or prohibitively expensive to dispose of harvested plant material. Resource managers sometimes have access to a nearby "high and dry" site where collected weeds can be stockpiled and allowed to desiccate and decay, but harvested material must often be transported off-site for disposal. This process can add significantly to the cost of the project, especially if the weeds must be disposed of in a landfill that charges tipping fees. As much as 95% of the fresh weight of aquatic weeds is water; a single acre of hydrilla can weigh as much as 24,000 pounds (10,886 kg), but only 1,200 pounds (544 kg) of that weight is plant material and the remaining 22,800 pounds (10,342 kg) is water [22]. Also, removal of weeds by volunteers or mechanical harvesters typically causes frag‐ mentation of plant material and fails to capture root crowns, tubers, seeds, and other propa‐ gules in the sediment. Many aquatic weeds – including hydrilla, curlyleaf pondweed, and Eurasian watermilfoil – easily root from fragments and quickly regrow from sediment- borne propagules. As a result, initial observations at many sites that are managed using hand or mechanical removal of aquatic weeds may suggest that these methods have successfully addressed the problem, but control of the new invader is often ephemeral and weed popula‐ tions regenerate in as little as a few weeks. A third factor to consider when hand-pulling or using mechanical harvesters to remove aquatic weeds is water depth. Volunteers are unlikely to be able to remove plants growing in water that is deeper than 3 feet (1 m) without diving gear and most traditional mechanical harvesters can only remove plant material in the upper 5 feet (1.5 m) of the water column, although newer equipment can harvest weeds in the upper 10 feet (3 m) of water. These factors should be considered before launching a weed removal program, regardless of whether weeds are taken out of the system by hand or by utilizing mechanical harvesters, but there are additional challenges inherent to each method. For example, volunteers tasked with hand-pulling invaders must be adequately trained to ensure that they will be able to successfully identify the target weed, especially when the invader is similar in appearance to desirable native plants that should be allowed to remain in the system. In contrast, mechanical harvesters are "non-selective" – they indiscriminately remove all plant material in the harvesting zone and are unable to distinguish between weeds and native species. Also, mechanical harvesters often result in bycatch, or the removal of fish and other aquatic fauna along with plant material. This problem is most pronounced when shallowwater (upper 5 feet; 1.5 m) harvesters are employed and can result in the removal of up to 28,000 fish per acre [23], but bycatch can be reduced by greater than 99% (removal of around 120 fish per acre) when deep-water (upper 10 feet; 3 m) harvesting is utilized [24].

vegetation in an aquatic system. Also, because the grass carp is a non-native introduced species, special precautions must be taken to reduce the likelihood of these biocontrol agents becoming invasive themselves. In Florida and many other states in the US, a permit must be issued by state resource managers before the introduction of grass carp into an aquatic system (although some states prohibit the use of grass carp as biocontrol agents altogether) [28]. In most cases, permit holders must ensure that stocked waters are secured (i.e., water intakes and outflows must be screened) to prevent the fish from escaping into other waters and all released grass carp must be triploid. Triploidy is the presence of an additional set of chromosomes, a condition that is induced by subjecting fish eggs to cold, heat, or pressure shock treatments immediately after artificial fertilization, and renders the grass carp unable to reproduce [29].

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 337

Other organisms have also been employed as biocontrol agents. For example, a number of insects and pathogens have been evaluated for control of various aquatic weeds, including the noxious aquatic invader alligatorweed [*Alternanthera philoxeroides* (Mart.) Griseb.]. The most promising of these agents, the alligatorweed flea beetle (*Agasicles hygrophila* Selman and Vogt) (Fig. 10), can reduce populations to the point that more aggressive weed control methods can be reduced or even eliminated, provided winter temperatures in the region are mild enough to allow overwintering of the beetles [30]. Although these and other biocontrol agents have some utility in aquatic weed control, they cannot be relied on to completely eliminate infesta‐ tions of invasive weeds. True biocontrol agents are host-specific; therefore, populations of the target weed must always be present in order to serve as a host or food source for the agent. As a result, weedy species cannot be eradicated through the actions of a biocontrol agent. When more complete control of aquatic weeds is necessary, resource managers rely heavily on

**Figure 9.** Asian grass carp. Photo courtesy William Haller.

chemical control, or the use of herbicides.

**Figure 8.** Mechanical harvesting of hydrilla. Photo courtesy William Haller.

Another method that can provide some control of unwanted aquatic species is biological control, or the use of organisms to reduce weed populations. This technique, often referred to as biocontrol, is based on the concept that most species that become weedy after introduction to a new region are not problematic in their native range due to the presence of endemic predators that keep their growth in check. Identifying and evaluating potential biocontrol agents is an arduous, time-consuming, expensive process. The process typically begins with researchers travelling to the invader's center of origin and collecting insects, pathogens, or other organisms that are found in association with the target weed species. These biological agents are quarantined and subjected to a battery of tests to determine whether they fit the criteria and requirements of successful biocontrol agents. A hallmark of a biocontrol agent is host specificity; in other words, they must cause damage exclusively to the target weed species while leaving other plants untouched [25, 26]. Biocontrol agents should also be able to survive, grow, and reproduce in the invaded range of the weed and ideally, they should be able to form self-sustaining populations without augmentation. Some success has been realized using biocontrol organisms for aquatic weed control; for example, the Asian or Chinese grass carp (*Ctenopharyngodon idella* Val.) (Fig. 9) is well-known as a voracious consumer of hydrilla [27]. Unfortunately, grass carp are somewhat non-selective; although they are most frequently employed to control hydrilla, they will consume and eliminate virtually all submersed vegetation in an aquatic system. Also, because the grass carp is a non-native introduced species, special precautions must be taken to reduce the likelihood of these biocontrol agents becoming invasive themselves. In Florida and many other states in the US, a permit must be issued by state resource managers before the introduction of grass carp into an aquatic system (although some states prohibit the use of grass carp as biocontrol agents altogether) [28]. In most cases, permit holders must ensure that stocked waters are secured (i.e., water intakes and outflows must be screened) to prevent the fish from escaping into other waters and all released grass carp must be triploid. Triploidy is the presence of an additional set of chromosomes, a condition that is induced by subjecting fish eggs to cold, heat, or pressure shock treatments immediately after artificial fertilization, and renders the grass carp unable to reproduce [29].

**Figure 9.** Asian grass carp. Photo courtesy William Haller.

similar in appearance to desirable native plants that should be allowed to remain in the system. In contrast, mechanical harvesters are "non-selective" – they indiscriminately remove all plant material in the harvesting zone and are unable to distinguish between weeds and native species. Also, mechanical harvesters often result in bycatch, or the removal of fish and other aquatic fauna along with plant material. This problem is most pronounced when shallowwater (upper 5 feet; 1.5 m) harvesters are employed and can result in the removal of up to 28,000 fish per acre [23], but bycatch can be reduced by greater than 99% (removal of around

Another method that can provide some control of unwanted aquatic species is biological control, or the use of organisms to reduce weed populations. This technique, often referred to as biocontrol, is based on the concept that most species that become weedy after introduction to a new region are not problematic in their native range due to the presence of endemic predators that keep their growth in check. Identifying and evaluating potential biocontrol agents is an arduous, time-consuming, expensive process. The process typically begins with researchers travelling to the invader's center of origin and collecting insects, pathogens, or other organisms that are found in association with the target weed species. These biological agents are quarantined and subjected to a battery of tests to determine whether they fit the criteria and requirements of successful biocontrol agents. A hallmark of a biocontrol agent is host specificity; in other words, they must cause damage exclusively to the target weed species while leaving other plants untouched [25, 26]. Biocontrol agents should also be able to survive, grow, and reproduce in the invaded range of the weed and ideally, they should be able to form self-sustaining populations without augmentation. Some success has been realized using biocontrol organisms for aquatic weed control; for example, the Asian or Chinese grass carp (*Ctenopharyngodon idella* Val.) (Fig. 9) is well-known as a voracious consumer of hydrilla [27]. Unfortunately, grass carp are somewhat non-selective; although they are most frequently employed to control hydrilla, they will consume and eliminate virtually all submersed

120 fish per acre) when deep-water (upper 10 feet; 3 m) harvesting is utilized [24].

**Figure 8.** Mechanical harvesting of hydrilla. Photo courtesy William Haller.

336 Herbicides - Current Research and Case Studies in Use

Other organisms have also been employed as biocontrol agents. For example, a number of insects and pathogens have been evaluated for control of various aquatic weeds, including the noxious aquatic invader alligatorweed [*Alternanthera philoxeroides* (Mart.) Griseb.]. The most promising of these agents, the alligatorweed flea beetle (*Agasicles hygrophila* Selman and Vogt) (Fig. 10), can reduce populations to the point that more aggressive weed control methods can be reduced or even eliminated, provided winter temperatures in the region are mild enough to allow overwintering of the beetles [30]. Although these and other biocontrol agents have some utility in aquatic weed control, they cannot be relied on to completely eliminate infesta‐ tions of invasive weeds. True biocontrol agents are host-specific; therefore, populations of the target weed must always be present in order to serve as a host or food source for the agent. As a result, weedy species cannot be eradicated through the actions of a biocontrol agent. When more complete control of aquatic weeds is necessary, resource managers rely heavily on chemical control, or the use of herbicides.

mental Protection Agency (USEPA), and prosecution by the USEPA for failure to follow label

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 339

**Flood control canals** should be able to quickly move large volumes of water. These systems may be used only rarely for their true purpose; however, their ability to function as intended is critical when residential or developed areas are threatened by tropical storms, hurricanes or other extreme weather events. As such, it is critical that these canals be kept clear of aquatic vegetation that may impede the flow of water. A "scorched earth" philosophy and the use of a non-selective herbicide is sometimes employed to ensure that flood control canals remain free of aquatic weeds, and native plants are not exempt from weed control efforts in these systems. This is because even a small population of submersed plants – be it a weed such as hydrilla or a native plant such as eelgrass (*Vallisneria americana* Michx.) (Fig. 11) – can severely restrict water flow and increase the likelihood of flooding. Although the goal of weed control efforts in flood control canals is often to eliminate as much vegetation in the water column and surface as possible, canal banks should remain vegetated (ideally with a well-rooted, non-

**Recreational waters** are typically managed to facilitate anthropogenic activities such as fishing, duck hunting, boating, and swimming. As a result, stakeholders – along with expect‐ ations and concerns – are many and varied. For example, most research has shown that sport fish populations in natural areas are greatest when submersed plants inhabit 30-40% or less of the water column [31, 32], but many sportfishers believe that dense weeds are necessary to provide good habitat for sportfish such as largemouth bass [33-35]. Also, some aquatic plants – including native species such as pondweed (*Potamogeton* spp.) and invasive weeds such as hydrilla – are eaten by ducks and waterfowl (Fig. 12). In fact, many duck hunters (and some waterfowl scientists) are less than supportive of aquatic vegetation control operations because they say these efforts deplete duck and waterfowl feeding habitat [36, 37]. These and other stakeholders often protest when weed control efforts are undertaken because they suspect reductions in weed coverage will negatively impact their hunting and fishing activities. Although some sportsmen recognize that it is rarely possible to maintain low coverage rates of aquatic weeds, many others fail to appreciate that the unchecked growth characteristic of

invasive native species) to prevent erosion during periods of rapid flow.

**Figure 11.** Eelgrass. Photo courtesy Lyn Gettys.

guidelines.

**Figure 10.** Alligatorweed flea beetle. Photo courtesy Lyn Gettys.

### **4. Water use and its influence on herbicide selection**

A number of factors must be taken into consideration when selecting a herbicide for chemical control. Clearly, the most important criterion is efficacy of the product on the target weed. However, resource managers must also take into account how treated waters will be used. Although some aquatic systems are used for fisheries or crop production (e.g., rice cultivation), most are not used to grow food. Non-production waters targeted for aquatic weed control efforts can be categorized in a number of different ways, but the most common broad group‐ ings include agricultural waters, flood control canals, recreational waters, retention ponds, and "development" waters (man-made lakes and ponds created for aesthetic reasons). Many waters are multi-use and span several of these categories, but this discussion will focus on the primary purpose of each grouping.

**Agricultural waters** are typically used for crop irrigation and for watering of livestock. A number of herbicides labeled for use in aquatic systems have irrigation and/or livestock watering restrictions. These restrictions preclude the use of treated water for a specific period of time or until the concentration of the herbicide is below a specified level. These restrictions vary among products and may also vary among products with the same active ingredient. Irrigation and livestock watering restrictions are clearly listed on the product label; compliance may be as simple as not using treated water for the appropriate length of time or may require laboratory tests to determine the concentration of herbicide in the water. Intentional or accidental failure to adhere to irrigation restrictions may result in a number of consequences, including – but not limited to – damage to livestock and non-target crop plants, herbicide residues in crops that exceed the allowed tolerance established by the United States Environ‐ mental Protection Agency (USEPA), and prosecution by the USEPA for failure to follow label guidelines.

**Flood control canals** should be able to quickly move large volumes of water. These systems may be used only rarely for their true purpose; however, their ability to function as intended is critical when residential or developed areas are threatened by tropical storms, hurricanes or other extreme weather events. As such, it is critical that these canals be kept clear of aquatic vegetation that may impede the flow of water. A "scorched earth" philosophy and the use of a non-selective herbicide is sometimes employed to ensure that flood control canals remain free of aquatic weeds, and native plants are not exempt from weed control efforts in these systems. This is because even a small population of submersed plants – be it a weed such as hydrilla or a native plant such as eelgrass (*Vallisneria americana* Michx.) (Fig. 11) – can severely restrict water flow and increase the likelihood of flooding. Although the goal of weed control efforts in flood control canals is often to eliminate as much vegetation in the water column and surface as possible, canal banks should remain vegetated (ideally with a well-rooted, noninvasive native species) to prevent erosion during periods of rapid flow.

**Figure 10.** Alligatorweed flea beetle. Photo courtesy Lyn Gettys.

338 Herbicides - Current Research and Case Studies in Use

primary purpose of each grouping.

**4. Water use and its influence on herbicide selection**

A number of factors must be taken into consideration when selecting a herbicide for chemical control. Clearly, the most important criterion is efficacy of the product on the target weed. However, resource managers must also take into account how treated waters will be used. Although some aquatic systems are used for fisheries or crop production (e.g., rice cultivation), most are not used to grow food. Non-production waters targeted for aquatic weed control efforts can be categorized in a number of different ways, but the most common broad group‐ ings include agricultural waters, flood control canals, recreational waters, retention ponds, and "development" waters (man-made lakes and ponds created for aesthetic reasons). Many waters are multi-use and span several of these categories, but this discussion will focus on the

**Agricultural waters** are typically used for crop irrigation and for watering of livestock. A number of herbicides labeled for use in aquatic systems have irrigation and/or livestock watering restrictions. These restrictions preclude the use of treated water for a specific period of time or until the concentration of the herbicide is below a specified level. These restrictions vary among products and may also vary among products with the same active ingredient. Irrigation and livestock watering restrictions are clearly listed on the product label; compliance may be as simple as not using treated water for the appropriate length of time or may require laboratory tests to determine the concentration of herbicide in the water. Intentional or accidental failure to adhere to irrigation restrictions may result in a number of consequences, including – but not limited to – damage to livestock and non-target crop plants, herbicide residues in crops that exceed the allowed tolerance established by the United States Environ‐ **Recreational waters** are typically managed to facilitate anthropogenic activities such as fishing, duck hunting, boating, and swimming. As a result, stakeholders – along with expect‐ ations and concerns – are many and varied. For example, most research has shown that sport fish populations in natural areas are greatest when submersed plants inhabit 30-40% or less of the water column [31, 32], but many sportfishers believe that dense weeds are necessary to provide good habitat for sportfish such as largemouth bass [33-35]. Also, some aquatic plants – including native species such as pondweed (*Potamogeton* spp.) and invasive weeds such as hydrilla – are eaten by ducks and waterfowl (Fig. 12). In fact, many duck hunters (and some waterfowl scientists) are less than supportive of aquatic vegetation control operations because they say these efforts deplete duck and waterfowl feeding habitat [36, 37]. These and other stakeholders often protest when weed control efforts are undertaken because they suspect reductions in weed coverage will negatively impact their hunting and fishing activities. Although some sportsmen recognize that it is rarely possible to maintain low coverage rates of aquatic weeds, many others fail to appreciate that the unchecked growth characteristic of submersed weeds necessitates weed control efforts that focus on eliminating as much vegeta‐ tion as possible.

**Figure 13.** Herbicide applicator wearing personal protective equipment. Photo courtesy Lyn Gettys.

qualities are optimized.

**Florida (USA)**

**"Development" waters** are man-made lakes and ponds that are created with the primary goal of increased aesthetics. These artificial bodies of water provide residential developers with a source of fill dirt, after which they are able to market adjacent homesites as desirable waterfront property, which are often sold at a premium. They also increase the value of the entire development, which can now be advertised as including ponds and water features. Some development waters are maintained in a pristine, plant-free state and rely on fountains or other hardscape features to provide an attractive visage. Others are planted or aquascaped, either to simulate natural bodies of water or to mimic large-scale water gardens with showy orna‐ mental plants (Fig. 14). Because development waters are rarely connected to public waters, weed problems in these systems are typically the result of introduction by humans, or less often, by waterfowl and wildlife that have visited the development waters after spending time in nearby weed-infested aquatic systems. Anthropogenic introduction of aquatic weeds is frequently intentional, as when property owners dump unwanted aquarium or water garden plants into the development waters. However, the introduction of aquatic weeds can occur inadvertently when invasive species are misidentified and sold as desirable native plants or when propagules of invasive species "hitchhike" as contaminants on the desirable plants that are used for aquascaping [38-40]. Because development waters are considered valuable components of the landscape, they are often intensively managed to ensure that their aesthetic

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 341

**5. Herbicide usage and labeling in aquatic systems — Case studies from**

Herbicides are used extensively to control weeds in crop production and agricultural systems. The terrestrial agrichemical industry in the US is robust; estimated sales in 2007 were \$12.454 billion, with 40% of the market (\$5.856 billion) attributable to herbicides [41]. In contrast, the

**Figure 12.** Ducks consuming seeds and vegetation on a pond bank. Photo courtesy Lyn Gettys.

Other recreational activities – such as boating and the use of personal watercrafts such as jet skis – are also directly impacted by aquatic weeds. Access to boat ramps can be restricted by overabundant growth of macrophytes in and around the littoral zone, while dense submersed vegetation can wrap around the propellers of outboard motors and hinder or halt boat operation. In addition, dense submersed vegetation can make swimming and waterskiing difficult, dangerous, or nearly impossible, and can increase the risk of drowning if individuals become entangled in dense weeds.

**Retention ponds** are by definition designed to be ephemeral; their ultimate purpose is to retain storm water, capture runoff, filter nutrients, and lessen or prevent flooding. Nevertheless, many stakeholders consider retention ponds to be long-term "water features" that enhance the aesthetics of urban and suburban areas. Retention ponds may be used on a limited basis for recreational purposes (e.g., fishing and swimming), but these activities are often restricted by the resource owner to limit liability. Aquatic weed control efforts in retention ponds must take into account stakeholder expectations; for example, if the goal is to reduce or eliminate unsightly algae or submersed weeds while leaving a fringe of ornamental flowering plants in the littoral zone, care must be taken to choose a selective herbicide that will control the target species without causing unacceptable levels of damage to desirable vegetation. Weed control efforts in retention ponds may also be challenging for resource managers due to the high visibility of these sites. Many stakeholders become alarmed at the sight of herbicide applicators wearing "moon suits" (Fig. 13) – a common name for personal protective equipment specified on the herbicide label – and assume that the water is being poisoned. Therefore, it can be useful to ensure that applicators are able to communicate with the public and to assuage fears regarding the toxicity of herbicides labeled for use in aquatic systems.

**Figure 13.** Herbicide applicator wearing personal protective equipment. Photo courtesy Lyn Gettys.

submersed weeds necessitates weed control efforts that focus on eliminating as much vegeta‐

**Figure 12.** Ducks consuming seeds and vegetation on a pond bank. Photo courtesy Lyn Gettys.

regarding the toxicity of herbicides labeled for use in aquatic systems.

become entangled in dense weeds.

Other recreational activities – such as boating and the use of personal watercrafts such as jet skis – are also directly impacted by aquatic weeds. Access to boat ramps can be restricted by overabundant growth of macrophytes in and around the littoral zone, while dense submersed vegetation can wrap around the propellers of outboard motors and hinder or halt boat operation. In addition, dense submersed vegetation can make swimming and waterskiing difficult, dangerous, or nearly impossible, and can increase the risk of drowning if individuals

**Retention ponds** are by definition designed to be ephemeral; their ultimate purpose is to retain storm water, capture runoff, filter nutrients, and lessen or prevent flooding. Nevertheless, many stakeholders consider retention ponds to be long-term "water features" that enhance the aesthetics of urban and suburban areas. Retention ponds may be used on a limited basis for recreational purposes (e.g., fishing and swimming), but these activities are often restricted by the resource owner to limit liability. Aquatic weed control efforts in retention ponds must take into account stakeholder expectations; for example, if the goal is to reduce or eliminate unsightly algae or submersed weeds while leaving a fringe of ornamental flowering plants in the littoral zone, care must be taken to choose a selective herbicide that will control the target species without causing unacceptable levels of damage to desirable vegetation. Weed control efforts in retention ponds may also be challenging for resource managers due to the high visibility of these sites. Many stakeholders become alarmed at the sight of herbicide applicators wearing "moon suits" (Fig. 13) – a common name for personal protective equipment specified on the herbicide label – and assume that the water is being poisoned. Therefore, it can be useful to ensure that applicators are able to communicate with the public and to assuage fears

tion as possible.

340 Herbicides - Current Research and Case Studies in Use

**"Development" waters** are man-made lakes and ponds that are created with the primary goal of increased aesthetics. These artificial bodies of water provide residential developers with a source of fill dirt, after which they are able to market adjacent homesites as desirable waterfront property, which are often sold at a premium. They also increase the value of the entire development, which can now be advertised as including ponds and water features. Some development waters are maintained in a pristine, plant-free state and rely on fountains or other hardscape features to provide an attractive visage. Others are planted or aquascaped, either to simulate natural bodies of water or to mimic large-scale water gardens with showy orna‐ mental plants (Fig. 14). Because development waters are rarely connected to public waters, weed problems in these systems are typically the result of introduction by humans, or less often, by waterfowl and wildlife that have visited the development waters after spending time in nearby weed-infested aquatic systems. Anthropogenic introduction of aquatic weeds is frequently intentional, as when property owners dump unwanted aquarium or water garden plants into the development waters. However, the introduction of aquatic weeds can occur inadvertently when invasive species are misidentified and sold as desirable native plants or when propagules of invasive species "hitchhike" as contaminants on the desirable plants that are used for aquascaping [38-40]. Because development waters are considered valuable components of the landscape, they are often intensively managed to ensure that their aesthetic qualities are optimized.

### **5. Herbicide usage and labeling in aquatic systems — Case studies from Florida (USA)**

Herbicides are used extensively to control weeds in crop production and agricultural systems. The terrestrial agrichemical industry in the US is robust; estimated sales in 2007 were \$12.454 billion, with 40% of the market (\$5.856 billion) attributable to herbicides [41]. In contrast, the

(Fig. 15). If these preliminary experiments suggest that a herbicide shows promise as an aquatic weed control agent, the registrant may pursue an aquatic label for the product. Registrants seeking an aquatic label must submit additional data to the Agency, including how the product affects target and non-target aquatic flora and fauna, its persistence in aquatic sediments and water, and the nature and impacts of its decomposition components. These tests are conducted under Experimental Use Permits (EUPs) issued by the USEPA and by state regulatory agencies. For example, the testing of pesticides in Florida waters is conducted under EUPs issued by the USEPA and by the Florida Department of Agriculture and Consumer Services (FDACS). There are a number of restrictions on waters that are treated with experimental products; for example, treated waters may not be used for fishing, swimming, irrigation, drinking, or watering of livestock. Evaluation of an EUP herbicide typically continues for several years until the registrant has sufficient data to submit to the USEPA, along with a proposed aquatic label [43]. The aquatic label includes all of the information found on terrestrial labels, such as the personal protective equipment that is required to handle and apply the herbicide. In addition, aquatic herbicide labels include water use restrictions to prevent harm to human health and the environment. Some products have no limitations on the use of treated waters; however, others may specify that water from the system may be not be used for various purposes until either a certain period of time has elapsed or until the concentration of the herbicide falls below a

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 343

specified set point.

**Figure 15.** Efficacy testing in the greenhouse. Photo courtesy William Haller.

It is important to note that all herbicides labeled for aquatic weed control by the USEPA in the US are "general use" pesticides that can be purchased and applied by anyone, including homeowners and unlicensed applicators. However, the USEPA allows states to apply addi‐

**Figure 14.** Waterlilies in a development pond. Photo courtesy Lyn Gettys.

market for aquatic weed control products is much smaller; for example, public agencies in Florida spent around \$22.5 million in 2005 to manage aquatic invaders in public waters [42]. Any product that is marketed in the US to control pests – including weeds – must first be labeled by the US Environmental Protection Agency (USEPA or the Agency). Obtaining a pesticide label from the USEPA is a time-consuming and expensive undertaking; the Agency requires registrants (the manufacturer or group seeking a pesticide label) to submit data from more than 100 tests before a product can be evaluated for possible labeling, and the testing process typically requires the investment of tens of millions of dollars [43]. These tests are conducted to determine the effects of the experimental pesticide on the organism targeted for control, but also to assess its impact on non-target organisms, human health, and the environ‐ ment as well. USEPA regulation of pesticides began with the adoption of the Federal Insecti‐ cide, Fungicide, and Rodenticide Act (FIFRA) in 1947; FIFRA has since been amended multiple times, most notably by the Federal Environmental Pesticide Control Act of 1972, and continues to serve as the primary process to ensure that human health and the environment are not negatively impacted by the use of pesticides [43].

Because obtaining a pesticide label from the USEPA requires significant financial resources, registrants only request Agency evaluation of products that are likely to capture a market large enough to offset the costs associated with obtaining a pesticide label. As outlined above, aquatic herbicides constitute a small niche market, with limited potential to allow registrants to recoup the funds required for initial labeling of a pesticide. Therefore, most herbicides that are labeled by the USEPA for use in aquatic systems have already been approved by the Agency for terrestrial use. Small-scale testing – such as greenhouse studies evaluating the efficacy of a product on aquatic weeds – may be conducted on a limited basis under specific conditions (Fig. 15). If these preliminary experiments suggest that a herbicide shows promise as an aquatic weed control agent, the registrant may pursue an aquatic label for the product. Registrants seeking an aquatic label must submit additional data to the Agency, including how the product affects target and non-target aquatic flora and fauna, its persistence in aquatic sediments and water, and the nature and impacts of its decomposition components. These tests are conducted under Experimental Use Permits (EUPs) issued by the USEPA and by state regulatory agencies. For example, the testing of pesticides in Florida waters is conducted under EUPs issued by the USEPA and by the Florida Department of Agriculture and Consumer Services (FDACS). There are a number of restrictions on waters that are treated with experimental products; for example, treated waters may not be used for fishing, swimming, irrigation, drinking, or watering of livestock. Evaluation of an EUP herbicide typically continues for several years until the registrant has sufficient data to submit to the USEPA, along with a proposed aquatic label [43]. The aquatic label includes all of the information found on terrestrial labels, such as the personal protective equipment that is required to handle and apply the herbicide. In addition, aquatic herbicide labels include water use restrictions to prevent harm to human health and the environment. Some products have no limitations on the use of treated waters; however, others may specify that water from the system may be not be used for various purposes until either a certain period of time has elapsed or until the concentration of the herbicide falls below a specified set point.

**Figure 15.** Efficacy testing in the greenhouse. Photo courtesy William Haller.

market for aquatic weed control products is much smaller; for example, public agencies in Florida spent around \$22.5 million in 2005 to manage aquatic invaders in public waters [42]. Any product that is marketed in the US to control pests – including weeds – must first be labeled by the US Environmental Protection Agency (USEPA or the Agency). Obtaining a pesticide label from the USEPA is a time-consuming and expensive undertaking; the Agency requires registrants (the manufacturer or group seeking a pesticide label) to submit data from more than 100 tests before a product can be evaluated for possible labeling, and the testing process typically requires the investment of tens of millions of dollars [43]. These tests are conducted to determine the effects of the experimental pesticide on the organism targeted for control, but also to assess its impact on non-target organisms, human health, and the environ‐ ment as well. USEPA regulation of pesticides began with the adoption of the Federal Insecti‐ cide, Fungicide, and Rodenticide Act (FIFRA) in 1947; FIFRA has since been amended multiple times, most notably by the Federal Environmental Pesticide Control Act of 1972, and continues to serve as the primary process to ensure that human health and the environment are not

Because obtaining a pesticide label from the USEPA requires significant financial resources, registrants only request Agency evaluation of products that are likely to capture a market large enough to offset the costs associated with obtaining a pesticide label. As outlined above, aquatic herbicides constitute a small niche market, with limited potential to allow registrants to recoup the funds required for initial labeling of a pesticide. Therefore, most herbicides that are labeled by the USEPA for use in aquatic systems have already been approved by the Agency for terrestrial use. Small-scale testing – such as greenhouse studies evaluating the efficacy of a product on aquatic weeds – may be conducted on a limited basis under specific conditions

negatively impacted by the use of pesticides [43].

**Figure 14.** Waterlilies in a development pond. Photo courtesy Lyn Gettys.

342 Herbicides - Current Research and Case Studies in Use

It is important to note that all herbicides labeled for aquatic weed control by the USEPA in the US are "general use" pesticides that can be purchased and applied by anyone, including homeowners and unlicensed applicators. However, the USEPA allows states to apply addi‐ tional restrictions to pesticides; in fact, a number of states classify aquatic herbicides as "restricted use" products that can only be purchased and applied by individuals that have received a state-issued license. Any and all individuals using a pesticide must comply with all of the requirements outlined on the pesticide label. The label is a legally binding document and misuse of a pesticide can result in serious consequences, up to and including the levy of fines and incarceration [43]. Although licensing is not required by federal law to purchase or apply aquatic herbicides, the vast majority of public agencies and private companies that employ applicators to manage aquatic systems specify that all personnel using these products obtain an aquatic pesticide applicator license from the state in which they are employed. This ensures that applicators have been trained and have shown competency in a number of important areas, including label interpretation, proper application techniques, equipment calibration, use of personal protective equipment, and proper disposal methods. Each state has its own requirements for obtaining and keeping a pesticide license. For example, all certified pesticide applicators in Florida must pass at least two written examinations – one that tests core competency and one that evaluates competence in a specific area or category [44]. A number of categories are offered to individuals seeking certification in Florida, and applicators may become licensed in as many categories as desired after the core competency examination has been successfully completed. Most licensees that are charged with applying pesticides in aquatic systems have multiple category certifications, the most common being aquatics, natural areas, and right-of-way. Florida pesticide applicator licenses are valid for four years from the date of issuance, and a license can be renewed in one of two ways. Applicators may submit proof that they have attended training sessions and earned a specified number of continuing education units (CEUs) in core and category areas during the four-year period since the license was issued or last renewed. Alternatively, applicators may re-take core competency and category examinations every four years [44].

epilimnion and hypolimnion, respectively. Water in the epilimnion is directly exposed to ambient air temperatures and therefore tends to be very warm in the summer and cold or frozen during winter. In contrast, water in the hypolimnion maintains a more or less constant temperature all year. The epilimnion and hypolimnion are separated by the thermocline, a layer characterized by drastic temperature changes [45, 46]. The effect of stratification may have little effect on efforts to manage emergent or floating aquatic weeds. However, this phenomenon can have a substantial effect on treatment of submersed invaders, because herbicides applied to the epilimnion are unlikely to penetrate through the thermocline to reach

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 345

**Figure 16.** Stratified lake (summer, with warm epilimnion). Illustration courtesy UF/IFAS Center for Aquatic and Inva‐

Another important consideration in the treatment of aquatic systems is the effect of weed control activities on fish that reside in waters targeted for herbicide application. Although the presence of fish does not affect herbicide efficacy, special precautions must be taken to ensure that these and other aquatic denizens are not harmed as a result of weed control efforts. Only a few herbicides labeled for use in bodies of water are inherently dangerous to fish, but fish kills are nonetheless a major concern for applicators working in aquatic systems. The primary reason fish kills occur after weed control activities are undertaken is a reduction in dissolved oxygen (DO), which results from a number of factors [47]. Primary among these factors is the decomposition of vegetative material that has been killed by herbicides and is broken down by aerobic organisms, which deplete DO during the process. Also, photosynthesis by plants that have been killed by herbicides is eliminated and the DO they previously contributed to the water column is no longer produced, further reducing levels of DO. In order to reduce the likelihood of fish kills, most labels for aquatic products specify that herbicides be applied to only a portion of a weed-infested body of water at a time to allow fish to escape from treated areas and to prevent the extreme drop in DO that accompanies the elimination of all vegetation

target weeds growing in the hypolimnion.

sive Plants.

from an aquatic system.

### **6. How environmental factors influence herbicide applications**

Herbicide applications to the aquatic environment share some of the challenges associated with treatment of agricultural lands, including drift (the unintended aerial dispersal of herbicides from the treatment area) and damage to desirable non-target plants. However, aquatic herbicide applications are further complicated by a number of factors unique to aquatic systems. For example, herbicides used for weed control in crop production typically reach the target plant at the concentration in which they are applied. In contrast, products employed to control submersed aquatic weeds must travel through the water column to reach their target and thus undergo substantial dilution before coming into contact with the plant. In addition, flow and currents result in the movement of the herbicide out of the treated area, which reduces contact exposure time (the period in which the product maintains contact with the target weed) and further limits efficacy of the treatment [45]. Another factor that complicates herbicide application in aquatic systems is the stratification of waters (Fig. 16), especially within systems in temperate regions. Most bodies of water have three distinct zones or layers, with little mixing among the layers. The upper and lower portions of a body of water are referred to as the epilimnion and hypolimnion, respectively. Water in the epilimnion is directly exposed to ambient air temperatures and therefore tends to be very warm in the summer and cold or frozen during winter. In contrast, water in the hypolimnion maintains a more or less constant temperature all year. The epilimnion and hypolimnion are separated by the thermocline, a layer characterized by drastic temperature changes [45, 46]. The effect of stratification may have little effect on efforts to manage emergent or floating aquatic weeds. However, this phenomenon can have a substantial effect on treatment of submersed invaders, because herbicides applied to the epilimnion are unlikely to penetrate through the thermocline to reach target weeds growing in the hypolimnion.

tional restrictions to pesticides; in fact, a number of states classify aquatic herbicides as "restricted use" products that can only be purchased and applied by individuals that have received a state-issued license. Any and all individuals using a pesticide must comply with all of the requirements outlined on the pesticide label. The label is a legally binding document and misuse of a pesticide can result in serious consequences, up to and including the levy of fines and incarceration [43]. Although licensing is not required by federal law to purchase or apply aquatic herbicides, the vast majority of public agencies and private companies that employ applicators to manage aquatic systems specify that all personnel using these products obtain an aquatic pesticide applicator license from the state in which they are employed. This ensures that applicators have been trained and have shown competency in a number of important areas, including label interpretation, proper application techniques, equipment calibration, use of personal protective equipment, and proper disposal methods. Each state has its own requirements for obtaining and keeping a pesticide license. For example, all certified pesticide applicators in Florida must pass at least two written examinations – one that tests core competency and one that evaluates competence in a specific area or category [44]. A number of categories are offered to individuals seeking certification in Florida, and applicators may become licensed in as many categories as desired after the core competency examination has been successfully completed. Most licensees that are charged with applying pesticides in aquatic systems have multiple category certifications, the most common being aquatics, natural areas, and right-of-way. Florida pesticide applicator licenses are valid for four years from the date of issuance, and a license can be renewed in one of two ways. Applicators may submit proof that they have attended training sessions and earned a specified number of continuing education units (CEUs) in core and category areas during the four-year period since the license was issued or last renewed. Alternatively, applicators may re-take core competency

and category examinations every four years [44].

344 Herbicides - Current Research and Case Studies in Use

**6. How environmental factors influence herbicide applications**

Herbicide applications to the aquatic environment share some of the challenges associated with treatment of agricultural lands, including drift (the unintended aerial dispersal of herbicides from the treatment area) and damage to desirable non-target plants. However, aquatic herbicide applications are further complicated by a number of factors unique to aquatic systems. For example, herbicides used for weed control in crop production typically reach the target plant at the concentration in which they are applied. In contrast, products employed to control submersed aquatic weeds must travel through the water column to reach their target and thus undergo substantial dilution before coming into contact with the plant. In addition, flow and currents result in the movement of the herbicide out of the treated area, which reduces contact exposure time (the period in which the product maintains contact with the target weed) and further limits efficacy of the treatment [45]. Another factor that complicates herbicide application in aquatic systems is the stratification of waters (Fig. 16), especially within systems in temperate regions. Most bodies of water have three distinct zones or layers, with little mixing among the layers. The upper and lower portions of a body of water are referred to as the

**Figure 16.** Stratified lake (summer, with warm epilimnion). Illustration courtesy UF/IFAS Center for Aquatic and Inva‐ sive Plants.

Another important consideration in the treatment of aquatic systems is the effect of weed control activities on fish that reside in waters targeted for herbicide application. Although the presence of fish does not affect herbicide efficacy, special precautions must be taken to ensure that these and other aquatic denizens are not harmed as a result of weed control efforts. Only a few herbicides labeled for use in bodies of water are inherently dangerous to fish, but fish kills are nonetheless a major concern for applicators working in aquatic systems. The primary reason fish kills occur after weed control activities are undertaken is a reduction in dissolved oxygen (DO), which results from a number of factors [47]. Primary among these factors is the decomposition of vegetative material that has been killed by herbicides and is broken down by aerobic organisms, which deplete DO during the process. Also, photosynthesis by plants that have been killed by herbicides is eliminated and the DO they previously contributed to the water column is no longer produced, further reducing levels of DO. In order to reduce the likelihood of fish kills, most labels for aquatic products specify that herbicides be applied to only a portion of a weed-infested body of water at a time to allow fish to escape from treated areas and to prevent the extreme drop in DO that accompanies the elimination of all vegetation from an aquatic system.

### **7. Application methods in aquatic systems**

Some of the techniques for applying herbicides in aquatic systems are similar to those used for weed control in crop production. This is especially true when the target aquatic invaders are growing along ditchbanks or shorelines or in narrow canals that can be treated using a backpack sprayer or a truck, tractor or other wheeled vehicle. However, herbicide applications to open waters require specialized equipment and tools in order to effectively reach the aquatic weeds that are targeted for control, and the primary vehicle required for aquatic weed control is a boat. The size and disposition of the treatment boat varies and is dependent on the application method to be employed, which is often dictated by the target weed and the form of herbicide being utilized. Aquatic herbicides are typically sold in liquid and granular formulations, and some active ingredients are available in both forms [48]. Granular formu‐ lations are most often applied using a boat-mounted spreader (Fig. 17). Most liquid formula‐ tions are packaged as concentrates and are applied in dilute form. Dilution is frequently accomplished by adding the concentrate to a boat-mounted tank filled with water. A variety of equipment exists to apply herbicides that have been diluted in an onboard tank; these include handguns (for treating emergent and floating weeds), booms (for treatment of surface water), and trailing weighted hoses (for subsurface treatments) [45, 49]. Regardless of the formulation and application method employed, calibration of application equipment is critically important to ensure that the correct amount of herbicide is introduced to the system. Poorly calibrated equipment may result in the application of too little herbicide, which will likely yield poor weed control and reduced product efficacy. Using an excess amount of herbicide will increase costs associated with the treatment and may result in concentrations above those specified on the product label; as outlined above, this is a violation of federal law and may have serious legal consequences.

**8. Conclusions**

**Acknowledgements**

**Author details**

my, Gainesville, FL, USA

Fresh-water resources are extremely important components of global and local ecosystems. The introduction of exotic invasive species to these systems limits their ability to function as healthy, diverse habitats for native flora and fauna; in addition, anthropogenic uses such as flood control, public safety, and recreation are hindered as well. The most effective method to reduce the impact of aquatic invaders is to prevent their introduction to these valuable and important systems, but invasive species continue to become established in aquatic systems throughout the world. The primary method used to control introduced aquatic weeds in the US is the application of registered aquatic herbicides. Pesticides that are applied to waters in the US are labeled and registered by the USEPA after extensive testing, and most states – including Florida – require that the use of these products be regulated by state agencies as well. Aquatic herbicides represent a small subset of the pesticides labeled by the USEPA and registrants only pursue aquatic labeling of products if there is a market large enough to offset the costs associated with additional registration requirements. A number of unique challenges are associated with weed control in aquatic systems, including the effects of dilution, current, and stratification of water within systems. These challenges can be overcome through the

Herbicides in Aquatic Systems http://dx.doi.org/10.5772/56015 347

This publication is a contribution of the University of Florida Institute for Food and Agricul‐

1 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐

2 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐

3 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐

selection of proper herbicides and application methods.

tural Sciences and the Florida Agricultural Experiment Station.

Lyn A. Gettys1\*, William T. Haller2\* and Gregory E. MacDonald3\*

my, Fort Lauderdale Research and Education Center, Davie, FL, USA

my, Center for Aquatic and Invasive Plants, Gainesville, FL, USA

\*Address all correspondence to: lgettys@ufl.edu

\*Address all correspondence to: whaller@ufl.edu

\*Address all correspondence to: pineacre@ufl.edu

**Figure 17.** Application of granular herbicide using a boat-mounted spreader. Photo courtesy UF/IFAS Center for Aquatic and Invasive Plants.

### **8. Conclusions**

**7. Application methods in aquatic systems**

346 Herbicides - Current Research and Case Studies in Use

and may have serious legal consequences.

Aquatic and Invasive Plants.

Some of the techniques for applying herbicides in aquatic systems are similar to those used for weed control in crop production. This is especially true when the target aquatic invaders are growing along ditchbanks or shorelines or in narrow canals that can be treated using a backpack sprayer or a truck, tractor or other wheeled vehicle. However, herbicide applications to open waters require specialized equipment and tools in order to effectively reach the aquatic weeds that are targeted for control, and the primary vehicle required for aquatic weed control is a boat. The size and disposition of the treatment boat varies and is dependent on the application method to be employed, which is often dictated by the target weed and the form of herbicide being utilized. Aquatic herbicides are typically sold in liquid and granular formulations, and some active ingredients are available in both forms [48]. Granular formu‐ lations are most often applied using a boat-mounted spreader (Fig. 17). Most liquid formula‐ tions are packaged as concentrates and are applied in dilute form. Dilution is frequently accomplished by adding the concentrate to a boat-mounted tank filled with water. A variety of equipment exists to apply herbicides that have been diluted in an onboard tank; these include handguns (for treating emergent and floating weeds), booms (for treatment of surface water), and trailing weighted hoses (for subsurface treatments) [45, 49]. Regardless of the formulation and application method employed, calibration of application equipment is critically important to ensure that the correct amount of herbicide is introduced to the system. Poorly calibrated equipment may result in the application of too little herbicide, which will likely yield poor weed control and reduced product efficacy. Using an excess amount of herbicide will increase costs associated with the treatment and may result in concentrations above those specified on the product label; as outlined above, this is a violation of federal law

**Figure 17.** Application of granular herbicide using a boat-mounted spreader. Photo courtesy UF/IFAS Center for

Fresh-water resources are extremely important components of global and local ecosystems. The introduction of exotic invasive species to these systems limits their ability to function as healthy, diverse habitats for native flora and fauna; in addition, anthropogenic uses such as flood control, public safety, and recreation are hindered as well. The most effective method to reduce the impact of aquatic invaders is to prevent their introduction to these valuable and important systems, but invasive species continue to become established in aquatic systems throughout the world. The primary method used to control introduced aquatic weeds in the US is the application of registered aquatic herbicides. Pesticides that are applied to waters in the US are labeled and registered by the USEPA after extensive testing, and most states – including Florida – require that the use of these products be regulated by state agencies as well. Aquatic herbicides represent a small subset of the pesticides labeled by the USEPA and registrants only pursue aquatic labeling of products if there is a market large enough to offset the costs associated with additional registration requirements. A number of unique challenges are associated with weed control in aquatic systems, including the effects of dilution, current, and stratification of water within systems. These challenges can be overcome through the selection of proper herbicides and application methods.

### **Acknowledgements**

This publication is a contribution of the University of Florida Institute for Food and Agricul‐ tural Sciences and the Florida Agricultural Experiment Station.

### **Author details**

Lyn A. Gettys1\*, William T. Haller2\* and Gregory E. MacDonald3\*

\*Address all correspondence to: lgettys@ufl.edu

\*Address all correspondence to: whaller@ufl.edu

\*Address all correspondence to: pineacre@ufl.edu

1 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Fort Lauderdale Research and Education Center, Davie, FL, USA

2 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Center for Aquatic and Invasive Plants, Gainesville, FL, USA

3 University of Florida Institute of Food and Agricultural Sciences, Department of Agrono‐ my, Gainesville, FL, USA

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[30] Buckingham GR. 2002. Alligatorweed. In: Van Driesche R, Blossey B, Hoddle M, Lyon S, Reardon R (eds.). Biological control of invasive plants in the eastern United

[31] Canfield DE Jr., Hoyer MV. 1992. Aquatic macrophytes and their relationships to Florida lakes. Final report to the Bureau of Aquatic Plants, Florida Department of

[32] Colle DE, Shireman JV. 1980. Coefficients of condition for largemouth bass, bluegill, and redear sunfish in hydrilla-infested lakes. Transactions of the American Fisheries

[33] Estes JR, Sheaffer WA, Hall EP. 1990. Study I. Fisheries studies of the Orange Lake chain of lakes. Florida Game and Fresh Water Fish Commission. Tallahassee, Florida,

[34] Porak WF, Crawford S, Renfro D, Cailteux RL, Chadwick J. 1990. Study XII. Large‐ mouth bass population responses to aquatic plant management strategies. Florida

[36] Johnson FA, Montalbano F III. 1987. Considering waterfowl habitat in hydrilla con‐

[37] Anonymous. 2011. Background information for the Fish and Wildlife Conservation Commission's position on hydrilla management. Florida Fish and Wildlife Conserva‐ tion Commission, Tallahassee, Florida, USA. http://myfwc.com/media/1386747/ hydrilla-mgmt-position-background-information.pdf (accessed 31 October 2012).

[38] Les DH. 1996. Hydrilla verticillata threatens New England. Aquatic Exotic News

Game and Fresh Water Fish Commission. Tallahassee, Florida, USA.

[35] Tucker T. 1987. How to fish hydrilla. Bassmaster 20(9):30-34.

trol policies. Wildlife Society Bulletin 15(3):466-469.

yngodon idella) utilizing hydrostatic pressure. Aquaculture 55(1):43-50.

States, pp. 5-15. USDA Forest Service Publication FHTET-2002-04, 413 p.

Journal of Aquatic Plant Management 46:15-32.

Natural Resources. Tallahassee, Florida, USA.

Society 109:521-531.

USA.

3(1):1-2.

USA.

350 Herbicides - Current Research and Case Studies in Use

USA.


**Chapter 14**

**Herbicide Impact on Seagrass Communities**

Anthropogenic chemical contamination is of concern due to the continuous decline of ecosys‐ tems. Pesticide use impacts whole environmental matrices, especially aquatic ones, because of collecting watershed pollution in streams, rivers, and finally coastal areas. The coastal environment is one of the most vulnerable: global changes (current sea level rising, ocean acidification, global warming) add to land use disruption (soil erosion, chemical uses, urban sprawl) in coastline areas. Moreover, population growth mainly affects this endangered environment because of rural flight and city growth –75% of billions of human beings will live in 100km-large belt around global seas in 2035 [1] imposing urban lifestyle demands. Envi‐ ronmental stress due to such a heterogeneous population repartition will be acute (1) on freshwater, in order to provide it for drinking, industrial and agricultural needs, and (2) on coastal ecosystems because of waste waters and coastline management. Such environmental concerns are critical for tropical countries because of some that are being discovered to be

Human impact is partly due to pesticide use [3-5]. In order to feed a growing population and to manage urban areas, herbicides are often used profusely [6]. These herbicides affect wild fauna and flora through improper use, inefficient (even lack of) wastewater treatment plant effluents or the direct input of herbicide contained by sewage sludge into aquatic environments [7]. For hydrophobic pesticides, contaminated solid phases transfer downstream, in an erosive

Thus, herbicides will contaminate coastal environments [9-10]. In shallow water, such residues will expose remarkable biocoenosis, especially in tropical contexts, because of conserved biodiversity compared to temperate ones, i.e. exposed for decades to aquatic pollution from

> © 2013 Devault and Pascaline; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Devault and Pascaline; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

context due to deforestation and agricultural intensification is involved [8].

A. Damien Devault and Hélène Pascaline

http://dx.doi.org/10.5772/55973

**1. Introduction**

biodiversity hot spots [2].

developed countries' activities.

Additional information is available at the end of the chapter

## **Herbicide Impact on Seagrass Communities**

A. Damien Devault and Hélène Pascaline

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55973

### **1. Introduction**

Anthropogenic chemical contamination is of concern due to the continuous decline of ecosys‐ tems. Pesticide use impacts whole environmental matrices, especially aquatic ones, because of collecting watershed pollution in streams, rivers, and finally coastal areas. The coastal environment is one of the most vulnerable: global changes (current sea level rising, ocean acidification, global warming) add to land use disruption (soil erosion, chemical uses, urban sprawl) in coastline areas. Moreover, population growth mainly affects this endangered environment because of rural flight and city growth –75% of billions of human beings will live in 100km-large belt around global seas in 2035 [1] imposing urban lifestyle demands. Envi‐ ronmental stress due to such a heterogeneous population repartition will be acute (1) on freshwater, in order to provide it for drinking, industrial and agricultural needs, and (2) on coastal ecosystems because of waste waters and coastline management. Such environmental concerns are critical for tropical countries because of some that are being discovered to be biodiversity hot spots [2].

Human impact is partly due to pesticide use [3-5]. In order to feed a growing population and to manage urban areas, herbicides are often used profusely [6]. These herbicides affect wild fauna and flora through improper use, inefficient (even lack of) wastewater treatment plant effluents or the direct input of herbicide contained by sewage sludge into aquatic environments [7]. For hydrophobic pesticides, contaminated solid phases transfer downstream, in an erosive context due to deforestation and agricultural intensification is involved [8].

Thus, herbicides will contaminate coastal environments [9-10]. In shallow water, such residues will expose remarkable biocoenosis, especially in tropical contexts, because of conserved biodiversity compared to temperate ones, i.e. exposed for decades to aquatic pollution from developed countries' activities.

© 2013 Devault and Pascaline; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Devault and Pascaline; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Considering such shallow waters, herbicides could influence seagrasses. Such tracheophyte flowering plants are the result of terrestrial grass adaptation to marine environments. These monocotyledons colonize shallow water bottoms, especially in silty-sandy substrata because of roots, unlike bryophytes and algae. Thus, in opposition to those non-tracheophyte ones, seagrass meadows could limit sediment erosion and stabilise navigation channels.

As marine species, seagrasses are vulnerable to low salinity events and cannot colonize upstream estuaries and freshwater shallows. A 5‰ salinity is the smallest salinity amount compatible with seagrass development (Iversen, 1931 cited by Vermaat et al. [23];[24-26]), whereas seagrass communities can stand waters which are more salty than the global ocean salinity (35‰): Depending on species, seagrasses could stand a salty environment up to 42‰ [27]. Euryhaline seagrass species, i.e. large scale salinity ones, colonize all the climatic areas except polar ones. But, in all of them, these remarkable adaptations are balanced by severe

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973 355

Seagrasses are present in all the marine ecotones, except polar ones ([28]; Figures 1 and 2).

**Figure 1.** Global seagrass diversity and distribution. Shades of green indicate numbers of species reported for an area;

Temperate areas are marked by seasons with different temperatures, light and precipitation regimes. Land and sea weathers provide extreme wind and flow conditions. Nutrient inputs occur by pulses which seagrass meadows must cope with [29]. Seagrass meadows will consume nutrients in perennial vegetative growth, limiting eutrophication conditions [30]. Ecosystems including seagrasses are listed in Figure 3. In temperate marine water, seagrasses are associated with marshes and kelp beds, and have been providing for centuries ecosystem services to coastal lands [31]. Human use of kelp began as picking fodder, fertilizer –even organic matter- and food on shore. During the late modern time period, dried kelp was used

blue points and polygons indicate documented reports of seagrass occurrence (from 2005 UNEP-WCMC).

**3. Seagrass meadows repartition and involved landscape**

seagrass meadow regression.

### **2. Seagrasses: Unique origin, physiology and performances**

Seagrass is a taxonomic group of about 60 species worldwide likely evolving from a single monocotyledonous flowering plant ancestor (70-100 million years ago), divided into three independent lineages: Hydrocharitaceae, Cymodoceaceae and Zosteraceae [11]. Seagrass species have strong physiological similitude and low interspecies diversity.

As flowering plants, they are anchored in sediments by their roots –what non-tracheophyte marine plant species don't have. Notwithstanding, seagrass only live in submarine environ‐ ments, even for pollination or other critical steps, unlike other aquatic flowering plants who should use an emerged organ or pass by a terrestrial stage [12]. Seagrasses have some of the highest light requirements (25% of incident radiation when 1% is the average requirement of angiosperm species [13]) even if epidermal chloroplast and internal gas transport systems have been developed, in order to maintain oxidative conditions, despite highly reducing sediment, including toxic sulphide levels, for large amounts of non-photosynthetic tissues [14]. Sea‐ grasses are especially vulnerable to lack of light, mainly due to erosion or eutrophication.

While algae, whose growth is proportioned to the eutrophication level and thus could lead to a dystrophic crisis due to algal necromass decomposition, seagrass growth biomass is sus‐ tainable. Seagrass bed increase due to nutrient input makes seagrass meadows, for carbon trapping and storing [15], like corn or sugar cane, among the most efficient trapping plants [16]. Seagrass meadows are a more efficient carbon sink than trees: with an equivalent carbon sequestration per year (about 27 million tons [17]), carbon sequestered in meadows will be buried and therefore partly avoid decomposition in the matte [18]; Pergent et al. [19] estimated this stored amount about a third of the primary production. Living seagrass biomass actually reaches 19.9 billion tons [17].

Moreover, to this biomass should be added suspended matters that seagrass leaves could efficiently sequester because of the blade effect on suspended matter, i.e. acting like a mat, trapping suspended matter and inherent organic matter, and because seagrass decomposition is too long for inducing dead zones [12]. Seagrass blades could drift to the abyss where they are an indispensable carbon contribution for poor-carbon deep sea biocoenosis [20].

Seagrass could be susceptible to exondation because of tides. Such events could be fatal depending on shore temperature. Temperatures of 35°C and greater, not found in the marine environment but possible in pools or during extreme low tide coefficient, could kill seagrass [21, 22] because of photosynthesis interruption; irreparable structural alterations to the PhotoSystem II (PSII) reaction centres induce chloroplast dysfunction, leading the plants to insufficiently jugulate of the reductive conditions in roots.

As marine species, seagrasses are vulnerable to low salinity events and cannot colonize upstream estuaries and freshwater shallows. A 5‰ salinity is the smallest salinity amount compatible with seagrass development (Iversen, 1931 cited by Vermaat et al. [23];[24-26]), whereas seagrass communities can stand waters which are more salty than the global ocean salinity (35‰): Depending on species, seagrasses could stand a salty environment up to 42‰ [27]. Euryhaline seagrass species, i.e. large scale salinity ones, colonize all the climatic areas except polar ones. But, in all of them, these remarkable adaptations are balanced by severe seagrass meadow regression.

### **3. Seagrass meadows repartition and involved landscape**

Considering such shallow waters, herbicides could influence seagrasses. Such tracheophyte flowering plants are the result of terrestrial grass adaptation to marine environments. These monocotyledons colonize shallow water bottoms, especially in silty-sandy substrata because of roots, unlike bryophytes and algae. Thus, in opposition to those non-tracheophyte ones,

Seagrass is a taxonomic group of about 60 species worldwide likely evolving from a single monocotyledonous flowering plant ancestor (70-100 million years ago), divided into three independent lineages: Hydrocharitaceae, Cymodoceaceae and Zosteraceae [11]. Seagrass

As flowering plants, they are anchored in sediments by their roots –what non-tracheophyte marine plant species don't have. Notwithstanding, seagrass only live in submarine environ‐ ments, even for pollination or other critical steps, unlike other aquatic flowering plants who should use an emerged organ or pass by a terrestrial stage [12]. Seagrasses have some of the highest light requirements (25% of incident radiation when 1% is the average requirement of angiosperm species [13]) even if epidermal chloroplast and internal gas transport systems have been developed, in order to maintain oxidative conditions, despite highly reducing sediment, including toxic sulphide levels, for large amounts of non-photosynthetic tissues [14]. Sea‐ grasses are especially vulnerable to lack of light, mainly due to erosion or eutrophication. While algae, whose growth is proportioned to the eutrophication level and thus could lead to a dystrophic crisis due to algal necromass decomposition, seagrass growth biomass is sus‐ tainable. Seagrass bed increase due to nutrient input makes seagrass meadows, for carbon trapping and storing [15], like corn or sugar cane, among the most efficient trapping plants [16]. Seagrass meadows are a more efficient carbon sink than trees: with an equivalent carbon sequestration per year (about 27 million tons [17]), carbon sequestered in meadows will be buried and therefore partly avoid decomposition in the matte [18]; Pergent et al. [19] estimated this stored amount about a third of the primary production. Living seagrass biomass actually

Moreover, to this biomass should be added suspended matters that seagrass leaves could efficiently sequester because of the blade effect on suspended matter, i.e. acting like a mat, trapping suspended matter and inherent organic matter, and because seagrass decomposition is too long for inducing dead zones [12]. Seagrass blades could drift to the abyss where they

Seagrass could be susceptible to exondation because of tides. Such events could be fatal depending on shore temperature. Temperatures of 35°C and greater, not found in the marine environment but possible in pools or during extreme low tide coefficient, could kill seagrass [21, 22] because of photosynthesis interruption; irreparable structural alterations to the PhotoSystem II (PSII) reaction centres induce chloroplast dysfunction, leading the plants to

are an indispensable carbon contribution for poor-carbon deep sea biocoenosis [20].

insufficiently jugulate of the reductive conditions in roots.

seagrass meadows could limit sediment erosion and stabilise navigation channels.

**2. Seagrasses: Unique origin, physiology and performances**

species have strong physiological similitude and low interspecies diversity.

reaches 19.9 billion tons [17].

354 Herbicides - Current Research and Case Studies in Use

Seagrasses are present in all the marine ecotones, except polar ones ([28]; Figures 1 and 2).

**Figure 1.** Global seagrass diversity and distribution. Shades of green indicate numbers of species reported for an area; blue points and polygons indicate documented reports of seagrass occurrence (from 2005 UNEP-WCMC).

Temperate areas are marked by seasons with different temperatures, light and precipitation regimes. Land and sea weathers provide extreme wind and flow conditions. Nutrient inputs occur by pulses which seagrass meadows must cope with [29]. Seagrass meadows will consume nutrients in perennial vegetative growth, limiting eutrophication conditions [30].

Ecosystems including seagrasses are listed in Figure 3. In temperate marine water, seagrasses are associated with marshes and kelp beds, and have been providing for centuries ecosystem services to coastal lands [31]. Human use of kelp began as picking fodder, fertilizer –even organic matter- and food on shore. During the late modern time period, dried kelp was used

**Figure 2.** Current global distribution of seagrass in relation to mean ocean temperature. Regional divisions are based on polar (<4 degrees Celsius [°C]), temperate (4°C-24°C), and tropical (>24°C) climate [52].

in order to provide "non-caustic" or "commercial" soda (in fact, sodium carbonate Na2CO3) for early industries (glass, photograph, soap, etc.). Seagrass meadows are colonizing nearshore environment mixing or not with seaweeds, i.e. seagrasses mainly settle on movable substrata where their roots could anchor them and seaweeds only settle on hard substrata (rocks or shingles) needed by their basal adhesive organ. Seaweeds are sessile, macroscopic, benthic and multi-cellular algae [32] constituting a polyphyletic community. Seagrass mead‐ ows and seaweeds live in adjacent environments and could marginally be in competition. However, the relationship between them does not present the same cooperative side as in tropical areas.

In tropical areas, marine seagrasses are associated with preserved triptych mangrovemeadows-corals [33]: (1) Mangrove stabilises and protects the coastline, limits sediment input in marine environments and holds tidal biodiversity [34]; (2) seagrass meadows, because of blades and roots, limits current movements, enhances suspended particular matter deposition, provides food for endangered species like sea turtles and manatees [35]; (3) coral reefs protect the shore from waves, acting like living breakwater, an especially acute property in tropical areas subjected to typhoons and tsunamis [36]. Triptych partners have a mutual service relationship, i.e. corals are vulnerable to sedimentation limited by mangroves, which are vulnerable to large waves buffered by coral reefs. Moreover, each partner has a nursing role for marine species [37, 38]: for example, considering the eastern area of the Caribbean Sea, 80% of fisheries are located in mangrove, meadow or coral areas for juvenile stages [39]. However, significant landscapes of corresponding environments are limited to some French West Indies bays, exposed to land speculation. Thus, because each triptych partner has its own vulnera‐

**Figure 3.** Seagrass habitat diagrams for (a) Bioregions 1–3 and (b) Bioregions 4–6. Major species for each bioregion

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973 357

listed according to dominance within habitats. Maximum reported depths [113].

in order to provide "non-caustic" or "commercial" soda (in fact, sodium carbonate Na2CO3) for early industries (glass, photograph, soap, etc.). Seagrass meadows are colonizing nearshore environment mixing or not with seaweeds, i.e. seagrasses mainly settle on movable substrata where their roots could anchor them and seaweeds only settle on hard substrata (rocks or shingles) needed by their basal adhesive organ. Seaweeds are sessile, macroscopic, benthic and multi-cellular algae [32] constituting a polyphyletic community. Seagrass mead‐ ows and seaweeds live in adjacent environments and could marginally be in competition. However, the relationship between them does not present the same cooperative side as in

**Figure 2.** Current global distribution of seagrass in relation to mean ocean temperature. Regional divisions are based

on polar (<4 degrees Celsius [°C]), temperate (4°C-24°C), and tropical (>24°C) climate [52].

356 Herbicides - Current Research and Case Studies in Use

In tropical areas, marine seagrasses are associated with preserved triptych mangrovemeadows-corals [33]: (1) Mangrove stabilises and protects the coastline, limits sediment input in marine environments and holds tidal biodiversity [34]; (2) seagrass meadows, because of blades and roots, limits current movements, enhances suspended particular matter deposition, provides food for endangered species like sea turtles and manatees [35]; (3) coral reefs protect the shore from waves, acting like living breakwater, an especially acute property in tropical areas subjected to typhoons and tsunamis [36]. Triptych partners have a mutual service relationship, i.e. corals are vulnerable to sedimentation limited by mangroves, which are vulnerable to large waves buffered by coral reefs. Moreover, each partner has a nursing role for marine species [37, 38]: for example, considering the eastern area of the Caribbean Sea, 80% of fisheries are located in mangrove, meadow or coral areas for juvenile stages [39]. However, significant landscapes of corresponding environments are limited to some French West Indies bays, exposed to land speculation. Thus, because each triptych partner has its own vulnera‐

tropical areas.

**Figure 3.** Seagrass habitat diagrams for (a) Bioregions 1–3 and (b) Bioregions 4–6. Major species for each bioregion listed according to dominance within habitats. Maximum reported depths [113].

bilities, this ecosystemic symbiosis is acutely vulnerable; if mangroves are largely unaffected by water quality [40], seagrass meadows are highly susceptible to chemical inputs [12] when corals are mainly sensitive to herbicides because of endosymbiosis [41], i.e. corals include algal symbionts which are highly vulnerable to PSII herbicides, in a few minutes and for ng/L contamination range [42]. The Achilles' heel of each ecological partner endangers the whole ecosystem equilibrium.

Notwithstanding ecological and fishery services, rendered in tropical and temperate zones, seagrass meadows support the detrital food web, perpetuates navigation supports, and have a key position considering carbon and nutrient cycles. Each seagrass annual services have been estimated between \$9,000 and \$28,000 per acre [43] –globally \$1.9 trillion per year by Watson et al. [44] describing seagrass meadows as constituting an endangered capital.

### **4. Seagrass: The silent fall**

Indeed, because of local triptych disruption due to anthropogenic needs or airiness in tropical areas or due to the global environmental decline, seagrass meadows are threatened (Figure 4). All over the world, this unique biocoenosis is regressing; during the last decade, between 20 and 100% in the Gulf of Mexico, depending on the coastal zone, 85% in Florida, 40% in the bay of Arcachon [45], an accelerating loss process [28] leading to an evaluated total loss since 1980 of about 30% of global seagrass meadows, i.e. at the same scale as mangrove regression (-1.8% yr-1 [46]). Thus, seagrass meadows are more endangered than the tropical rain forest (-0.5 yr-1 [47]) and as precious as it for carbon storage (cf. *supra*). Mangrove and seagrass regression undermine coral reefs, more sensitive to the seagrass meadows' regression and sensitive to another threats (-0.72 to -9% yr-1 [48-50]). Each year, about 177,000 km² of seagrass meadows, i.e. 1.5% of global seagrass meadows (Ibid.), are lost -about 299 million tonnes of carbon trapped [12].

meadows [12] even though estimated monetary value of seagrass meadows is more than twofold more important than mangrove or marshes, and four-fold more than coral reefs [43].

34 sites in Europe, and 40 sites in Australia [28].

**Figure 4.** Global map indicating changes in seagrass area plotted by coastline regions. Changes in seagrass areal ex‐ tent at each site are defined as declining (red) or increasing (green) when areal extent changed by >10%, or no de‐ tectable change (yellow) when final area was within ±10% of the initial area. There were 131 sites in North America,

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973 359

The seagrass loss origin is actually unknown. First of all because, due to perennial growth of seagrass meadows, environmental disease impact is more obviously observed than for other marine plants, and particularly unicellular organisms. Moreover, because of the macroscopic size of seagrass specimens and their eminent services for the environment and for human activity, seagrass meadow loss critically alters coastal activities [51]. Lastly, seagrass meadow loss reveals a long-term impact on the environment when planktonic or fugacious species providing short-term environmental status; such outcomes need high-frequency monitoring for a valuable putting into perspective. Orth et al. [12] defined seagrass as "coastal canaries".

Chemical content in seawater is directly, but locally, altered by port uses, i.e. antifouling coatings and urban pesticide uses, and indirectly, but globally, altered by agricultural and urban chemical input due to landscape runoff and subsequent river pollution [55] and when groundwater tables well up through the soil directly at the sea (phenomenon known as Submarine Groundwater Discharge [56]). Their terrestrial impact is well known, as on soils, then on groundwater, surface water [3], sediments [57], biota [4] and human health [58].

Vulnerability of aquatic environments to organic chemicals, and especially pesticides, has been asserted for decades. In many countries, pesticide monitoring is performed for groundwater,

**5. Predominant pesticide effects applied to seagrass physiology**

Worldwide seagrass meadow loss is not balanced by seeding or planting campaigns. First, restoration scales are largely smaller than the seagrass meadows loss; most of them are <1 ha because of costs –even if restoration cost is still less expensive than seagrass loss consequences themselves. Secondly, restoration success rate is low: about 30% [51] or more [52] –but some seagrass species are not transplantable [51] leading them to a more acute endangered situation in lineage, vulnerable because of poor genetic diversity [53]. But restoration initiatives are induced by information about seagrass loss and its consequences. Actually, information lacks in order to know the impact of seagrass meadow fragmentation; such interconnectivity loss is due to human activity because of the declining chemical quality of seawater as well as to building or coastline management [54].

Notwithstanding the alarming situation and issues, the publication rate about seagrass meadow loss remains low; the actual increase of publication numbers and quality about this concern should be proportioned to the global ocean crisis. Mangrove, salt marsh, and coral reefs, in particular, have are three- to one hundred-fold more publications than seagrass

bilities, this ecosystemic symbiosis is acutely vulnerable; if mangroves are largely unaffected by water quality [40], seagrass meadows are highly susceptible to chemical inputs [12] when corals are mainly sensitive to herbicides because of endosymbiosis [41], i.e. corals include algal symbionts which are highly vulnerable to PSII herbicides, in a few minutes and for ng/L contamination range [42]. The Achilles' heel of each ecological partner endangers the whole

Notwithstanding ecological and fishery services, rendered in tropical and temperate zones, seagrass meadows support the detrital food web, perpetuates navigation supports, and have a key position considering carbon and nutrient cycles. Each seagrass annual services have been estimated between \$9,000 and \$28,000 per acre [43] –globally \$1.9 trillion per year by Watson

Indeed, because of local triptych disruption due to anthropogenic needs or airiness in tropical areas or due to the global environmental decline, seagrass meadows are threatened (Figure 4). All over the world, this unique biocoenosis is regressing; during the last decade, between 20 and 100% in the Gulf of Mexico, depending on the coastal zone, 85% in Florida, 40% in the bay of Arcachon [45], an accelerating loss process [28] leading to an evaluated total loss since 1980 of about 30% of global seagrass meadows, i.e. at the same scale as mangrove regression (-1.8% yr-1 [46]). Thus, seagrass meadows are more endangered than the tropical rain forest (-0.5 yr-1 [47]) and as precious as it for carbon storage (cf. *supra*). Mangrove and seagrass regression undermine coral reefs, more sensitive to the seagrass meadows' regression and sensitive to another threats (-0.72 to -9% yr-1 [48-50]). Each year, about 177,000 km² of seagrass meadows, i.e. 1.5% of global seagrass meadows (Ibid.), are lost -about 299 million tonnes of

Worldwide seagrass meadow loss is not balanced by seeding or planting campaigns. First, restoration scales are largely smaller than the seagrass meadows loss; most of them are <1 ha because of costs –even if restoration cost is still less expensive than seagrass loss consequences themselves. Secondly, restoration success rate is low: about 30% [51] or more [52] –but some seagrass species are not transplantable [51] leading them to a more acute endangered situation in lineage, vulnerable because of poor genetic diversity [53]. But restoration initiatives are induced by information about seagrass loss and its consequences. Actually, information lacks in order to know the impact of seagrass meadow fragmentation; such interconnectivity loss is due to human activity because of the declining chemical quality of seawater as well as to

Notwithstanding the alarming situation and issues, the publication rate about seagrass meadow loss remains low; the actual increase of publication numbers and quality about this concern should be proportioned to the global ocean crisis. Mangrove, salt marsh, and coral reefs, in particular, have are three- to one hundred-fold more publications than seagrass

et al. [44] describing seagrass meadows as constituting an endangered capital.

ecosystem equilibrium.

358 Herbicides - Current Research and Case Studies in Use

**4. Seagrass: The silent fall**

carbon trapped [12].

building or coastline management [54].

**Figure 4.** Global map indicating changes in seagrass area plotted by coastline regions. Changes in seagrass areal ex‐ tent at each site are defined as declining (red) or increasing (green) when areal extent changed by >10%, or no de‐ tectable change (yellow) when final area was within ±10% of the initial area. There were 131 sites in North America, 34 sites in Europe, and 40 sites in Australia [28].

meadows [12] even though estimated monetary value of seagrass meadows is more than twofold more important than mangrove or marshes, and four-fold more than coral reefs [43].

The seagrass loss origin is actually unknown. First of all because, due to perennial growth of seagrass meadows, environmental disease impact is more obviously observed than for other marine plants, and particularly unicellular organisms. Moreover, because of the macroscopic size of seagrass specimens and their eminent services for the environment and for human activity, seagrass meadow loss critically alters coastal activities [51]. Lastly, seagrass meadow loss reveals a long-term impact on the environment when planktonic or fugacious species providing short-term environmental status; such outcomes need high-frequency monitoring for a valuable putting into perspective. Orth et al. [12] defined seagrass as "coastal canaries".

### **5. Predominant pesticide effects applied to seagrass physiology**

Chemical content in seawater is directly, but locally, altered by port uses, i.e. antifouling coatings and urban pesticide uses, and indirectly, but globally, altered by agricultural and urban chemical input due to landscape runoff and subsequent river pollution [55] and when groundwater tables well up through the soil directly at the sea (phenomenon known as Submarine Groundwater Discharge [56]). Their terrestrial impact is well known, as on soils, then on groundwater, surface water [3], sediments [57], biota [4] and human health [58].

Vulnerability of aquatic environments to organic chemicals, and especially pesticides, has been asserted for decades. In many countries, pesticide monitoring is performed for groundwater, often in order to ensure drinking water, as well as for surface water. Incidentally, it is possible to determine the most frequently used herbicides. According to Gilliom [3] for the U.S.A. and to Schäfer et al. [4] in the European Union, 21 herbicides could be identified as being the most frequently used in developed countries, leading them to probably being significantly used in developing ones (Table 1). Haynes et al. [59-61] consider diuron as the most threatening herbicide, even pesticide, for seagrass meadows, partnering the corals of the Great Barrier Reef. However, aquatic plant toxicology is not well-known: BCPC [62] only informs us about two aquatic plant toxicological tests: EC50 (120h) for diuron on *Selenastrum capricornutum*(0.002mg/ L) and atrazine EC50 (96h) for *S. capricornutum* (0.01mg/L). Lewis & Devereux [55] provided the first review on non-nutrient anthropogenic chemicals in seagrass ecosystems, summarizing all publications on seagrass –and finding only ten on herbicide impact on seagrass.

**Solubility Koc Kow Application Effects/metabolic target Notes**

483 (1) 0.5-1 microtubule

norflurazon 34 218-635 2,45 (pH6,5) 0,5-2 photosensible; high

2,4D 311 60 (calc) -0.75(sp) 0.28-2.3 synthetic auxin turns crystal in hard water

Herbicide effects have been summarized by Jurado et al. [63]. The biochemical target of many herbicides is PhotoSystem II (P680), acting in photosynthesis as a photon-electron converting disruptor. More precisely, the inhibition of Hill reaction (photosynthetic electron transport) is performed in A site by triazines, uraciles pyridazines, in B site by ureas. Acylanilides, diphenyl ethers and nitriles inhibit the Hill reaction too. In the 21 predominant herbicides compilation list [3, 4], 11 herbicides uncouple the biochemical cascade in PSII leading to plastoquinone terminal electron acceptor. Instead of this outcome, formation of unmanaged singlet oxygen provokes lesions proportioned to photosynthesis. Biosynthesis of carotenoids, used to manage singlet oxygen, could be a collateral damage facilitating herbicide effects (pyridazines).

On a plant scale, PSII herbicides lead to a more marked leaf yellowing in new leaves than in old ones, i.e. leafs where photosynthesis has been active, and in places of intense photosyn‐ thesis i.e. between leaf veins. Such symptomology should impact on shallow water depth seagrass communities and even save seagrass communities in turbid water. However, light provides food for seagrasses [64] like for the other photosynthetic taxa, but it is a way of detoxifying too. Over time, depending on contamination by such herbicides, light conditions favourable to seagrass communities could be limited by (1) minimal photosynthesis needs, especially high for seagrasses in order to confront sediment anaerobic conditions, and (2) lethal photosynthetic induction, due to poisonous singlet oxygen produced by incident radiation. Indeed, seagrasses reach a 25% requirement of incident radiation [65], due to their submarine

**Table 1.** Summary of properties of the predominant herbicides in Europe [4] and in U.S.A.[3]. Solubility is expressed in mg/L, Koc in mL/g, application in kg/ha. Experimental temperature is 25°C without complementary information: (1): 20C; (2): 23°C; (3): Experimental pH is 7 without complementary information: a: pH 5; b: pH 9; c: pH 10; d: pH 4. EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme involved in aromatic amino acids phenylalanine, tyrosine and tryptophan biosynthesis. PSII: inhibition of Hill reaction in photosynthetic electron transport. Triazole: probable interference with carotenoid biosynthesis leading to photooxidation of chlorophyll. Sp: 2,4-D Kow: 2.58-2.83 (pH 1); 0.04-0.33 (pH 5); -0.75 (pH 7) Calc: Value obtained by calculation. Total: total

polymerisation inhibition in roots

inhibition germination

2.5-5 lipid synthesis disruption:

not hydrolyzable

361

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973

> Photosensible; no degradation after 2 years); not hydrolyzable

volatilizable; shelf-life: 4 years and more

trifluraline 0.221

(calc) 0.395 (field)

molinate 1100 121-252 2.86

weed killing –in order to obtain a bare soil.

4400-4000 0

> (pH7.85-7.94 (2))

tebuthiuron 2500 (1) 1,82 (1) 0,6-6,87 PSII

bromacil 807a; 700; 1287b 1,88a 1.5-8; 5-15 total PSII



often in order to ensure drinking water, as well as for surface water. Incidentally, it is possible to determine the most frequently used herbicides. According to Gilliom [3] for the U.S.A. and to Schäfer et al. [4] in the European Union, 21 herbicides could be identified as being the most frequently used in developed countries, leading them to probably being significantly used in developing ones (Table 1). Haynes et al. [59-61] consider diuron as the most threatening herbicide, even pesticide, for seagrass meadows, partnering the corals of the Great Barrier Reef. However, aquatic plant toxicology is not well-known: BCPC [62] only informs us about two aquatic plant toxicological tests: EC50 (120h) for diuron on *Selenastrum capricornutum*(0.002mg/ L) and atrazine EC50 (96h) for *S. capricornutum* (0.01mg/L). Lewis & Devereux [55] provided the first review on non-nutrient anthropogenic chemicals in seagrass ecosystems, summarizing

all publications on seagrass –and finding only ten on herbicide impact on seagrass.

glyphosate 10500 -3.2 (1) 2 block EPSPS-catabolic

diuron 37.4 400 2.85 6-30

360 Herbicides - Current Research and Case Studies in Use

*160*

*142*

"/>1384000 d

prometon 750 2.69 10-20 PSII

cyanazine 171 1-3 selectif

acetochlor 282 3 No definitively known metribuzin 1050 0.07-1.45 species-specific; PSII

> 0.77 (a); -0.46; -0.55 (b)

EPTC 375 3.2 4.5-6.7 Inhibit lipid synthesis

simazine 6.2 (1) 103-277:

amitrole 26000 pH7-c;

bentazone 570 (1) 13.3-176

Smetolachlor **Solubility Koc Kow Application Effects/metabolic target Notes**

(10-30 total)

tropical)

isoproturon 65 2.5 (1) 1.15 PSII DT50: 1560d

linuron 63.8 (1) 500-600 3 PSII DT50: "/>1000d for all pH metolachlor 488 121-309 1-2,5 PSII DT50 hydrolysis: "/>200d

480 61-369 0.6-1.6 PSII non hydrolyzable

atrazine 33 2.5 1,5 PSII, especially on

2,1 1,5 (3

crossroad for proteins

PSII, especially on dichotyledons

PSII


1-2,2 species-specific; PSII winter herbicide; low

Very easily complexed

DT50: 50-180d depending to humidity Hill et al., 1955

> hydrolysis but high photolysis

dichotyledons

**Table 1.** Summary of properties of the predominant herbicides in Europe [4] and in U.S.A.[3]. Solubility is expressed in mg/L, Koc in mL/g, application in kg/ha. Experimental temperature is 25°C without complementary information: (1): 20C; (2): 23°C; (3): Experimental pH is 7 without complementary information: a: pH 5; b: pH 9; c: pH 10; d: pH 4. EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme involved in aromatic amino acids phenylalanine, tyrosine and tryptophan biosynthesis. PSII: inhibition of Hill reaction in photosynthetic electron transport. Triazole: probable interference with carotenoid biosynthesis leading to photooxidation of chlorophyll. Sp: 2,4-D Kow: 2.58-2.83 (pH 1); 0.04-0.33 (pH 5); -0.75 (pH 7) Calc: Value obtained by calculation. Total: total weed killing –in order to obtain a bare soil.

Herbicide effects have been summarized by Jurado et al. [63]. The biochemical target of many herbicides is PhotoSystem II (P680), acting in photosynthesis as a photon-electron converting disruptor. More precisely, the inhibition of Hill reaction (photosynthetic electron transport) is performed in A site by triazines, uraciles pyridazines, in B site by ureas. Acylanilides, diphenyl ethers and nitriles inhibit the Hill reaction too. In the 21 predominant herbicides compilation list [3, 4], 11 herbicides uncouple the biochemical cascade in PSII leading to plastoquinone terminal electron acceptor. Instead of this outcome, formation of unmanaged singlet oxygen provokes lesions proportioned to photosynthesis. Biosynthesis of carotenoids, used to manage singlet oxygen, could be a collateral damage facilitating herbicide effects (pyridazines).

On a plant scale, PSII herbicides lead to a more marked leaf yellowing in new leaves than in old ones, i.e. leafs where photosynthesis has been active, and in places of intense photosyn‐ thesis i.e. between leaf veins. Such symptomology should impact on shallow water depth seagrass communities and even save seagrass communities in turbid water. However, light provides food for seagrasses [64] like for the other photosynthetic taxa, but it is a way of detoxifying too. Over time, depending on contamination by such herbicides, light conditions favourable to seagrass communities could be limited by (1) minimal photosynthesis needs, especially high for seagrasses in order to confront sediment anaerobic conditions, and (2) lethal photosynthetic induction, due to poisonous singlet oxygen produced by incident radiation. Indeed, seagrasses reach a 25% requirement of incident radiation [65], due to their submarine adaptation and because of anoxia containment in buried non-photosynthetic tissues [66]. Seagrass incident radiation requirement is to compare with 1% or less of the requirements for other angiosperm species [65]. Seagrasses will physiologically enhance their incident radiation: chloroplast efficiency is modulated for better light capture: adaptation at biochemical level [67] and at organelle scale by conditioning its position in the cell and orientation relatively to the light source [68]. Acting for maximizing photon capture, seagrasses could maximize the risk of lesions due to electron transport alteration by herbicides especially if PSII disruptors mimic a lacks of light because of induced low photosynthetic yield.

However, some remarks are necessary: (1) like for many environmental monitoring, metrology improvement since decades enhances phenomena perception: concentrations reached 30 years ago could be regarded as overestimated, (2) interspecies heterogeneity could be important: *Halodule wrightii* growth is enhanced at 120μg/L [75] when, at the same concentration *Ruppia maritima* photosynthesis IC50 is reached [42] and *Zostera marina* presents mortalities [76] (3) *Zostera marina* mortalities [76] are noted for concentrations considered as stimulating [69], but in the second case after much more exposition time, (5) the effect is less and recovery is greater

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973 363

Seagrasses are mainly regarded as shadow plants [77]. Indeed, the situation is critical during summer low tides when photoinhibition is a threat. Photoinhibition is defined by Touchette & Burkholder [78] as a reduction in the photosynthetic rate due to other processes such as the toxicological impact of herbicides [79]. Photoinhibition is primarly a photoprotective tool, avoiding PSII excessive photophosphorylation, and dissipating energy as heat [80, 81]. Photoinhibition is obtained by PSII centres rarefaction [81] or inactivation because of D1 protein photolysis surpassing D1 synthesis [80-84] and the increase of xanthophylls cycle's violaxanthin de-epoxidation agent leading to energy dissipation: zeaxanthin. Protein D1 is known to be influenced by ATP in thylakoid lumen; in the case of a lack of ATP production, for example as herbicide impact occurs, seagrass could maintain a high photosynthetic level, initiating a vicious circle leading to the herbicide effect. Such an herbicide trajectory could lead

The second critical target for seagrass, i.e. especially critical due to aquatic life, is a photores‐ piration process which is, like for several topics, innovative in the case of seagrasses [78]. Indeed, respiration provides to seagrasses oxidative conditions propitious to life in their partially reductive environment. In order to maintain a redox potential suitable for whole enzymatic activity even in tissues buried into the sediment, seagrasses will actively manage inner gas exchanges. A large part of tissue volume will be due to lacunae or *aerarium*, empty spaces allowing to preserve terrestrial-like conditions for cells, providing oxidative conditions and leaving the leafs erect. Respiration will produce CO2 for the plant –which will mainly consume its own CO2 production in order to limit exchanges with external marine environ‐ ments. Such C3-C4 intermediate plants present, moreover, concentrating carbon systems strengthening ribulose 1,5-bisphosphate carboxylase (Rubisco) in carbon acquisition. 2,4-D photosynthetic pathways are negatively impacted [86], even in micromolar concentrations, by auxin like 2,4-D, leading to up-regulating growth. Because of gas flows to the external environment are more strictly controlled by seagrasses' stomata, which are less numerous than terrestrial plants' ones, auxin-like activity of 2,4-D causes an up - regulation in oxygen production and a subsequent oxygen-inhibition of a key enzyme Rubisco. Lack of carbonsequestering photosynthetic processes leads to carbon and energy deficits aggravated by messy leaf creation induced by 2,4-D, exhausting the plant, drawing on belowground stocks,

Photorespiration leads to consume O2 and is considered as protective for photosynthetic electron transport, limiting damage to the photosynthetic apparatus to photo-inactivation during periods of low CO2 availability and high light intensity [87]. Rates of photorespiration

first to photosuppression with excessive UV radiations [85].

and limiting photosynthesis efficiency.

*in situ* [74].

The photosynthetic stress hypothesis is strengthened by ten publications (Table 2) observing oxygen production alteration [69-71] and especially oxygen production stimulation for low atrazine concentration (75μg/L) for EC50 at 320μg/L [69]. In the same way (and at same scale, leading to suggest that it is the same phenomenon but with a different descriptor), photosyn‐ thesis alteration [72] reaches 120μg/L for immediate (2h) IC50 [42] when pigments, chlorophyll and fluorescence are altered from 10μg/L [73, 74].


**Table 2.** Example of toxic effect concentrations reported for atrazine and seagrasses. EC50, IC50 –concentration reducing effect parameter 50% relative to control. rec: recovery; i: inhibition; ti: total inhibition; e: enhancement; However, some remarks are necessary: (1) like for many environmental monitoring, metrology improvement since decades enhances phenomena perception: concentrations reached 30 years ago could be regarded as overestimated, (2) interspecies heterogeneity could be important: *Halodule wrightii* growth is enhanced at 120μg/L [75] when, at the same concentration *Ruppia maritima* photosynthesis IC50 is reached [42] and *Zostera marina* presents mortalities [76] (3) *Zostera marina* mortalities [76] are noted for concentrations considered as stimulating [69], but in the second case after much more exposition time, (5) the effect is less and recovery is greater *in situ* [74].

adaptation and because of anoxia containment in buried non-photosynthetic tissues [66]. Seagrass incident radiation requirement is to compare with 1% or less of the requirements for other angiosperm species [65]. Seagrasses will physiologically enhance their incident radiation: chloroplast efficiency is modulated for better light capture: adaptation at biochemical level [67] and at organelle scale by conditioning its position in the cell and orientation relatively to the light source [68]. Acting for maximizing photon capture, seagrasses could maximize the risk of lesions due to electron transport alteration by herbicides especially if PSII disruptors mimic

The photosynthetic stress hypothesis is strengthened by ten publications (Table 2) observing oxygen production alteration [69-71] and especially oxygen production stimulation for low atrazine concentration (75μg/L) for EC50 at 320μg/L [69]. In the same way (and at same scale, leading to suggest that it is the same phenomenon but with a different descriptor), photosyn‐ thesis alteration [72] reaches 120μg/L for immediate (2h) IC50 [42] when pigments, chlorophyll

**Test species Response parameters Test duration Effect concentration (µg/L) References**

*Thalassia testudinum* Oxygen production 40h, 88h 320 (EC50) [71] *Zostera marina* Oxygen production 24h 100i, 1000ti [70] *Zostera marina* Oxygen production 21-42d 75e, 650i [69] *Zostera marina* Adenine nucleotides 6h, 21d 10, 100 [76]

*Zostera marina* Growth 10-40d 1900 (first effect, whole plant) [114]

*Zostera capricorni* Chlorophyll 10h, 4d (rec) 10, 100 [74]

*Halophila ovalis* Chlorophyll 4d 10 [73]

*Ruppia maritima* Photosynthesis 2h 120 (IC50) [42] *Ruppia maritima* Growth 35d 2,500, 44,700 (EC50) [72]

*Halodule whrightii* Growth 22d 10e, 40e, 120e, 420i [75]

**Table 2.** Example of toxic effect concentrations reported for atrazine and seagrasses. EC50, IC50 –concentration reducing effect parameter 50% relative to control. rec: recovery; i: inhibition; ti: total inhibition; e: enhancement;

a lacks of light because of induced low photosynthetic yield.

Growth Mortality

Mortality Chlorophyll

Fluorescence Pigments

Fluorescence Pigments

Photosynthesis

and fluorescence are altered from 10μg/L [73, 74].

362 Herbicides - Current Research and Case Studies in Use

Seagrasses are mainly regarded as shadow plants [77]. Indeed, the situation is critical during summer low tides when photoinhibition is a threat. Photoinhibition is defined by Touchette & Burkholder [78] as a reduction in the photosynthetic rate due to other processes such as the toxicological impact of herbicides [79]. Photoinhibition is primarly a photoprotective tool, avoiding PSII excessive photophosphorylation, and dissipating energy as heat [80, 81]. Photoinhibition is obtained by PSII centres rarefaction [81] or inactivation because of D1 protein photolysis surpassing D1 synthesis [80-84] and the increase of xanthophylls cycle's violaxanthin de-epoxidation agent leading to energy dissipation: zeaxanthin. Protein D1 is known to be influenced by ATP in thylakoid lumen; in the case of a lack of ATP production, for example as herbicide impact occurs, seagrass could maintain a high photosynthetic level, initiating a vicious circle leading to the herbicide effect. Such an herbicide trajectory could lead first to photosuppression with excessive UV radiations [85].

The second critical target for seagrass, i.e. especially critical due to aquatic life, is a photores‐ piration process which is, like for several topics, innovative in the case of seagrasses [78]. Indeed, respiration provides to seagrasses oxidative conditions propitious to life in their partially reductive environment. In order to maintain a redox potential suitable for whole enzymatic activity even in tissues buried into the sediment, seagrasses will actively manage inner gas exchanges. A large part of tissue volume will be due to lacunae or *aerarium*, empty spaces allowing to preserve terrestrial-like conditions for cells, providing oxidative conditions and leaving the leafs erect. Respiration will produce CO2 for the plant –which will mainly consume its own CO2 production in order to limit exchanges with external marine environ‐ ments. Such C3-C4 intermediate plants present, moreover, concentrating carbon systems strengthening ribulose 1,5-bisphosphate carboxylase (Rubisco) in carbon acquisition. 2,4-D photosynthetic pathways are negatively impacted [86], even in micromolar concentrations, by auxin like 2,4-D, leading to up-regulating growth. Because of gas flows to the external environment are more strictly controlled by seagrasses' stomata, which are less numerous than terrestrial plants' ones, auxin-like activity of 2,4-D causes an up - regulation in oxygen production and a subsequent oxygen-inhibition of a key enzyme Rubisco. Lack of carbonsequestering photosynthetic processes leads to carbon and energy deficits aggravated by messy leaf creation induced by 2,4-D, exhausting the plant, drawing on belowground stocks, and limiting photosynthesis efficiency.

Photorespiration leads to consume O2 and is considered as protective for photosynthetic electron transport, limiting damage to the photosynthetic apparatus to photo-inactivation during periods of low CO2 availability and high light intensity [87]. Rates of photorespiration activity are considerably lower in most submersed aquatic plants than in terrestrial ones [78]; if O2 depletion is too great, anaerobic conditions rule: Krebs cycle's NAD+ reduction, leading to energy storage in mitochondria by NADH production driving ATP synthesis, is interrupted. NADH accumulates and NAD+ lacks for critical metabolic processes [88]. Parenthetically, pyruvate is metabolized, leading to fermentation (Davies, 1980) and alcohol content increases, altering whole tissues and thus removing the main obstacle to reductive conditions which are unfit for seagrass life.

–Sucrose-P synthase (SPS), in the opposite way of terrestrial plants: increasing temperature leads to the increase of SPS activity, which is also influenced by salinity, photosynthesis,

Herbicide Impact on Seagrass Communities http://dx.doi.org/10.5772/55973 365

–Stomata function: stomata will be closed in high temperatures, in order to avoid dehydration.

Without herbicide impact, 40-45°C is considered as the threshold temperature [22]: for higher ones, irreversible effects are observed, especially at PSII scale. The herbicide presence, even at limited concentrations, could lower such threshold temperature, considering the complex physiologic equilibrium that herbicides could disrupt; such temperature sensibility leads to an enhanced impact of herbicides in warm conditions [105]. Metal accumulation is enhanced by temperature increases [106]. Metals are toxic for seagrasses and especially for PSII [107]. Cu, used in this way as an antifouling alternative, early impacts PSII complex [108-111] in a few days after contamination. The cocktail effect is highlighted [74] for Cu and Irgarol 1051. Gamain [112] shows that herbicide impact is increased in presence of Cu and following temperature: at a temperature for which *Z. noltii* when free of herbicide alteration, even on a biochemical scale, seagrass presents damages in presence of this cocktail. However, these cocktail and summer temperatures are more close to field conditions, especially in tropical

Seagrass decline is actually misunderstood. If nutrients increase, it leads to epiphyte proliferation which limits seagrass photosynthesis [104]. Erosion, burying meadows and inducing turbidity limiting photosynthesis, are evoked as the main threat on the seagrass community, chemical interactions could be regarded as underestimated. Even if seagrass‐ es seem to be resilient to herbicide pollution, and even if seagrass recovery has been shown to be better *in situ* than *in vitro*, the cocktail impact seems to be a promising study field. Data concerning seagrass contamination are dramatically scarce despite the precious services that the seagrass community provides, as for economical activity as for environ‐ mental concerns like biodiversity preservation and carbon fixation. In the field, seagrass meadows are regressing, and their resilience seem to be altered. Impacts of herbicides on the minimal requirement and on the adaptation to high irradiances are not sufficiently studied leading to observe regression without understanding underlying phenomenology [45]. Seagrass originality involves more largely trans-disciplinarily in order preventing the meadows' decline –but if such a consistent *pièce de résistance* will need appropriate research efforts, the seagrass crisis, taken into account by environmental monitoring like Water

and grazing [93, 101, 102].

–C metabolism, i.e. C-sink or C-source depending on temperature [103],

Thus, temperature impacts seagrass growth independently to insulation [104].

CO2 availability, NH4

**7. Conclusion**

+

waters, than cold conditions and isolate herbicides.

Framework, allows, after all, hope for a remediation.

Unlike photosynthesis which increases with temperature up to 5–10°C above ambient, respiration rates continue to increase with increasing temperatures in excess of 40°C [78, 89-91]. Light, then depth [92] can also significantly influence respiration; water-column nitrate enrichment tissue NR activity enhances respiration rates in *Z. marina* [93].

## **6.** *In situ***: Chemical cocktail, interaction with metals and temperature increase**

If Lewis & Devereux [55] rightly indicated that seagrass are quite non-sensitive to herbicides, based on scientific literature showing the high herbicide concentration reached in order to observe seagrass alteration *in vitro* (Table 2), such results *ex situ* should be weighted by monitoring results, showing everywhere a variegated contamination in space, in time and, moreover, in impact. In the same way, limited impact of herbicides and organic chemicals is mentioned by Waycott et al. [94].

Seagrass meadow contamination by herbicides is well known, as from rivers, as from anti‐ fouling coatings [55, 95 and therein]. The impact of herbicides on seagrass is more scarcely noted [60, 96, 97], even on a limited scale (3% inhibition of photosynthetic biomaterial assay [98]). But seagrass vulnerability to short but intense contamination has been highlighted [99] and such events could be difficult to monitor. Moreover, short term contamination could be integrated by passive samplers, deployed for weeks, and weighted by the mean concentration in the aquatic environment: depending to monitoring protocol, fugacious pollution could be neglected. Then, seagrass could be resistant to long-term herbicide contamination with severe concentration [61] but vulnerable to toxic pulses [99].

Seagrass physiology is temperature dependant. Seagrass growth is enhanced by temperature increases; the optimal temperature for temperate species is between 11.5°C and 26°C when tropical ones' *preferenda* is between 23°C and 32°C (Lee et al., 2007). Temperature conditions:

–Respiration (see supra); temperature is the predominant factor for respiration control [66, 100],

–Rubisco oxygenase fonction (increased by increasing temperature [88]),

–Sucrose synthase (SS) activity, enhanced in belowground tissues with O2 decrease and temperature increase [78],

–Sucrose-P synthase (SPS), in the opposite way of terrestrial plants: increasing temperature leads to the increase of SPS activity, which is also influenced by salinity, photosynthesis, CO2 availability, NH4 + and grazing [93, 101, 102].

–C metabolism, i.e. C-sink or C-source depending on temperature [103],

–Stomata function: stomata will be closed in high temperatures, in order to avoid dehydration.

Thus, temperature impacts seagrass growth independently to insulation [104].

Without herbicide impact, 40-45°C is considered as the threshold temperature [22]: for higher ones, irreversible effects are observed, especially at PSII scale. The herbicide presence, even at limited concentrations, could lower such threshold temperature, considering the complex physiologic equilibrium that herbicides could disrupt; such temperature sensibility leads to an enhanced impact of herbicides in warm conditions [105]. Metal accumulation is enhanced by temperature increases [106]. Metals are toxic for seagrasses and especially for PSII [107]. Cu, used in this way as an antifouling alternative, early impacts PSII complex [108-111] in a few days after contamination. The cocktail effect is highlighted [74] for Cu and Irgarol 1051. Gamain [112] shows that herbicide impact is increased in presence of Cu and following temperature: at a temperature for which *Z. noltii* when free of herbicide alteration, even on a biochemical scale, seagrass presents damages in presence of this cocktail. However, these cocktail and summer temperatures are more close to field conditions, especially in tropical waters, than cold conditions and isolate herbicides.

### **7. Conclusion**

activity are considerably lower in most submersed aquatic plants than in terrestrial ones [78];

to energy storage in mitochondria by NADH production driving ATP synthesis, is interrupted.

pyruvate is metabolized, leading to fermentation (Davies, 1980) and alcohol content increases, altering whole tissues and thus removing the main obstacle to reductive conditions which are

Unlike photosynthesis which increases with temperature up to 5–10°C above ambient, respiration rates continue to increase with increasing temperatures in excess of 40°C [78, 89-91]. Light, then depth [92] can also significantly influence respiration; water-column nitrate

**6.** *In situ***: Chemical cocktail, interaction with metals and temperature**

If Lewis & Devereux [55] rightly indicated that seagrass are quite non-sensitive to herbicides, based on scientific literature showing the high herbicide concentration reached in order to observe seagrass alteration *in vitro* (Table 2), such results *ex situ* should be weighted by monitoring results, showing everywhere a variegated contamination in space, in time and, moreover, in impact. In the same way, limited impact of herbicides and organic chemicals is

Seagrass meadow contamination by herbicides is well known, as from rivers, as from anti‐ fouling coatings [55, 95 and therein]. The impact of herbicides on seagrass is more scarcely noted [60, 96, 97], even on a limited scale (3% inhibition of photosynthetic biomaterial assay [98]). But seagrass vulnerability to short but intense contamination has been highlighted [99] and such events could be difficult to monitor. Moreover, short term contamination could be integrated by passive samplers, deployed for weeks, and weighted by the mean concentration in the aquatic environment: depending to monitoring protocol, fugacious pollution could be neglected. Then, seagrass could be resistant to long-term herbicide contamination with severe

Seagrass physiology is temperature dependant. Seagrass growth is enhanced by temperature increases; the optimal temperature for temperate species is between 11.5°C and 26°C when tropical ones' *preferenda* is between 23°C and 32°C (Lee et al., 2007). Temperature conditions: –Respiration (see supra); temperature is the predominant factor for respiration control [66,

–Sucrose synthase (SS) activity, enhanced in belowground tissues with O2 decrease and

–Rubisco oxygenase fonction (increased by increasing temperature [88]),

lacks for critical metabolic processes [88]. Parenthetically,

reduction, leading

if O2 depletion is too great, anaerobic conditions rule: Krebs cycle's NAD+

enrichment tissue NR activity enhances respiration rates in *Z. marina* [93].

NADH accumulates and NAD+

364 Herbicides - Current Research and Case Studies in Use

mentioned by Waycott et al. [94].

concentration [61] but vulnerable to toxic pulses [99].

unfit for seagrass life.

**increase**

100],

temperature increase [78],

Seagrass decline is actually misunderstood. If nutrients increase, it leads to epiphyte proliferation which limits seagrass photosynthesis [104]. Erosion, burying meadows and inducing turbidity limiting photosynthesis, are evoked as the main threat on the seagrass community, chemical interactions could be regarded as underestimated. Even if seagrass‐ es seem to be resilient to herbicide pollution, and even if seagrass recovery has been shown to be better *in situ* than *in vitro*, the cocktail impact seems to be a promising study field. Data concerning seagrass contamination are dramatically scarce despite the precious services that the seagrass community provides, as for economical activity as for environ‐ mental concerns like biodiversity preservation and carbon fixation. In the field, seagrass meadows are regressing, and their resilience seem to be altered. Impacts of herbicides on the minimal requirement and on the adaptation to high irradiances are not sufficiently studied leading to observe regression without understanding underlying phenomenology [45]. Seagrass originality involves more largely trans-disciplinarily in order preventing the meadows' decline –but if such a consistent *pièce de résistance* will need appropriate research efforts, the seagrass crisis, taken into account by environmental monitoring like Water Framework, allows, after all, hope for a remediation.

### **Acknowledgements**

Authors wish to thank William and Diana R. Corby for their contributions to their English improvement.

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22, 843-853.

(1997). , 34-179.

NOAA, Silver Springs, MD, , 26-37.

gy and Ecology (2007). , 350-144.

synthesis Research (1985). , 7-31.

Plant Physiology (1984). , 74-717.

dissertation. Université de Bordeaux 1. 40 pp.

[114] Schwartzschild, A. C. MacIntyre W.G., Moore K.A., Libelo E.L. *Zostera marina* L. growth response to atrazine in rootrhizome and whole plant exposure experiments. Journal of Experimental Marine Biology and Ecology (1994). , 183-77.

**Chapter 15**

**Transgenic Herbicide-Resistant Turfgrasses**

Turfgrasses grow in different habitats for numerous purposes worldwide. They are cultivated for their agronomical, environmental, ornamental, recreational and stock feeding values [1, 2]. Various turfgrasses are used for environmental beautification and for the protection of resources such as land, soil and water. Many varieties of turfgrasses cover home yards, golf courses, parks, soccer fields, and roadsides, etc. To cite a few examples of renewed interest in turfgrasses, they play a significant environmental role in photosynthetically fixing carbon dioxide to evolve oxygen into the atmosphere. In addition to their vast acreage of widespread forage, planting of the grasses in urban areas such as rooftops, parks and, more recently automobile parking lots, contributes to the suppression of urban heat island phenomena [3]. Various causes of soil erosion and losses due to flood washout and landslide can also be circumvented and managed, as the damages are greatly reduced and the conservation of soil moisture and underground water is effectively sustained by the planting of turfgrass varieties. Recreational and sporting activities on the natural turfgrass field, compared to an artificial turf, greatly reduce the risk of personal injuries, thus contributing to the wellbeing of people in

Not surprisingly, the worldwide turfgrass market and its associated herbicide sales are substantial; in the United States alone, turfgrass is one of the four major staple crops, second only to corn [4, 5]. In facing the challenge of global warming, turfgrasses are gaining attention of both environmentalists and agronomists for their role in the certified emission reductions. Relatively high production costs of cultivating and maintaining turfgrasses concerns them, however. Healthy swarth growth and well-maintained turf habitats entail herbicide spraying because otherwise dominant weed varieties easily overtake the sward. Annually, their

> © 2013 Song et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Song et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

In-Ja Song, Tae-Woong Bae, Markkandan Ganesan,

Jeong-Il Kim, Hyo-Yeon Lee and Pill-Soon Song

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56096

**1. Introduction**

general.

## **Transgenic Herbicide-Resistant Turfgrasses**

In-Ja Song, Tae-Woong Bae, Markkandan Ganesan, Jeong-Il Kim, Hyo-Yeon Lee and Pill-Soon Song

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56096

### **1. Introduction**

Turfgrasses grow in different habitats for numerous purposes worldwide. They are cultivated for their agronomical, environmental, ornamental, recreational and stock feeding values [1, 2]. Various turfgrasses are used for environmental beautification and for the protection of resources such as land, soil and water. Many varieties of turfgrasses cover home yards, golf courses, parks, soccer fields, and roadsides, etc. To cite a few examples of renewed interest in turfgrasses, they play a significant environmental role in photosynthetically fixing carbon dioxide to evolve oxygen into the atmosphere. In addition to their vast acreage of widespread forage, planting of the grasses in urban areas such as rooftops, parks and, more recently automobile parking lots, contributes to the suppression of urban heat island phenomena [3]. Various causes of soil erosion and losses due to flood washout and landslide can also be circumvented and managed, as the damages are greatly reduced and the conservation of soil moisture and underground water is effectively sustained by the planting of turfgrass varieties. Recreational and sporting activities on the natural turfgrass field, compared to an artificial turf, greatly reduce the risk of personal injuries, thus contributing to the wellbeing of people in general.

Not surprisingly, the worldwide turfgrass market and its associated herbicide sales are substantial; in the United States alone, turfgrass is one of the four major staple crops, second only to corn [4, 5]. In facing the challenge of global warming, turfgrasses are gaining attention of both environmentalists and agronomists for their role in the certified emission reductions. Relatively high production costs of cultivating and maintaining turfgrasses concerns them, however. Healthy swarth growth and well-maintained turf habitats entail herbicide spraying because otherwise dominant weed varieties easily overtake the sward. Annually, their

© 2013 Song et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Song et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

maintenance costs alone run around 4.5 billion dollars in the United States [4, 6]. One of the major costs is certainly herbicidal requirement.

**Plant species Cultivar Method Marker gene Target gene Target trait References**

*EPSPS*

Penncross *Agrobacterium bar bar/ZjLsL* Herbicide resistance/

Penn-A-4 *Agrobacterium hph/gus, bar* bar Herbicide resistance [43] Penn-A-4 *Agrobacterium bar bar/Pen4-1* Herbicide resistance/

Regent Tiger *Agrobacterium bar/gfp bar* Herbicide resistance [47] Cobra Electroporation *bar bar* Herbicide resistance [48]

TifEagle Biolistics *bar bar* Herbicide resistance [50]

TifEagle *Agrobacterium bar/gus bar* Herbicide resistance [51]

Embryogen-P Biolistics *bar/gus bar* Herbicide resistance [52]

Rapido Biolistics *bar/hph/gus bar* Herbicide resistance [53]

Alley Biolistics *bar bar/Ipt* Herbicide resistance/

Riikka Biolistics *bar bar/wft1/wft2* Herbicide resistance/

TopGun *Agrobacterium bar bar/OsNHX1* Herbicide resistance/

Alamo Biolistics *bar/gfp bar* Herbicide resistance [59]

Penn-A-4 *Agrobacterium bar bar/AVP1* Herbicide resistance/

*VuNCED*1

*+glucanase*

Biolistics *bar/gus bar* Herbicide resistance [46]

Biolistics *bar bar/hs2* Herbicide resistance [49]

Protoplasts *bar/hph bar* Herbicide resistance [54]

Protoplasts *bar bar* Herbicide resistance [56]

Drought/salt tolerance

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096

*bar/CP4-EPSPS* Herbicide resistance [22]

dwarf

Herbicide resistance/ Disease resistance

Disease resistance

Salt tolerance

Cole tolerance

Freezing tolerance

Salt tolerance

[40]

379

[41]

[42]

[44]

[45]

[55]

[57]

[58]

Penncross *Agrobacterium bar bar/*Cowpea

Province Penn-A-4 Biolistics *bar/gus bar/chitinase*

Penncross *Agrobacterium bar/CP4-*

*Agrostis palustris* (creeping bentgrass)

*Cynodon* spp. (bermudagrass)

*Dactylis glomerata* (orchardgrass)

*Festuca arundinacea* (tall fescue)

*Festuca rubra* (red fescue)

*Lolium perenne* (perennial ryegrass)

*Panicum virgatum* (switchgrass) Suthshore Emerald

Herbicidal agrochemicals are classified into two categories, selective and non-selective herbicides. The latter kills all plant species, whereas the former is targeted at specific plant(s)/ weed(s) for herbicidal action. The biochemical mechanisms of herbicides include the disrup‐ tions of (i) the photosynthesis by blocking the photosynthetic reaction centers, electron transport system or photo-oxidative membrane damages, (ii) cell division and root develop‐ ment, (iii) energy transduction and metabolism, (iv) plant growth hormones, (v) biosynthesis of amino acids/proteins and (vi) disruption of other physiologically significant molecules such as chlorophylls and carotenoids, as discussed elsewhere in this volume.

Frequent herbicide applications also pose serious environmental and health concerns, for example, to the authors' residential island of Jeju where there are 30 golf courses open for business. In spite of the current difficulties arising from the public objections, genetically modified turfgrasses with a herbicide-resistant gene provide an effective alternative to the wide applications of agrochemical herbicides. Since the development and ecological impact studies of transgenic herbicide-resistant creeping bentgrass [7, 8] and zoysiagrass [9, 10], several GM varieties of turfgrasses including those of herbicide-resistant cultivars have been developed (see Table 1). Most recently, in reference [11] bentgrass ASR-368 has been patented for its commercial rights. With an increasing number of reports on transgenic herbicideresistant turfgrasses, it is appropriate to review the subject at this time. Discussion in this chapter focuses on the transgenic herbicide-resistant turfgrasses developed primarily in our laboratory here in Jeju and Gwangju, Korea. For a review of other transgenic grasses with herbicide-resistance traits, see Table 1 and references therein.



maintenance costs alone run around 4.5 billion dollars in the United States [4, 6]. One of the

Herbicidal agrochemicals are classified into two categories, selective and non-selective herbicides. The latter kills all plant species, whereas the former is targeted at specific plant(s)/ weed(s) for herbicidal action. The biochemical mechanisms of herbicides include the disrup‐ tions of (i) the photosynthesis by blocking the photosynthetic reaction centers, electron transport system or photo-oxidative membrane damages, (ii) cell division and root develop‐ ment, (iii) energy transduction and metabolism, (iv) plant growth hormones, (v) biosynthesis of amino acids/proteins and (vi) disruption of other physiologically significant molecules such

Frequent herbicide applications also pose serious environmental and health concerns, for example, to the authors' residential island of Jeju where there are 30 golf courses open for business. In spite of the current difficulties arising from the public objections, genetically modified turfgrasses with a herbicide-resistant gene provide an effective alternative to the wide applications of agrochemical herbicides. Since the development and ecological impact studies of transgenic herbicide-resistant creeping bentgrass [7, 8] and zoysiagrass [9, 10], several GM varieties of turfgrasses including those of herbicide-resistant cultivars have been developed (see Table 1). Most recently, in reference [11] bentgrass ASR-368 has been patented for its commercial rights. With an increasing number of reports on transgenic herbicideresistant turfgrasses, it is appropriate to review the subject at this time. Discussion in this chapter focuses on the transgenic herbicide-resistant turfgrasses developed primarily in our laboratory here in Jeju and Gwangju, Korea. For a review of other transgenic grasses with

**Plant species Cultivar Method Marker gene Target gene Target trait References**

Crenshaw *Agrobacterium bar bar/*Rice *tlpd34* Disease resistance [16]

Crenshaw *Agrobacterium bar bar/*Barley *hva1* Drought tolerance [33] Crenshaw *Agrobacterium bar/gus bar/PepEST* Herbicide resistance/

Crenshaw *Agrobacterium bar/gus bar*/Maize *Lc+Pl* Purple-color [35] Crenshaw *Agrobacterium bar/gus bar/AtBG1* Herbicide resistance/

Penncross Electroporation *bar bar* Herbicide resistance [38] Penncross Electroporation *bar/gus bar* Herbicide resistance [39]

*Agrobacterium bar/gus* bar Herbicide resistance [37]

Disease resistance

Drought tolerance/

dwarf

[34]

[36]

as chlorophylls and carotenoids, as discussed elsewhere in this volume.

herbicide-resistance traits, see Table 1 and references therein.

*Agrostis stolonifera* (creeping bentgrass)

> Crenshaw, Penncross

major costs is certainly herbicidal requirement.

378 Herbicides - Current Research and Case Studies in Use


**3. Transgenes and mechanisms of herbicidal action**

and will be reviewed in this chapter.

[19] (See Figure 1).

tase by the herbicide.

Turfgrass has been a subject of classical breeding for trait improvement over decades, espe‐ cially in Japan and United States. However, conventional breeding suffers from such draw‐ backs as low efficiency, time consuming and labor intensiveness. With an increasing trend in turfgrass cultivation worldwide, excessive applications of herbicides and other agrochemicals over the grass habitats adversely impact the environment, biodiversity and human health [13, 14]. Several attempts to develop GM turfgrass lines with improved traits have been reported; for example, herbicide-resistant turfgrass varieties in references [15], [16], 17] and [10] and insect-resistant turfgrass in reference [18]. A number of laboratories are developing herbicideresistant and other transgenic turfgrasses with biotic and abiotic stress tolerances (Table 1).

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096 381

So far, several genes including the two widely adopted ones, *CP4 EPSPS* encoding 5-enolpyr‐ uvylshikimate-3-phosphate synthase (EPSPs) and *BAR or PAT* encoding a phosphinothricin acetyl transferase (PAT), have been introduced to generate herbicide-resistant turfgrasses. Other target genes for herbicide resistance include *BXN* (bromoxylnil nitrilase gene), *DHPS* (dihydropteroate synthase gene), *ALS* (acetolactate synthase gene) and others (Table 1). Transgenic bentgrass and zoysiagrass stacked with *BAR* and *PHYA* (phytochrome A) genes conferring herbicide- and shade-resistance traits, respectively, have also been developed [10]

The widely used herbicide, bialaphos (also phosphinothricin-alanyl-alanine tripeptide, PTT), is an antibiotic produced by certain *Streptomyces* genera and used as an agrochemical, which has been commercialized under the trade name Basta by Bayer Crop Science. It kills plants non-selectively. Bialaphos itself is an inactive compound as a herbicide, but it is cleaved by intracellular peptidases to phosphinothricin (L-PPT), Phosphinothricin (glufosinate) so produced *in situ* binds glutamine synthetase (GS), the key enzyme in the nitrogen fixation in plants, inhibiting its catalytic activity to fix the ammonium with L-glutamate to form glutamine

**Figure 1.** Biochemical mechanism for the herbicidal action of glufosinate through the inhibition of glutamine synthe‐

bar: bialaphos resistance gene, gus: β-glucuronidase, hph: hygromycin phosphotransferase. gfp: green fluorescent protein

**Table 1.** Transgenic herbicide-resistant turfgrasses

### **2. Turfgrass species**

There are some 7,500 turfgrass species of more than 600 genera distributed worldwide. Of these, 30~40 species are cultivated as agronomic plants [1]. Turfgrasses are generally classified into two major species, warm and cold season grasses. The plants are also divided into two groups based on their mechanism of photosynthetic carbon dioxide fixation, C3 and C4 plants. As representative C4 warm season turfgrasses with optimal growth temperatures of 27~35°C, zoysiagrass and Bermuda grass species are widely used for sports fields because of their strong traits such as swarth growth, vegetative propagation and drought tolerance as they are cultivated widely, especially in China, Japan and Korea. However, they tend to grow relatively slowly and particularly with zoysiagrasses prematurely lose their greenness by late autumn. Typical C3 cold season turfgrasses with optimal temperatures in the 15~25°C range include blue grass and bentgrass varieties. The latter is particularly advantageous for the putting greens [1, 4, 5, 12]. In this chapter, the review will be concerned with two main varieties, zoysiagrass (*Zoysia japonica* Steud.) and bentgrass (*Agrostis palustris* L., Crenshaw and Penn‐ cross varieties), focusing on their herbicide resistant transgenic cultivars.

### **3. Transgenes and mechanisms of herbicidal action**

**Plant species Cultivar Method Marker gene Target gene Target trait References**

*Paspalum notatum* (bahiagrass)

*Paspalum vaginatum* Swartz (Seashore Paspalum)

*Zoysia japonica* (zoysiagrass)

*Zoysia sinica* (Chinese lawngrass)

protein

**Table 1.** Transgenic herbicide-resistant turfgrasses

380 Herbicides - Current Research and Case Studies in Use

**2. Turfgrass species**

Alamo *Agrobacterium bar/gus bar* Herbicide resistance [60]

Tifton-7 Biolistics *bar bar* Herbicide resistance [61]

Pensacola Biolistics *bar/gus bar* Herbicide resistance [62]

Zenith Biolistics *bar/hpt bar* Herbicide resistance [64]

bar: bialaphos resistance gene, gus: β-glucuronidase, hph: hygromycin phosphotransferase. gfp: green fluorescent

There are some 7,500 turfgrass species of more than 600 genera distributed worldwide. Of these, 30~40 species are cultivated as agronomic plants [1]. Turfgrasses are generally classified into two major species, warm and cold season grasses. The plants are also divided into two groups based on their mechanism of photosynthetic carbon dioxide fixation, C3 and C4 plants. As representative C4 warm season turfgrasses with optimal growth temperatures of 27~35°C, zoysiagrass and Bermuda grass species are widely used for sports fields because of their strong traits such as swarth growth, vegetative propagation and drought tolerance as they are cultivated widely, especially in China, Japan and Korea. However, they tend to grow relatively slowly and particularly with zoysiagrasses prematurely lose their greenness by late autumn. Typical C3 cold season turfgrasses with optimal temperatures in the 15~25°C range include blue grass and bentgrass varieties. The latter is particularly advantageous for the putting greens [1, 4, 5, 12]. In this chapter, the review will be concerned with two main varieties, zoysiagrass (*Zoysia japonica* Steud.) and bentgrass (*Agrostis palustris* L., Crenshaw and Penn‐

cross varieties), focusing on their herbicide resistant transgenic cultivars.

*Agrobacterium bar/gus bar* Herbicide resistance [63]

*Agrobacterium bar/gus bar* Herbicide resistance [15]

Shade tolerance

Chilling tolerance

[10]

[65]

*Agrobacterium bar bar/phyA* Herbicide resistance/

*Agrobacterium bar bar/CBF1* Herbicide resistance/

Turfgrass has been a subject of classical breeding for trait improvement over decades, espe‐ cially in Japan and United States. However, conventional breeding suffers from such draw‐ backs as low efficiency, time consuming and labor intensiveness. With an increasing trend in turfgrass cultivation worldwide, excessive applications of herbicides and other agrochemicals over the grass habitats adversely impact the environment, biodiversity and human health [13, 14]. Several attempts to develop GM turfgrass lines with improved traits have been reported; for example, herbicide-resistant turfgrass varieties in references [15], [16], 17] and [10] and insect-resistant turfgrass in reference [18]. A number of laboratories are developing herbicideresistant and other transgenic turfgrasses with biotic and abiotic stress tolerances (Table 1).

So far, several genes including the two widely adopted ones, *CP4 EPSPS* encoding 5-enolpyr‐ uvylshikimate-3-phosphate synthase (EPSPs) and *BAR or PAT* encoding a phosphinothricin acetyl transferase (PAT), have been introduced to generate herbicide-resistant turfgrasses. Other target genes for herbicide resistance include *BXN* (bromoxylnil nitrilase gene), *DHPS* (dihydropteroate synthase gene), *ALS* (acetolactate synthase gene) and others (Table 1). Transgenic bentgrass and zoysiagrass stacked with *BAR* and *PHYA* (phytochrome A) genes conferring herbicide- and shade-resistance traits, respectively, have also been developed [10] and will be reviewed in this chapter.

The widely used herbicide, bialaphos (also phosphinothricin-alanyl-alanine tripeptide, PTT), is an antibiotic produced by certain *Streptomyces* genera and used as an agrochemical, which has been commercialized under the trade name Basta by Bayer Crop Science. It kills plants non-selectively. Bialaphos itself is an inactive compound as a herbicide, but it is cleaved by intracellular peptidases to phosphinothricin (L-PPT), Phosphinothricin (glufosinate) so produced *in situ* binds glutamine synthetase (GS), the key enzyme in the nitrogen fixation in plants, inhibiting its catalytic activity to fix the ammonium with L-glutamate to form glutamine [19] (See Figure 1).

**Figure 1.** Biochemical mechanism for the herbicidal action of glufosinate through the inhibition of glutamine synthe‐ tase by the herbicide.

The glufosinate herbicide causes accumulation of lethal levels of ammonia in both soil bacteria and plant cells. The GS inhibiting activity of glufosinate is lost when its amino group is acetylated by a phosphinothricin acetyl transferase (PAT encoded by *PAT;* also known as *bar or BAR* for bialaphos resistance) (Figure. 2).

**Figure 2.** Detoxication of glufosinate by phosphinothricin acetyl transferase (*BAR* or *PAT*).

Thus, a transgenic turfgrass transformed with *BAR* gene becomes resistant to the Basta spray, as glufosinate from the Basta is effectively detoxicated in the plant. The transgenic zoysiagrass and bentgrass developed in our laboratories carry the *BAR* gene isolated from *Streptomyces hygroscopicus* in the soil [10].

tolerance to both Basta and Roundup, respectively. While such dual transgene herbicide resistance may counter for a single-transgene plant to lose tolerance to the herbicide and/or for the weeds to develop tolerance to the herbicide, it remains to be seen if this expectation is

**Figure 3.** The reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate synthase(EPSPS) (Modified from reference [32])

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096 383

One of the most promising herbicide-resistant traits can be conferred by dicamba monooxy‐ genase gene (*DMO*). Dicamba (3, 6-dichloro-2-methoxybenzoic acid) is an active auxin analog and its presence in the plant cells exaggerate the hormonal effects that lead to the cell and plant death. It is widely used in the Unites States for over four decades. It is a relatively non-toxic and environment-friendly herbicide. Its herbicidal activity is lost in a *DMO*-transgenic crop as dicamba is detoxified to its inactive 3, 6-DCSA (3, 6-dichlorosalicylic acid) [23]. Attempts are

In a previous report, we discussed the development of the *BAR*-transgenic *Zoysia japonica* Steud., currently undergoing a regulatory approval process under the cultivar name "Jeju Green 21" and compared its phenotypic traits with those of non-transgenic control [9]. Figure 4 (A, B) illustrates the effect of spraying Basta on the test plot containing both control and herbicide-resistant zoysiagrasses. In Figure 4(A), the herbicide-resistant runners were planted in the GMO-spelled area, which continued to grow healthily after Basta spray, showing "Jeju

being made to generate DMO-transgenic turfgrass plants in several laboratories.

**4. Herbicide-resistant zoysiagrass and bentgrass**

borne out in natural habitats.

Glyphosate is a non-selective herbicidal agent commercialized under the trade name "Round‐ up" by Monsanto. It exerts its herbicidal action by competitively inhibiting the 5-enolpyru‐ vylshikimate-3-phosphate synthase (EPSPs) centrally involved in the biosynthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine). Plants treated with glyphosate are killed for the lack of these amino acids in protein biosynthesis. Accumulation of shikimate also leads to cell death, thus contributing to the herbicidal action of glyphosate [20] (Figure. 3).

A transgenic bentgrass carrying the EPSPS gene ("Roundup Ready") then develops resistance to Roundup [7, 21].

Although both *BAR*- and *EPSPS*-.transgenic turfgrasses are yet to be released for agronomic cultivations, second and third generation GM crops including turfgrasses are forthcoming to deal with the intolerance and tolerance being developed to the non-specific herbicides in the transgenic herbicide-resistant turfgrasses and weed plants, respectively. Such next generation crops are also being developed with the hope of leading consumer acceptance. In reference [22] the authors stacked both *BAR* and *CP4 EPSPS* genes in creeping bentgrass to generate dual (glufosinate and glyphosate) herbicide-resistant turfgrasses, hoping that less amounts of two herbicides together are required for weed necrosis than with the greater amount needed with one herbicide alone. The bentgrass species so developed showed an expected degree of

The glufosinate herbicide causes accumulation of lethal levels of ammonia in both soil bacteria and plant cells. The GS inhibiting activity of glufosinate is lost when its amino group is acetylated by a phosphinothricin acetyl transferase (PAT encoded by *PAT;* also known as *bar*

**Figure 2.** Detoxication of glufosinate by phosphinothricin acetyl transferase (*BAR* or *PAT*).

Thus, a transgenic turfgrass transformed with *BAR* gene becomes resistant to the Basta spray, as glufosinate from the Basta is effectively detoxicated in the plant. The transgenic zoysiagrass and bentgrass developed in our laboratories carry the *BAR* gene isolated from *Streptomyces*

Glyphosate is a non-selective herbicidal agent commercialized under the trade name "Round‐ up" by Monsanto. It exerts its herbicidal action by competitively inhibiting the 5-enolpyru‐ vylshikimate-3-phosphate synthase (EPSPs) centrally involved in the biosynthesis of aromatic amino acids (phenylalanine, tryptophan and tyrosine). Plants treated with glyphosate are killed for the lack of these amino acids in protein biosynthesis. Accumulation of shikimate also leads to cell death, thus contributing to the herbicidal action of glyphosate [20] (Figure. 3).

A transgenic bentgrass carrying the EPSPS gene ("Roundup Ready") then develops resistance

Although both *BAR*- and *EPSPS*-.transgenic turfgrasses are yet to be released for agronomic cultivations, second and third generation GM crops including turfgrasses are forthcoming to deal with the intolerance and tolerance being developed to the non-specific herbicides in the transgenic herbicide-resistant turfgrasses and weed plants, respectively. Such next generation crops are also being developed with the hope of leading consumer acceptance. In reference [22] the authors stacked both *BAR* and *CP4 EPSPS* genes in creeping bentgrass to generate dual (glufosinate and glyphosate) herbicide-resistant turfgrasses, hoping that less amounts of two herbicides together are required for weed necrosis than with the greater amount needed with one herbicide alone. The bentgrass species so developed showed an expected degree of

*or BAR* for bialaphos resistance) (Figure. 2).

382 Herbicides - Current Research and Case Studies in Use

*hygroscopicus* in the soil [10].

to Roundup [7, 21].

**Figure 3.** The reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate synthase(EPSPS) (Modified from reference [32])

tolerance to both Basta and Roundup, respectively. While such dual transgene herbicide resistance may counter for a single-transgene plant to lose tolerance to the herbicide and/or for the weeds to develop tolerance to the herbicide, it remains to be seen if this expectation is borne out in natural habitats.

One of the most promising herbicide-resistant traits can be conferred by dicamba monooxy‐ genase gene (*DMO*). Dicamba (3, 6-dichloro-2-methoxybenzoic acid) is an active auxin analog and its presence in the plant cells exaggerate the hormonal effects that lead to the cell and plant death. It is widely used in the Unites States for over four decades. It is a relatively non-toxic and environment-friendly herbicide. Its herbicidal activity is lost in a *DMO*-transgenic crop as dicamba is detoxified to its inactive 3, 6-DCSA (3, 6-dichlorosalicylic acid) [23]. Attempts are being made to generate DMO-transgenic turfgrass plants in several laboratories.

### **4. Herbicide-resistant zoysiagrass and bentgrass**

In a previous report, we discussed the development of the *BAR*-transgenic *Zoysia japonica* Steud., currently undergoing a regulatory approval process under the cultivar name "Jeju Green 21" and compared its phenotypic traits with those of non-transgenic control [9]. Figure 4 (A, B) illustrates the effect of spraying Basta on the test plot containing both control and herbicide-resistant zoysiagrasses. In Figure 4(A), the herbicide-resistant runners were planted in the GMO-spelled area, which continued to grow healthily after Basta spray, showing "Jeju Green 21" plants growing in "GMO" spell pattern before and after the herbicide treatment at a concentration of 0.1% (w/v) glufosinate. Figure 4(B) shows the mixed turfgrass/weed habitat treated with a 0.5% Basta spray, showing an effective herbicidal killing of the weeds. Nontransgenic grasses are effectively wilted out, whereas the resistant plants remain healthy and indistinguishable from their non-transgenic counterparts physiologically and phenotypically [9]. Figure 5 displays the herbicidal performance of *BAR-*transgenic creeping bentgrass in which a wild type or mutant *PHYA* (*Ser599Ala PHYA*) gene is stacked with the *BAR* gene, *vide infra*. The results show that the gene stacking has not compromised the herbicide-resistance function conferred by the *BAR* gene. Qualitatively, both *BAR*- and *EPSPS-*transgenic bent‐ grasses effectively tolerate the herbicides, Basta and Roundup, respectively, but quantitative comparisons of the herbicide resistances exhibited by different transgenic zoysiagrass and bentgrass varieties entail further study.

**Figure 5.** Herbicide resistance assay of putative transgenic creeping bentgrass plants. 0.8% BASTA® was sprayed onto non-transgenic plants (NT) and transgenic plants over-expressing *Wt-PHYA* or *Ser599Ala-PHYA*, and the herbicide re‐ sistance of the plants was determined 10 days after the spraying. *Wt-PHYA*, transgenic bentgrass plants with wild-type

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096 385

When zoysiagrass and possibly other turfgrass species are left unmanaged under natural habitats, their populations and swarth growth are easily overtaken by the dominant weed plants. Figure 6 shows our own observations of herbicide-resistant zoysiagrass plants growing in natural habitats during the four consecutive years (2006~2009). In four years, the ground coverage of zoysiagrass was dominated by the weeds when the grass plot was left unmanaged. On the other hand, the herbicide-resistant plants continued healthy population and swarth growths under managed conditions involving fertilizer applications, herbicide sprays and

Recently, we reported the development and morphological characterization of transgenic *Zoysia japonica* and *Agrostis stolonifera* plants transformed with both *BAR* and *PHYA* genes [1]. The two transgenes confer herbicide resistance and shade tolerance to the grass, respectively. We developed these turfgrass plants by harboring wild-type *Avena PHYA* or *Ser599Ala PHYA* mutant (*S599A-phytochrome A hyperactive mutant* gene [24]) on the *BAR*-decked *pCAM‐ BIA3301* vector in order to confer both herbicide and shade tolerant phenotypes to them. The transgenic plants with *Ser599Ala-PHYA* and *Wt-PHYA* also displayed the shorter phenotypes

*PHYA* gene; *Ser599Ala-PHYA*, transgenic bentgrass plants with *Ser599Ala-PHYA* mutant.

desired, in addition to their herbicide resistance trait (Figure 7).

timely mowings.

**Figure 4.** Herbicide resistance assay of putative transgenic zoysiagrass plants. A. 0.8% BASTA® was sprayed onto nontransgenic plants (NT) and bialaphos-resistant zoysiagrass, "GMO" was spelled by removing the plants; GM grass was then planted into the letters, B. 0.5% BASTA® was sprayed onto the weed and bialaphos-resistance zoysiagrass plants.

Green 21" plants growing in "GMO" spell pattern before and after the herbicide treatment at a concentration of 0.1% (w/v) glufosinate. Figure 4(B) shows the mixed turfgrass/weed habitat treated with a 0.5% Basta spray, showing an effective herbicidal killing of the weeds. Nontransgenic grasses are effectively wilted out, whereas the resistant plants remain healthy and indistinguishable from their non-transgenic counterparts physiologically and phenotypically [9]. Figure 5 displays the herbicidal performance of *BAR-*transgenic creeping bentgrass in which a wild type or mutant *PHYA* (*Ser599Ala PHYA*) gene is stacked with the *BAR* gene, *vide infra*. The results show that the gene stacking has not compromised the herbicide-resistance function conferred by the *BAR* gene. Qualitatively, both *BAR*- and *EPSPS-*transgenic bent‐ grasses effectively tolerate the herbicides, Basta and Roundup, respectively, but quantitative comparisons of the herbicide resistances exhibited by different transgenic zoysiagrass and

**Figure 4.** Herbicide resistance assay of putative transgenic zoysiagrass plants. A. 0.8% BASTA® was sprayed onto nontransgenic plants (NT) and bialaphos-resistant zoysiagrass, "GMO" was spelled by removing the plants; GM grass was

was sprayed onto the weed and bialaphos-resistance zoysiagrass plants.

bentgrass varieties entail further study.

384 Herbicides - Current Research and Case Studies in Use

then planted into the letters, B. 0.5% BASTA®

**Figure 5.** Herbicide resistance assay of putative transgenic creeping bentgrass plants. 0.8% BASTA® was sprayed onto non-transgenic plants (NT) and transgenic plants over-expressing *Wt-PHYA* or *Ser599Ala-PHYA*, and the herbicide re‐ sistance of the plants was determined 10 days after the spraying. *Wt-PHYA*, transgenic bentgrass plants with wild-type *PHYA* gene; *Ser599Ala-PHYA*, transgenic bentgrass plants with *Ser599Ala-PHYA* mutant.

When zoysiagrass and possibly other turfgrass species are left unmanaged under natural habitats, their populations and swarth growth are easily overtaken by the dominant weed plants. Figure 6 shows our own observations of herbicide-resistant zoysiagrass plants growing in natural habitats during the four consecutive years (2006~2009). In four years, the ground coverage of zoysiagrass was dominated by the weeds when the grass plot was left unmanaged. On the other hand, the herbicide-resistant plants continued healthy population and swarth growths under managed conditions involving fertilizer applications, herbicide sprays and timely mowings.

Recently, we reported the development and morphological characterization of transgenic *Zoysia japonica* and *Agrostis stolonifera* plants transformed with both *BAR* and *PHYA* genes [1]. The two transgenes confer herbicide resistance and shade tolerance to the grass, respectively. We developed these turfgrass plants by harboring wild-type *Avena PHYA* or *Ser599Ala PHYA* mutant (*S599A-phytochrome A hyperactive mutant* gene [24]) on the *BAR*-decked *pCAM‐ BIA3301* vector in order to confer both herbicide and shade tolerant phenotypes to them. The transgenic plants with *Ser599Ala-PHYA* and *Wt-PHYA* also displayed the shorter phenotypes desired, in addition to their herbicide resistance trait (Figure 7).

We observed a delay in necrosis (senescence) of *Ser599Ala-PHYA* leaves under outdoor conditions in early winter (Figure 8). During the rejuvenation of zoysiagrass after the winter season, various weeds began to dominate over the transgenic turfgrass habitats. However, zoysiagrass plants expressing both *BAR* and *Ser599Ala-PHYA* genes exhibited a significant increase in tiller number and runner length relative to the non-transgenic controls [10]. These traits will be helpful for the zoysiagrass plants to compete effectively with the weeds, especially

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096 387

**Figure 8.** Photographic view of browning (necrosis) in zoysiagrass transformant lines in early winter. NT, non-trans‐ genic zoysiagrass plants; HR, herbicide-resistant zoysiagrass plants with *BAR* gene; *Wt-PHYA*, transgenic zoysiagrass plants with wild-type *PHYA* gene; *Ser599Ala-PHYA* 2-14 & 2-18 transformant lines, transgenic zoysiagrass plants with

To commercialize any of the transgenic turfgrass varieties listed in Table 1, their environ‐ mental risks must be assessed under their natural habitats [7, 8, 9, 25]. This chapter briefly reviews our own studies and discusses attempts to block or minimize the risks of gene flow from the transgenic turfgrass habitats to the plants at neighboring and remote sites. For example, in reference [26] and [27] the workers introduced a male-sterility gene into GM crops to block the escape of a transgene from the latter, and this strategy may be applied to turfgrasses. We developed a sterile herbicide-resistant zoysiagrass through γ-radiation mutation, making the latter unbolting and deficient in fertile pollens [28, 29]. The γradiation generated herbicide-resistant zoysiagrass can be cultivated in agronomic habi‐

A preliminary study showed that the transgene (*BAR*) of herbicide-resistant *Zoysia japonica* unintentionally escaped from the test plants to the close neighbored non-transgenic zoysia‐ grass species [9]. However, the introgression is likely to be suppressed under natural condi‐ tions (see Figure. 6) and can be easily terminated by applying non-specific herbicides such as

According to the "Weed risk assessments for Hawaii and Pacific Islands" database (http:// www.botany.hawaii.edu/faculty/daehler/wra/default.htm), transgenic *Zoysia japonica and Zoysia tenuifolia* are classified as being L grade, i.e. not currently recognized as invasive in

in disrupting the germination of unwanted weeds.

*Ser599Ala-PHYA* mutant gene.

**5. Environmental risk assessment**

tats for eventual commercialization [25].

glyphosate and paraquat [25].

**Figure 6.** Survival of the transgenic herbicide-resistant zoysiagrass during 4 years (2006-2009) in natural habitats. A. Natural habitats during 4 years, B. Managed field, C. Plant height of zoysiagrass, D. Grass coverage of zoysiagrass, E. Grass density of zoysiagrass. Blue bar, natural habitat; red bar, managed field.

**Figure 7.** Growth performance of transgenic zoysiagrass plants over-expressing *Ser599Ala-PHYA* showed short phe‐ notypes compared with control plants *(BAR* gene) under field conditions. Bar in insert 1 cm.

We observed a delay in necrosis (senescence) of *Ser599Ala-PHYA* leaves under outdoor conditions in early winter (Figure 8). During the rejuvenation of zoysiagrass after the winter season, various weeds began to dominate over the transgenic turfgrass habitats. However, zoysiagrass plants expressing both *BAR* and *Ser599Ala-PHYA* genes exhibited a significant increase in tiller number and runner length relative to the non-transgenic controls [10]. These traits will be helpful for the zoysiagrass plants to compete effectively with the weeds, especially in disrupting the germination of unwanted weeds.

**Figure 8.** Photographic view of browning (necrosis) in zoysiagrass transformant lines in early winter. NT, non-trans‐ genic zoysiagrass plants; HR, herbicide-resistant zoysiagrass plants with *BAR* gene; *Wt-PHYA*, transgenic zoysiagrass plants with wild-type *PHYA* gene; *Ser599Ala-PHYA* 2-14 & 2-18 transformant lines, transgenic zoysiagrass plants with *Ser599Ala-PHYA* mutant gene.

### **5. Environmental risk assessment**

**Figure 7.** Growth performance of transgenic zoysiagrass plants over-expressing *Ser599Ala-PHYA* showed short phe‐

**Figure 6.** Survival of the transgenic herbicide-resistant zoysiagrass during 4 years (2006-2009) in natural habitats. A. Natural habitats during 4 years, B. Managed field, C. Plant height of zoysiagrass, D. Grass coverage of zoysiagrass, E.

Grass density of zoysiagrass. Blue bar, natural habitat; red bar, managed field.

386 Herbicides - Current Research and Case Studies in Use

notypes compared with control plants *(BAR* gene) under field conditions. Bar in insert 1 cm.

To commercialize any of the transgenic turfgrass varieties listed in Table 1, their environ‐ mental risks must be assessed under their natural habitats [7, 8, 9, 25]. This chapter briefly reviews our own studies and discusses attempts to block or minimize the risks of gene flow from the transgenic turfgrass habitats to the plants at neighboring and remote sites. For example, in reference [26] and [27] the workers introduced a male-sterility gene into GM crops to block the escape of a transgene from the latter, and this strategy may be applied to turfgrasses. We developed a sterile herbicide-resistant zoysiagrass through γ-radiation mutation, making the latter unbolting and deficient in fertile pollens [28, 29]. The γradiation generated herbicide-resistant zoysiagrass can be cultivated in agronomic habi‐ tats for eventual commercialization [25].

A preliminary study showed that the transgene (*BAR*) of herbicide-resistant *Zoysia japonica* unintentionally escaped from the test plants to the close neighbored non-transgenic zoysia‐ grass species [9]. However, the introgression is likely to be suppressed under natural condi‐ tions (see Figure. 6) and can be easily terminated by applying non-specific herbicides such as glyphosate and paraquat [25].

According to the "Weed risk assessments for Hawaii and Pacific Islands" database (http:// www.botany.hawaii.edu/faculty/daehler/wra/default.htm), transgenic *Zoysia japonica and Zoysia tenuifolia* are classified as being L grade, i.e. not currently recognized as invasive in Hawaii, and not likely to have major ecological or economic impacts on other Pacific Islands based on the HP-WRA screening process. On the other hand, bentgrass (*Agrostis stolonifera*) belongs to an H grade group of plants, suggesting that transgenic herbicide-resistant bentgrass is a higher risk turfgrass than the zoysiagrass; according to the Hawaii database, *Agrostis stolonifera* is likely to be invasive in Hawaii and on other Pacific Islands as determined by the HP-WRA screening process. In fact, the transgene of the Roundup Ready creeping bentgrass introgressed other recipient plant species 3.8 km away from the test plot [8]. In conclusion, the herbicide-resistant zoysiagrass developed in our laboratory poses substantially less risk of transgene flow than the bentgrass (Figure. 5).

Figure 9 shows the sites in Jeju Island monitored for the potential gene flow from the herbicideresistant *Zoysia japonica* to wild-type zoysiagrass within a 5-km radius in natural habitat. No

Transgenic Herbicide-Resistant Turfgrasses http://dx.doi.org/10.5772/56096 389

Turfgrass is a highly value-added crop in terms of commercial profits per land acreage, when compared to other crops. Turfgrasses sward vigorously through vegetative propagation and swarth growth. According to TPI data (Turfgrass Producers International), the turfgrass market size increased by 35% during the five year (2002-2007) period [31]. Based on the data available, transgenic zoysiagrasses pose considerably less risk of transgene escape than does bentgrass. Furthermore, the former can be effectively propagated vegetatively, and sterile herbicide-resistant zoysiagrass (and bentgrass) can be developed through γ-radiation treat‐ ment [30]. This will circumvent to a large extent the public's objections to genetically modified

We compiled a table of transgenic herbicide-resistant turfgrass varieties in various stages of development and eventual agronomic cultivations. As can be seen in Table 1 of this chapter, several transgenes have been introduced into zoysiagrass, bentgrass and other lawn grass species primarily through Agrobacterium-mediated transformation and biolistic transfection. These grasses all exhibit resistance to their intended herbicides such as Basta, Roundup and others, but how well each of the transgenics developed performs in test plots and natural habitats cannot be assessed at this point largely because quantitative data such as the doseresponse curves and the outdoor performances are lacking in most cases. In this chapter, we focused our discussion to the *BAR* transgenic *Zoysia japonica* and *Agrostis stolonifera* species. We conclude that these cultivars offer promising potentials as environmentally friendly and economically beneficial turfgrass varieties, especially the former, for Jeju Island and elsewhere.

This research was supported by Next-Generation Biogreen 21 Program, Rural Development Administration, Republic of Korea (Grant No. PJ00949901), Basic Science Research Program (NRF Grant No. 2012R1A1A2000706 to PSS, 2012-0004335) and the Priority Research Centers Program (2012048080) through the National Research Foundation of Korea (NRF) funded by

introgression was observed at these sites as of this writing.

**6. Commercial potentials and outlook**

plants and their unintended escapes.

**7. Conclusion**

**Acknowledgements**

the Ministry of Education, Science and Technology.

Although the risk of transgene escape and flow from the genetically modified zoysiagrass is low, pollen-induced gene flow cannot be completely discounted. In reference [30] we examined the pollen releases from the defined boundary of *BAR* –transgenic *Zoysia japonica* habitats as a function of physical variables including the boundary, temperature, atmospheric humidity, and lighting condition/duration. Results suggest that zoysiagrass' pollen escape is essentially limited to the close neighborhood, in contrast to bentgrass pollens.

**Figure 9.** Monitoring for the potential gene flow from the genetically modified zoysiagrass to wild-type zoysiagrass plants within a 5-km radius in natural habitat. Samples were taken from 112 zones (448 sites): *Zoysia japonica* 96 zones (384 sites) and *Zoysia matrella* 16 zones (64 sites).

Figure 9 shows the sites in Jeju Island monitored for the potential gene flow from the herbicideresistant *Zoysia japonica* to wild-type zoysiagrass within a 5-km radius in natural habitat. No introgression was observed at these sites as of this writing.

### **6. Commercial potentials and outlook**

Turfgrass is a highly value-added crop in terms of commercial profits per land acreage, when compared to other crops. Turfgrasses sward vigorously through vegetative propagation and swarth growth. According to TPI data (Turfgrass Producers International), the turfgrass market size increased by 35% during the five year (2002-2007) period [31]. Based on the data available, transgenic zoysiagrasses pose considerably less risk of transgene escape than does bentgrass. Furthermore, the former can be effectively propagated vegetatively, and sterile herbicide-resistant zoysiagrass (and bentgrass) can be developed through γ-radiation treat‐ ment [30]. This will circumvent to a large extent the public's objections to genetically modified plants and their unintended escapes.

### **7. Conclusion**

Hawaii, and not likely to have major ecological or economic impacts on other Pacific Islands based on the HP-WRA screening process. On the other hand, bentgrass (*Agrostis stolonifera*) belongs to an H grade group of plants, suggesting that transgenic herbicide-resistant bentgrass is a higher risk turfgrass than the zoysiagrass; according to the Hawaii database, *Agrostis stolonifera* is likely to be invasive in Hawaii and on other Pacific Islands as determined by the HP-WRA screening process. In fact, the transgene of the Roundup Ready creeping bentgrass introgressed other recipient plant species 3.8 km away from the test plot [8]. In conclusion, the herbicide-resistant zoysiagrass developed in our laboratory poses substantially less risk of

Although the risk of transgene escape and flow from the genetically modified zoysiagrass is low, pollen-induced gene flow cannot be completely discounted. In reference [30] we examined the pollen releases from the defined boundary of *BAR* –transgenic *Zoysia japonica* habitats as a function of physical variables including the boundary, temperature, atmospheric humidity, and lighting condition/duration. Results suggest that zoysiagrass' pollen escape is essentially

**Figure 9.** Monitoring for the potential gene flow from the genetically modified zoysiagrass to wild-type zoysiagrass plants within a 5-km radius in natural habitat. Samples were taken from 112 zones (448 sites): *Zoysia japonica* 96

transgene flow than the bentgrass (Figure. 5).

388 Herbicides - Current Research and Case Studies in Use

zones (384 sites) and *Zoysia matrella* 16 zones (64 sites).

limited to the close neighborhood, in contrast to bentgrass pollens.

We compiled a table of transgenic herbicide-resistant turfgrass varieties in various stages of development and eventual agronomic cultivations. As can be seen in Table 1 of this chapter, several transgenes have been introduced into zoysiagrass, bentgrass and other lawn grass species primarily through Agrobacterium-mediated transformation and biolistic transfection. These grasses all exhibit resistance to their intended herbicides such as Basta, Roundup and others, but how well each of the transgenics developed performs in test plots and natural habitats cannot be assessed at this point largely because quantitative data such as the doseresponse curves and the outdoor performances are lacking in most cases. In this chapter, we focused our discussion to the *BAR* transgenic *Zoysia japonica* and *Agrostis stolonifera* species. We conclude that these cultivars offer promising potentials as environmentally friendly and economically beneficial turfgrass varieties, especially the former, for Jeju Island and elsewhere.

### **Acknowledgements**

This research was supported by Next-Generation Biogreen 21 Program, Rural Development Administration, Republic of Korea (Grant No. PJ00949901), Basic Science Research Program (NRF Grant No. 2012R1A1A2000706 to PSS, 2012-0004335) and the Priority Research Centers Program (2012048080) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.

### **Author details**

In-Ja Song1 , Tae-Woong Bae1 , Markkandan Ganesan1 , Jeong-Il Kim2 , Hyo-Yeon Lee1\* and Pill-Soon Song1\*

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[35] Han Y.J., Kim Y.M., Lee J.Y, Kim S.J., Cho K.C., Chandrasekhar T., Song P.S., Woo Y.M., Kim J.I. Production of purple-colored creeping bentgrass using maize tran‐

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[59] Richards H.A., Rudas V.A., Sun H., McDaiel J.K., Tomaszewski Z., Conger B.V. Con‐ struction of a GFP-BAR plasmid and its use for switchgrass transformation. Plant

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[62] Gondo T., Tsurta S.I., Akashi R., Kawamura O., Hoffmann F. Green, herbicide-resist‐ ant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (*Pas‐*

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[64] Lim S.H., Kang B.C., Shin H.K.. Herbicide Resistant Turfgrass (*Zoysia japonica* cv. 'Ze‐ nith') Plants by Particle bombardment-mediated Transformation. 2004; Korean jour‐

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[54] Wang Z.Y., Takamizo T., Iglesias V.A., Osusky M., Nagel J., Potrykus I., Spangenberg G. Transgenic plants of tall fescue (*Festuca arundinacea* Schreb.) obtained by direct

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401-406.

394 Herbicides - Current Research and Case Studies in Use


**Section 3**

**Research Reviews**

**Section 3**

**Research Reviews**

**Chapter 16**

**Toxicity of Herbicides:**

Maria Aparecida Marin-Morales,

Márcia Miyuki Hoshina

http://dx.doi.org/10.5772/55851

**1. Introduction**

Bruna de Campos Ventura-Camargo and

Additional information is available at the end of the chapter

water, soil, food and organisms in general [10].

**Impact on Aquatic and Soil Biota and Human Health**

During the last decades, the scientific community, including government and non-government organizations have increased their interest in detecting and controlling the environmental agents responsible for damages to the human health and sustainability of the ecosystems. This interest has been intensified by the frightening increase on the reports of the anthropogenic action on the environment responsible for damages to the ozone layer, accidental release of wastes and radioactive gases, as well as contamination by pesticides used in agriculture. However, the growth of the human population and of the activities associated with agriculture, industrialization and urbanization have contributed to the depredation of the biodiversity and

genetic variability, resulting in the compromise of several species, including man [9].

After the industrial revolution, a great number of chemical substances have been released into the terrestrial and aquatic environments and in the atmosphere. These substances can be transported and transformed by different processes, whose transformation by-products can cause adverse effects on man, as well as damages to the terrestrial and aquatic ecosystems. Several studies have shown the presence of residues of several chemical substances in the air,

Environmental pollution by genotoxic and mutagenic products affects the exposed organism and its future generations, this fact is observed both for animals, and in this case man is included, and for the other groups of organisms such as plants and microorganisms. In order to evaluate the consequences of the anthropogenic activities on the ecosystem it is necessary that the scientific community pays a special attention in the search for understanding the

> © 2013 Marin-Morales et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Marin-Morales et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health**

Maria Aparecida Marin-Morales, Bruna de Campos Ventura-Camargo and Márcia Miyuki Hoshina

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55851

### **1. Introduction**

During the last decades, the scientific community, including government and non-government organizations have increased their interest in detecting and controlling the environmental agents responsible for damages to the human health and sustainability of the ecosystems. This interest has been intensified by the frightening increase on the reports of the anthropogenic action on the environment responsible for damages to the ozone layer, accidental release of wastes and radioactive gases, as well as contamination by pesticides used in agriculture. However, the growth of the human population and of the activities associated with agriculture, industrialization and urbanization have contributed to the depredation of the biodiversity and genetic variability, resulting in the compromise of several species, including man [9].

After the industrial revolution, a great number of chemical substances have been released into the terrestrial and aquatic environments and in the atmosphere. These substances can be transported and transformed by different processes, whose transformation by-products can cause adverse effects on man, as well as damages to the terrestrial and aquatic ecosystems. Several studies have shown the presence of residues of several chemical substances in the air, water, soil, food and organisms in general [10].

Environmental pollution by genotoxic and mutagenic products affects the exposed organism and its future generations, this fact is observed both for animals, and in this case man is included, and for the other groups of organisms such as plants and microorganisms. In order to evaluate the consequences of the anthropogenic activities on the ecosystem it is necessary that the scientific community pays a special attention in the search for understanding the

© 2013 Marin-Morales et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Marin-Morales et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

modes of action of xenobiotics present in the ecosystem in the biota exposed. For this, extensive, detailed and ordered studies of the contaminants must be developed with the purpose of preventing the biological impairment, such as inductions of alterations in the genetic materials of the organisms [11].

since many are harmful substances for man and the environment. The world practice of using agrochemicals for long periods, often indiscriminate and abusive, has raised concerns among the public authorities and experts of public health and sustainability of natural resources [16].

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

401

Many agrochemicals are very toxic substances whose absorption in man are almost exclusively oral and can also occur by inhalation or dermally. As a consequence of the human exposure to pesticides, a series of disturbances can be observed, such as gastric, neurological and

Among the pesticides, the main agents of intoxication are the herbicides and insecticides. According to Vasilescu and Medvedovici [19], herbicides are defined as any substance, individually or in mixtures, whose function is to control, destroy, repel or mitigate the growth

The use of herbicides, despite the fact that they are characterized as a highly effective tool in the control of weeds, has led to a change in the phytosociological composition of weeds and to a selection of biotypes resistant to herbicides, besides also causing impacts in the environment and human health. According to He et al. [20], herbicides are the most used chemical substances throughout the world. During the 90's, the global pesticide sales remained relatively constant, between 270 and 300 billions of US dollars, and 47% of this value corresponded to herbicides and 79% to insecticides. Since 2007, herbicides assumed the first place among the three major categories of pesticides (insecticides, fungicides/

The use of herbicides to control weeds has been a common practice in global agriculture, mainly with the objective to increase agricultural production. However, when these chemicals are used in an uncontrolled manner, they can cause impacts on non-target organisms,

According to Chevreuil et al. [23], Kim and Feagley [24] and Abdel-Ramham et al. [25], most of the toxic effects of the herbicides on animals and plants were insufficiently investigated. As a consequence of the lack of information about the action of herbicides in the biological environment, these chemical agents can also represent a problem to human health [26, 27]. The impact of a pesticide in the environment depends on its dispersion mode and its concentration, as well as its own toxicity [28]. The mutagenic effects of the herbicides can result from several reactions with the organism, as a direct action of the compound on the nuclear DNA; incor‐ poration in the DNA during cell replication; interference in the activity of the mitotic or meiotic

Some herbicides interfere directly in the cell division of plants, elongation and/or cell differ‐ entiation, causing disturbances in the functioning of the roots or vascular tissues [30]. In animals, herbicides can act in several tissues or organs and, sometimes, are associated with

Jurado et al. [32] listed the general advantages and disadvantages of using herbicides. In this list, the authors cited as advantages: kill unwanted plants; help crops grow since it eliminates weeds that compete with crops for water, nutrients and sunlight; can be safely used in

muscular [18].

of weeds in a crop.

bactericides, herbicides) [21].

tumorigenic processes [31].

especially on those that live in aquatic environments [22].

division, resulting in incorrect division of the cell [29].

Some studies have been performed in the attempt to evaluate the behaviour, transforma‐ tions and effects of chemical agents, both in the environment and in the organisms. Toxicology establishes the limits of concentration or quantity of chemical substances acceptable in the environment by studies on the toxic effects of these substances in the organism and ecosystems [12].

Considering that the use of agrochemicals, such as herbicides, have caused a great environ‐ mental contamination, due to their widespread use, it has become indispensable to perform the assessment of the toxicity of these compounds.

### **1.1. The importance of herbicides**

Living beings are exposed to the action of numerous agents that are potentially toxic. These agents can be physical, chemical or biological and can provoke in the organisms physiological, biochemical, pathological effects and, in some cases, genetic effects [13]. A great variety of chemical substances with mutagenic potential, both natural and synthetic, have been investi‐ gated. Many of these substances are found in food, pharmaceutical drugs, pesticides and in complexes of domestic and industrial effluents. It is known that these compounds can cause detrimental inheritable changes in the genetic material, without these changes being expressed immediately [14]. Thus, several compounds dispersed in the environment can represent danger to human health, since they present a potential to induce mutations [15].

The production of food can occur both by agricultural activities and by livestock. The yield of food production is directly related with the relationship established between the species of interest for production and the other plant, animal, microbial and parasitic biological systems that compete for resources available in the environment [16]. Among the species that jeopard‐ ize the agricultural production there are the weeds that, when invade crops, can cause significant loss in the yield and quality of the harvest [17]. Therefore, in order to enhance the productivity and the quality of crops, the removal of weeds from agriculture becomes important.

Before the introduction of selective herbicides as an agricultural practice, the removal of weeds was accomplished manually in an extremely laborious form. Thus, the farmers sought other forms to control weeds, such as, integrating other weed control practices such as crop rotation, tillage and fallow systems [17].

The introduction of selective herbicides in the late 40's and the constant production of new herbicides in the following decades gave farmers a new tool in the control of weeds [17]. Therefore, the process of modernization of agriculture introduced, in the 60's, the use of new biological varieties considered more productive, but dependent on chemical fertilizers and intensive use of pesticides, in order to increase productivity. The use of these chemical agents resulted in the increase of productivity, but, on the other hand, brought adverse consequences, since many are harmful substances for man and the environment. The world practice of using agrochemicals for long periods, often indiscriminate and abusive, has raised concerns among the public authorities and experts of public health and sustainability of natural resources [16].

modes of action of xenobiotics present in the ecosystem in the biota exposed. For this, extensive, detailed and ordered studies of the contaminants must be developed with the purpose of preventing the biological impairment, such as inductions of alterations in the genetic materials

Some studies have been performed in the attempt to evaluate the behaviour, transforma‐ tions and effects of chemical agents, both in the environment and in the organisms. Toxicology establishes the limits of concentration or quantity of chemical substances acceptable in the environment by studies on the toxic effects of these substances in the

Considering that the use of agrochemicals, such as herbicides, have caused a great environ‐ mental contamination, due to their widespread use, it has become indispensable to perform

Living beings are exposed to the action of numerous agents that are potentially toxic. These agents can be physical, chemical or biological and can provoke in the organisms physiological, biochemical, pathological effects and, in some cases, genetic effects [13]. A great variety of chemical substances with mutagenic potential, both natural and synthetic, have been investi‐ gated. Many of these substances are found in food, pharmaceutical drugs, pesticides and in complexes of domestic and industrial effluents. It is known that these compounds can cause detrimental inheritable changes in the genetic material, without these changes being expressed immediately [14]. Thus, several compounds dispersed in the environment can represent

The production of food can occur both by agricultural activities and by livestock. The yield of food production is directly related with the relationship established between the species of interest for production and the other plant, animal, microbial and parasitic biological systems that compete for resources available in the environment [16]. Among the species that jeopard‐ ize the agricultural production there are the weeds that, when invade crops, can cause significant loss in the yield and quality of the harvest [17]. Therefore, in order to enhance the productivity and the quality of crops, the removal of weeds from agriculture becomes

Before the introduction of selective herbicides as an agricultural practice, the removal of weeds was accomplished manually in an extremely laborious form. Thus, the farmers sought other forms to control weeds, such as, integrating other weed control practices such as crop rotation,

The introduction of selective herbicides in the late 40's and the constant production of new herbicides in the following decades gave farmers a new tool in the control of weeds [17]. Therefore, the process of modernization of agriculture introduced, in the 60's, the use of new biological varieties considered more productive, but dependent on chemical fertilizers and intensive use of pesticides, in order to increase productivity. The use of these chemical agents resulted in the increase of productivity, but, on the other hand, brought adverse consequences,

danger to human health, since they present a potential to induce mutations [15].

of the organisms [11].

organism and ecosystems [12].

400 Herbicides - Current Research and Case Studies in Use

**1.1. The importance of herbicides**

important.

tillage and fallow systems [17].

the assessment of the toxicity of these compounds.

Many agrochemicals are very toxic substances whose absorption in man are almost exclusively oral and can also occur by inhalation or dermally. As a consequence of the human exposure to pesticides, a series of disturbances can be observed, such as gastric, neurological and muscular [18].

Among the pesticides, the main agents of intoxication are the herbicides and insecticides. According to Vasilescu and Medvedovici [19], herbicides are defined as any substance, individually or in mixtures, whose function is to control, destroy, repel or mitigate the growth of weeds in a crop.

The use of herbicides, despite the fact that they are characterized as a highly effective tool in the control of weeds, has led to a change in the phytosociological composition of weeds and to a selection of biotypes resistant to herbicides, besides also causing impacts in the environment and human health. According to He et al. [20], herbicides are the most used chemical substances throughout the world. During the 90's, the global pesticide sales remained relatively constant, between 270 and 300 billions of US dollars, and 47% of this value corresponded to herbicides and 79% to insecticides. Since 2007, herbicides assumed the first place among the three major categories of pesticides (insecticides, fungicides/ bactericides, herbicides) [21].

The use of herbicides to control weeds has been a common practice in global agriculture, mainly with the objective to increase agricultural production. However, when these chemicals are used in an uncontrolled manner, they can cause impacts on non-target organisms, especially on those that live in aquatic environments [22].

According to Chevreuil et al. [23], Kim and Feagley [24] and Abdel-Ramham et al. [25], most of the toxic effects of the herbicides on animals and plants were insufficiently investigated. As a consequence of the lack of information about the action of herbicides in the biological environment, these chemical agents can also represent a problem to human health [26, 27]. The impact of a pesticide in the environment depends on its dispersion mode and its concentration, as well as its own toxicity [28]. The mutagenic effects of the herbicides can result from several reactions with the organism, as a direct action of the compound on the nuclear DNA; incor‐ poration in the DNA during cell replication; interference in the activity of the mitotic or meiotic division, resulting in incorrect division of the cell [29].

Some herbicides interfere directly in the cell division of plants, elongation and/or cell differ‐ entiation, causing disturbances in the functioning of the roots or vascular tissues [30]. In animals, herbicides can act in several tissues or organs and, sometimes, are associated with tumorigenic processes [31].

Jurado et al. [32] listed the general advantages and disadvantages of using herbicides. In this list, the authors cited as advantages: kill unwanted plants; help crops grow since it eliminates weeds that compete with crops for water, nutrients and sunlight; can be safely used in plantations, while the manual or mechanical removal processes of weeds can cause damages to crops; can be used in geographically close crops; in most cases, only one application of the herbicide is sufficient to control the weeds, while the other methods must be constantly used; are easy to use; have fast action; are relatively inexpensive and are economically more viable than manual removal; non-selective herbicides can be used to eliminate vegetation cover in areas intended for the construction of residences and/or roads; to eradicate plants bearing diseases; and since some herbicides are biodegradable, they can become relatively inert after some time. The disadvantages listed by the authors are: some herbicides are not biodegradable and, thus, can persist in the environment for a long period of time; all herbicides are, at least, mildly toxic; can cause diseases and even accidental death (case of paraquat); can be carried into rivers by rainwater or be leached to groundwater polluting these environments; some herbicides can accumulate in the food chain and are toxic for animals, including man.

**d.** inhibitory uncouplers: the term "inhibitory uncouplers" was used by Moreland [33] to indicate that the herbicides interfere in reactions affected by electron transport inhibitors and by uncouplers; These "inhibitory uncouplers" inhibit the basal transport, uncoupled and coupled of electrons. The herbicides classified in this group affect both the electron transport and the gradient of protons. Examples: acylanilides, dinitrophenols, imidazole,

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

403

**e.** electron acceptors: the compounds classified in this group are able to compete with some component of electron transport and consequently suffer reduction. Examples: diquat,

**f.** inhibitors of the carotenoid synthesis: this class of herbicides acts to inhibit the synthesis of carotenoids, resulting in accumulation of precursors of carotenoid devoid colour (phytoene and phytofluene). The inhibition of carotenoid synthesis leads to the degrada‐ tion of chlorophyll in the presence of light; degradation of 70s ribosomes; inhibition of the synthesis of proteins and loss of plastids. Examples: amitrole, dichlormate, SAN6706. **2.** *mitochondrial electron transport and phosphorylation:* herbicides that interfere in the mito‐

**a.** electron transport inhibitors: defined as substances that have the ability to interrupt the electron flow in some point of the respiratory chain, acting in one of the complexes.

**b.** uncouplers: in appropriate concentrations, the classic uncouplers, that are weak lipophilic acids or bases, prevent the phosphorylation of ADP without interfering in the electron transport. Generally, any compound that promotes the dissipation of the energy gener‐ ated by the electron transport, except for the production of ATP, can be considered as

**c.** energy transfer inhibitors: compounds of this group inhibit the phosphorylating electron transport, when the apparatus of energy conservation of the mitochondria is intact and the inhibition is circumvented by uncouplers. They combine with an intermediary in the coupling energy chain and, thus, block the phosphorylation sequence that leads to the

**d.** inhibitory uncouplers: most of the herbicides that interfere in the oxidative phosphory‐ lation present a great variety of responses and are classified as uncoupling inhibitors. At low molar concentrations, herbicides fulfil almost all, if not all, of the requirements established for uncouplers, but at high concentrations they act as electron transport inhibitors. Herbicides that present this behaviour are the same classified as uncoupler inhibitors of the photoinduced reactions in the chloroplast. Example: perfluidone.

**3.** *interactions with membrane:* herbicides can affect the structure and function of membranes directly or indirectly. When the herbicides disaggregates a membrane, they can influence directly the transport processes by interacting with the protein compounds, such as, ATPases and by altering the permeability by physicochemical interactions, or indirectly

ATP formation. No herbicide seems to act as an energy transfer inhibitor.

bromofenoxim.

chondrial system are classified as:

Examples: diphenylether herbicides.

uncoupler. Example: isopropyl ester glyphosate.

paraquat.

#### **1.2. Herbicides classification**

According to Moreland [33], herbicides are designated by common names approved by the Weed Science Society of America (WSSA) or by the British Standards Institution. Organic herbicides are classified according to their application method, chemical affinity, structural similarity, and by their mode of action [34]. In relation to the application methods, herbicides can be classified into two groups: soil application and foliar application. According to Jurado et al. [32], all the herbicides applied in the pre-planting (surface or incorporation) and preemergence (in crops, weeds or both) are classified as herbicides of soil application and those applied in the post-emergence are classified as foliar application.

Moreover, herbicides can be classified according to their mode of action. Following, it will be presented the classes of herbicides, according to their mode of action, based in the classification of Moreland [33] :


**d.** inhibitory uncouplers: the term "inhibitory uncouplers" was used by Moreland [33] to indicate that the herbicides interfere in reactions affected by electron transport inhibitors and by uncouplers; These "inhibitory uncouplers" inhibit the basal transport, uncoupled and coupled of electrons. The herbicides classified in this group affect both the electron transport and the gradient of protons. Examples: acylanilides, dinitrophenols, imidazole, bromofenoxim.

plantations, while the manual or mechanical removal processes of weeds can cause damages to crops; can be used in geographically close crops; in most cases, only one application of the herbicide is sufficient to control the weeds, while the other methods must be constantly used; are easy to use; have fast action; are relatively inexpensive and are economically more viable than manual removal; non-selective herbicides can be used to eliminate vegetation cover in areas intended for the construction of residences and/or roads; to eradicate plants bearing diseases; and since some herbicides are biodegradable, they can become relatively inert after some time. The disadvantages listed by the authors are: some herbicides are not biodegradable and, thus, can persist in the environment for a long period of time; all herbicides are, at least, mildly toxic; can cause diseases and even accidental death (case of paraquat); can be carried into rivers by rainwater or be leached to groundwater polluting these environments; some

herbicides can accumulate in the food chain and are toxic for animals, including man.

applied in the post-emergence are classified as foliar application.

are divided into the following classes:

the phosphorylation. Example: diuron, atrazine.

According to Moreland [33], herbicides are designated by common names approved by the Weed Science Society of America (WSSA) or by the British Standards Institution. Organic herbicides are classified according to their application method, chemical affinity, structural similarity, and by their mode of action [34]. In relation to the application methods, herbicides can be classified into two groups: soil application and foliar application. According to Jurado et al. [32], all the herbicides applied in the pre-planting (surface or incorporation) and preemergence (in crops, weeds or both) are classified as herbicides of soil application and those

Moreover, herbicides can be classified according to their mode of action. Following, it will be presented the classes of herbicides, according to their mode of action, based in the classification

**1.** *chloroplast-associated reactions:* photo-induced electron transport and reaction coupled to phosphorylation occur in the chloroplast, any interference in these reactions inhibit the photosynthetic activity. Herbicides that inhibit the photo-chemically induced reactions

**a.** electron transport inhibitors: electron transport is inhibited when one or more interme‐ diary electron carriers are removed or inactivated or even when there is interference in

**b.** uncouplers: uncouplers dissociate the electron transport of the ATP formation through the dissipation of the energetic state of the thylakoid membrane, before the energy can be used to perform the high endergonic reaction of ADP phosphorylation. Example:

**c.** energy transference inhibitors: inhibition of energy transference inhibitors acts directly in the phosphorylation, as well as inhibitors of the electron transport, which inhibit both the electron flow and the formation of ATP in coupled systems. Example: 1,2,3-thiadiazol-

**1.2. Herbicides classification**

402 Herbicides - Current Research and Case Studies in Use

of Moreland [33] :

perfluidone.

phenylurea, nitrofen.


by modulating the supply of ATP needed to energize the membrane. Interactions with the membrane can cause:

**Class of the herbicide Examples of herbicides**

Dinitroaniniles Benefin, ethalfluralin, fluchloralin, pendimethalin, prodiamine,

Diphenylethers Acifluorfen, bifenox, fluoroglycofen, fomesafen, lactofen,

Imidazolinones Imazapyr, imazaquin, imazethapyr, imazamethabenz

trifluralin

oxyfluorfen

2,4-D, MCPA, 2,4,5-T

Dichlorprop, diclofop, fenoxaprop, fluazifop-P, quizalofop-P

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

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Bensulfuron, chlorimuron, chlorsulfuron, halosulfuron, metsulfuron, nicosulfuron, primisulfuron, prosulfuron, sulfometuron, thifensulfuron, triasulfuron, tribenuron

2,4-DB

Cyclohexanediones (cyclohexenones) Clethodin, cycloxidim, sethoxydim, tralkoxydim

Cineoles Cinmethylin

Imidazolidinones Buthidazole

Imines CGA-248757 Isoxazolidinones Clomazone

Oxadiazoles Oxadiazon Oxadiazolidines Methazole Phenols Dinoseb

*N-*phenylphthalimides Flumiclorac Phenylpyridazines Pyridate Phenyl Triazinones (Aryl Triazinones) Sulfentrazone Phthalamates Naptalam Pyrazoliums Difenzoquat Pyridazinones Norflurazon, pyrazon Pyridinecarboxylic Acids Clopyralid, picloram, triclopyr Pyridines Dithiopyr, thiazopyr Pyridinones Fluridone Pyrimidinythio-benzoates (Benzoates) Pyrithiobac Quinolinecaryoxylic acids Quinclorac

Tetrahydropyrimidinones Yet to be commercialized

Uracils Bromacil, terbacil, UCC-C4243

Ureas Diuron, fluometuron, linuron, tebuthiuron,

Triazinones Metribuzin Triazoles Amitrole Triazolopyrimidine Sulfonanilides Flumetsulam

**Table 1.** Classification of the herbicides according to the chemical affinity.

Thiocarbamates Butylate, diallate, EPTC, molinate, pebulate, thiobencarb, triallate Triazines Ametryn, atrazine, cyanazine, hexazinone, prometryn, simazine

Unclassified herbicides Bensulide, ethofumesate, fosamine, glufosinate, glyphosate,

tridiphane

Phenoxyalkanoic acids Phenoxyacetics Phenoxybutyrics Arylophenoxy propionics

Sulfonylureas

Nitriles Bromoxynil, dichlobenil, ioxynil


The herbicides can still be classified according to the chemical affinity. Table 1 shows the chemical classes and examples of each class, according to Rao [34].



**Table 1.** Classification of the herbicides according to the chemical affinity.

by modulating the supply of ATP needed to energize the membrane. Interactions with

**a.** compositional alterations: can modify or alter the composition of lipids in the membrane and can also act in the metabolism and synthesis of lipids. Examples: dinoben, chloram‐

**b.** effects in the permeability and integrity. Examples: paraquat, diquat, oryfluorfen,

**4.** *cell division:* herbicides may suppress cell division by interfering in the synthesis or active transport of precursors into the nucleus, which are necessary for the synthesis of DNA during interphase; modify the physical or chemical properties of the DNA or of their complexes; interfere in the formation and function of the spindle; and/or inhibits the formation of the cell wall. Several of the processes mentioned previously need energy and, therefore, interferences in the amount of energy caused by an herbicide could modulate the mitotic activity. The effects of the inhibitors of the cell division are dependent on the concentration and vary according to the species and the type of tissue. There is a rela‐ tionship between cell division and cellular energy. In higher plants, cell division is prevented or suppressed in conditions in which the glycolysis or the oxidative phosphor‐ ylation is inhibited. Another form of the herbicide to alter cell division would be inter‐ acting with the microtubules, since these cellular structures are responsible for the orientation and movement of chromosomes during cell division. Examples of herbicides

that interfere in cell division: N-phenylcarbamates, ioxynil, trifluralin.

chemical classes and examples of each class, according to Rao [34].

Benzamides Isoxaben Benzoics Dicamba Benzothiadiazoles Bentazon Bipyridiliums Diquat, paraquat

**5.** *Synthesis ofDNA, RNA and protein:* there are correlations between inhibition of RNA and protein synthesis and low concentration of ATP in tissues and these correlations suggest that interferences in the energy production, necessary to perform biosynthetic reactions, could be the mechanism by which the herbicides could express their effects. Moreover, they can inhibit the synthesis of DNA or RNA by altering the chromatin integrity and, in these cases, the synthesis of proteins is also affected. Examples: glyphosate, trifluralin.

The herbicides can still be classified according to the chemical affinity. Table 1 shows the

**Class of the herbicide Examples of herbicides**

Aliphatics Chlorinated aliphatic acid (TCA), acrolein, dalapon

Carbamates Asulam, desmedipham, phenmedipham

Acetochlor, alachlor, butachlor, dimethenamid, metolachlor,

napropamide, pronamide, propachlor, propanil

Disodium methanearsonate (DSMA), monosodium methanearsonate (MSMA), cacodylic acid

the membrane can cause:

404 Herbicides - Current Research and Case Studies in Use

bem, perfluidone.

oryzalin.

Acetamides

Arsenicals

#### **1.3. Aquatic and soil contamination due to the presence of herbicides**

When a herbicide is used to control weeds, sometimes a majority of the compound ends up in the environment, whether it is in the soil, water, atmosphere or in the products harvested [17]. Due to the widespread use of these chemicals over the years, there has been an accumulation of these residues in the environment, which is causing alarming contaminations in the ecosystems [35] and negative damages to the biota. To Bolognesi and Merlo [3], the widespread use of herbicides has drawn the attention of researchers concerned with the risks that they can promote on the environment and human health, since they are chemicals considered contam‐ inants commonly present in hydric resources and soils. According to the same authors, herbicides represent a high toxicity to target species but it can be also toxic, at different levels, to non-target species, such as human beings. Herbicides can cause deleterious effects on organisms and human health, both by their direct and indirect action [2]. Among the biological effects of these chemicals, it can be cited genetic damages, diverse physiological alterations and even death of the organisms exposed. Some herbicides, when at low concentrations, cannot cause immediate detectable effects in the organisms, but, in long term can reduce their lifespan longevity [4]. Herbicides can affect the organisms in different ways. As with other pesticides, the accumulation rate of these chemicals on biota depends on the type of the associated food chain, besides the physicochemical characteristics (chemical stability, solubility, photodecomposition, sorption in the soil) of the herbicide [5-6]. Thus, despite the existence of several toxicological studies carried out with herbicides, in different organisms, to quantify the impacts of these pollutants and know their mechanisms of action [7, 8, 2], there is a great need to expand even more the knowledge about the effects of different herbicides in aquatic and terrestrial ecosystems. Data obtained from *in situ*, *ex situ*, *in vivo* and *in vitro* tests, derived from experiments of simulation, occupational exposure or environmental contaminations, need to enhance so that it is possible to obtain even more consistent information about the action of these compounds.

affirm that pesticides produce deleterious effects to the organisms and biochemical and enzymatic processes in the soil [37]. According to Hussain et al. [37], in general, the application of pesticides, and here it is also included herbicides, made long term, can cause a disturbance

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

407

Once in the soil, herbicides can suffer alteration in their structure and composition, due to the action of physical, chemical and biological processes. This action on the herbicides is the one that will determine their activity and persistence in the soil. Some molecules, when incorpo‐ rated into the soil, are reduced by volatilization and photo-decomposition. Once in the soil, herbicides can suffer the action of microorganisms, which, added to the high humidity and high temperature, can have their decomposition favoured [38]. If they are not absorbed by plants, they can become strongly adsorbed on the organic matter present in the colloidal fraction of the soil, be carried by rainwater and/or irrigation and even be leachate, thus reaching

The prediction of the availability of herbicides to plants has two purposes: 1. ensure that the herbicide reaches the roots in concentrations high enough to control weeds, without compro‐ mising the agricultural productivity; 2. predict if the compound is mobile in the soil to estimate

The contamination of aquatic environments by herbicides has been characterized as a major world concern. This aquatic contamination is due to the use of these products in the control of aquatic plants, leachate and runoff of agricultural areas [40]. According to He et al. [20], it is a growing public concern about the amount of herbicides that have been introduced into the environment by leachate and runoff, not to mention that the contaminations of the aquatic environments generally occur by a mixture of these compounds and not by isolated substances. Guzzella et al. [1] did a survey on the presence of herbicides in groundwater in a highly cultivated region of northern Italy. The researchers monitored for two years the presence of 5 active ingredients and 17 metabolites resulting from these compounds. The authors verified that atrazine, although banned in Italy since 1986, was the major contaminant of the ground‐ water of the sites studied, they also observed that the concentration of at least one of the compounds studied exceeded the maximum allowed concentration in 59% of the samples likely due in both cases to off-label herbicide use. This scenario could be, in long term, a serious

Toccalino et al. [41] carried out a study to verify the potential of chemical mixtures existing in samples of groundwater used for public supply. In these samples, the most common organic contaminants were herbicides, disinfection by-products and solvents. The authors concluded that the combined concentrations of the contaminants can be a potential concern for more than half of the samples studied and that, even though the water destined to public supply pass through treatments to reduce contaminations and meet the legislations, it can still contain

Saka [42] evaluated the toxicity of three herbicides (simetryn, mefenacet and thiobencarb) commonly used in rice planting in Japan, on the test organism *Silurana tropicalis* (tadpoles). The authors observed that the three herbicides, particularly thiobencarb, are toxic for tadpoles

how much of the herbicide can be leachate from the roots zone to groundwater [17].

problem for the quality of this water, which is used as drinking water.

in the biochemical balance of the soil, which can reduce its fertility and productivity.

surface or groundwater [39].

mixtures at worrying concentrations.

According to Jurado et al. [32], when herbicides are applied in agricultural areas they can have different destinations, since being degraded by microorganisms or by non- biological means or even be transported by water, to areas distant from the application site. Thus, according to the same authors, the organisms can be then exposed to a great number of these xenobiotics as well as their metabolites.

The fate of the compound in the soil depends on the characteristics of the compound and the soil. The hydrogenionic properties of a compound in the soil determines its sorption charac‐ teristics, such as, acid herbicides in soils with normal pH are negatively charged and conse‐ quently are movable in most of the soils [17]. Some groups of pesticides are neutral in soils with normal pH but due to electronic dislocations in the molecules, they can bind to soil colloids by several forms [36].

According to Kudsk and Streiberg [17], during the last two decades, several studies have been completed to predict the behaviour of pesticides in the soil. Despite the numerous efforts to assess the effects of herbicides in the soil, there are conflicting data in the literature on the subject, where some studies show that the residues of pesticides can be sources of carbon and energy to microorganisms, and then are degraded and assimilated by them, while other reports affirm that pesticides produce deleterious effects to the organisms and biochemical and enzymatic processes in the soil [37]. According to Hussain et al. [37], in general, the application of pesticides, and here it is also included herbicides, made long term, can cause a disturbance in the biochemical balance of the soil, which can reduce its fertility and productivity.

**1.3. Aquatic and soil contamination due to the presence of herbicides**

406 Herbicides - Current Research and Case Studies in Use

these compounds.

as well as their metabolites.

colloids by several forms [36].

When a herbicide is used to control weeds, sometimes a majority of the compound ends up in the environment, whether it is in the soil, water, atmosphere or in the products harvested [17]. Due to the widespread use of these chemicals over the years, there has been an accumulation of these residues in the environment, which is causing alarming contaminations in the ecosystems [35] and negative damages to the biota. To Bolognesi and Merlo [3], the widespread use of herbicides has drawn the attention of researchers concerned with the risks that they can promote on the environment and human health, since they are chemicals considered contam‐ inants commonly present in hydric resources and soils. According to the same authors, herbicides represent a high toxicity to target species but it can be also toxic, at different levels, to non-target species, such as human beings. Herbicides can cause deleterious effects on organisms and human health, both by their direct and indirect action [2]. Among the biological effects of these chemicals, it can be cited genetic damages, diverse physiological alterations and even death of the organisms exposed. Some herbicides, when at low concentrations, cannot cause immediate detectable effects in the organisms, but, in long term can reduce their lifespan longevity [4]. Herbicides can affect the organisms in different ways. As with other pesticides, the accumulation rate of these chemicals on biota depends on the type of the associated food chain, besides the physicochemical characteristics (chemical stability, solubility, photodecomposition, sorption in the soil) of the herbicide [5-6]. Thus, despite the existence of several toxicological studies carried out with herbicides, in different organisms, to quantify the impacts of these pollutants and know their mechanisms of action [7, 8, 2], there is a great need to expand even more the knowledge about the effects of different herbicides in aquatic and terrestrial ecosystems. Data obtained from *in situ*, *ex situ*, *in vivo* and *in vitro* tests, derived from experiments of simulation, occupational exposure or environmental contaminations, need to enhance so that it is possible to obtain even more consistent information about the action of

According to Jurado et al. [32], when herbicides are applied in agricultural areas they can have different destinations, since being degraded by microorganisms or by non- biological means or even be transported by water, to areas distant from the application site. Thus, according to the same authors, the organisms can be then exposed to a great number of these xenobiotics

The fate of the compound in the soil depends on the characteristics of the compound and the soil. The hydrogenionic properties of a compound in the soil determines its sorption charac‐ teristics, such as, acid herbicides in soils with normal pH are negatively charged and conse‐ quently are movable in most of the soils [17]. Some groups of pesticides are neutral in soils with normal pH but due to electronic dislocations in the molecules, they can bind to soil

According to Kudsk and Streiberg [17], during the last two decades, several studies have been completed to predict the behaviour of pesticides in the soil. Despite the numerous efforts to assess the effects of herbicides in the soil, there are conflicting data in the literature on the subject, where some studies show that the residues of pesticides can be sources of carbon and energy to microorganisms, and then are degraded and assimilated by them, while other reports Once in the soil, herbicides can suffer alteration in their structure and composition, due to the action of physical, chemical and biological processes. This action on the herbicides is the one that will determine their activity and persistence in the soil. Some molecules, when incorpo‐ rated into the soil, are reduced by volatilization and photo-decomposition. Once in the soil, herbicides can suffer the action of microorganisms, which, added to the high humidity and high temperature, can have their decomposition favoured [38]. If they are not absorbed by plants, they can become strongly adsorbed on the organic matter present in the colloidal fraction of the soil, be carried by rainwater and/or irrigation and even be leachate, thus reaching surface or groundwater [39].

The prediction of the availability of herbicides to plants has two purposes: 1. ensure that the herbicide reaches the roots in concentrations high enough to control weeds, without compro‐ mising the agricultural productivity; 2. predict if the compound is mobile in the soil to estimate how much of the herbicide can be leachate from the roots zone to groundwater [17].

The contamination of aquatic environments by herbicides has been characterized as a major world concern. This aquatic contamination is due to the use of these products in the control of aquatic plants, leachate and runoff of agricultural areas [40]. According to He et al. [20], it is a growing public concern about the amount of herbicides that have been introduced into the environment by leachate and runoff, not to mention that the contaminations of the aquatic environments generally occur by a mixture of these compounds and not by isolated substances.

Guzzella et al. [1] did a survey on the presence of herbicides in groundwater in a highly cultivated region of northern Italy. The researchers monitored for two years the presence of 5 active ingredients and 17 metabolites resulting from these compounds. The authors verified that atrazine, although banned in Italy since 1986, was the major contaminant of the ground‐ water of the sites studied, they also observed that the concentration of at least one of the compounds studied exceeded the maximum allowed concentration in 59% of the samples likely due in both cases to off-label herbicide use. This scenario could be, in long term, a serious problem for the quality of this water, which is used as drinking water.

Toccalino et al. [41] carried out a study to verify the potential of chemical mixtures existing in samples of groundwater used for public supply. In these samples, the most common organic contaminants were herbicides, disinfection by-products and solvents. The authors concluded that the combined concentrations of the contaminants can be a potential concern for more than half of the samples studied and that, even though the water destined to public supply pass through treatments to reduce contaminations and meet the legislations, it can still contain mixtures at worrying concentrations.

Saka [42] evaluated the toxicity of three herbicides (simetryn, mefenacet and thiobencarb) commonly used in rice planting in Japan, on the test organism *Silurana tropicalis* (tadpoles). The authors observed that the three herbicides, particularly thiobencarb, are toxic for tadpoles (LD50 test), even for concentrations found in waters where the rice is cultivated. In a similar study carried out by Liu et al. [43], it was observed that the effect of the herbicide butachlor (most used herbicide in rice planting in Taiwan and Southeast Asia) on the organism *Fejervarya limnocharis* (alpine cricket frog) exposed to concentrations used in the field. In this study no effect on the growth of tadpoles of *F. limnocharis* was observed, but there was a negative action on survival, development and time of metamorphosis. The authors suggested that the herbicide butachlor can cause serious impacts on anurans that reproduce in rice fields, but this impact varies from species to species.

suspended and bottom sediment [47]. The sorption of the herbicides in sediments in suspen‐ sion can reduce the degradation rate of the herbicides in water, and the movement of the sediment in suspension can transport the pesticides from one place to another, entering into

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

409

A study conducted by Jacomini et al. [48] evaluated the contamination of three matrices (water, sediment and bivalve molluscs) collected in rivers influenced by crops of sugar cane in São Paulo State-Brazil. In this study, the authors observed that the highest concentrations of residues of the herbicide ametrin were present in the sediment, showing the persistence of this compound in the sediments of rivers and its potential to mobilize between the compartments

When the herbicides are dispersed in the water or sediments in suspension of the rivers, they can end up in other ecosystems such as estuaries. Duke et al. [49], when studying the effect of herbicides on mangroves of the Mackay region, found out that diuron, and even other herbicides, are potentially responsible for the mangrove dieback. According to the authors, the consequences for this death would be the impoverishment of the quality of the coastal water with an increase of the turbidity, nutrients and sediment deposition, problems in the

In a review conducted by Jones [50], the author highlights the contamination of marine environments by herbicides (such as diuron), discussing that the contamination of these environments can occur by transport of these substances of agricultural or non cultivated areas (roadsides, sports fields, train tracks), runoff by storms and tailwater irrigation release), pulverizations and accidental spills. These contaminations mean that the photochemical efficiency of intracellular symbiotic algae of the coral, in long term, may be compromised, leading to a loss in the symbiotic relationship of the coral with the algae and a consequent bleaching of corals. Still considering the marine ecosystem, Lewis et al. [51] verified that the runoff of pesticides from agricultural areas influence the health of the Great Barrier Reef in

Considering the prior literature, it is likely possible that the effects of herbicides do not occur only at the places that they are applied but also in places distant from their application. Moreover, herbicides can induce alterations in non-target organisms, altering the survival and the equilibrium of the ecosystems, whether they are aquatic or terrestrial. Thus, much care must be taken when introducing these substances into the environment and more studies should be conducted in order to thoroughly understand the environmental consequences that

**2. The effects of herbicides using different bioassays and test-organisms**

Many studies have evaluated the impact of different chemical classes of herbicides using different doses, organisms and bioassays, focusing on toxic, cytotoxic, genotoxic, mutagenic,

the tissue of organisms or settling on the bottom [40].

of the aquatic system, such as water and biota.

fixation of seedlings and consequent erosion of the estuaries.

Australia and can disturb this sensitive ecosystem.

embryotoxic, teratogenic, carcinogenic and estrogenic effects.

herbicides can cause.

In a study conducted by Ventura et al. [8], it was observed that the herbicide atrazine has a genotoxic and mutagenic effect on the species *Oreochromis niloticus* (Nile tilapia). In this study, the authors observed that the herbicide can interfere in the genetic material of the organisms exposed, even at doses considered residual, which led the authors to suggest that residual doses of atrazine, resulting from leaching of soils of crops near water bodies, can interfere in a negative form in the stability of aquatic ecosystems.

Bouilly et al. [44] studied the impact of the herbicide diuron on *Crassostrea gigas* (Pacific oyster) and observed that the herbicide can cause irreversible damages to the genetic material of the organism studied. Moreover, the authors affirm that, due to the persistence of diuron in environments adjacent to its application site and that it is preferably used in spring, the pollution caused by its use causes negative impact in the aquatic organisms during the breeding season.

In general, when herbicides contaminate the aquatic ecosystem, they can cause deleterious effects on the organisms of this system. Thus, organisms that live in regions impacted by these substances, whose breeding period coincides with the application period of the herbicides, can suffer serious risks of development and survival of their offspring.

Hladik et al. [45] evaluated the presence of two herbicides (chloroacetamide and triazine), as well as their by-products, in drinking water samples of the Midwest region of the United States. The authors detected the presence of neutral chloroacetamide degradates in median concen‐ trations (1 to 50 ng/L) of the water samples. Furthermore, they found that neither the original chloroacetamide herbicides nor their degradation products were efficiently removed by conventional water treatment processes (coagulation/flocculation, filtration, chlorination). According to Bannink [46], about 40% of the drinking water from Netherlands is derived from surface water. The Dutch water companies are facing problems with the water quality due to contamination by herbicides used to eliminate ruderal plants. These data serve as alerts for the presence of herbicides and their degradation products in drinking water, pointing out the need for the development of new treatment systems that could be more efficient to eliminate this class of contaminants.

According to Ying and Williams [40], organic herbicides, when in aquatic ecosystems, can be distributed in several compartments depending on their solubility in water. These compart‐ ments include water, aquatic organisms, suspended sediment and bottom sediment. The more hydrophilic the organic pesticide, the more it is transported to the aqueous phase, and the more hydrophobic a pesticide is, the more it will be associated to the organic carbon of the suspended and bottom sediment [47]. The sorption of the herbicides in sediments in suspen‐ sion can reduce the degradation rate of the herbicides in water, and the movement of the sediment in suspension can transport the pesticides from one place to another, entering into the tissue of organisms or settling on the bottom [40].

(LD50 test), even for concentrations found in waters where the rice is cultivated. In a similar study carried out by Liu et al. [43], it was observed that the effect of the herbicide butachlor (most used herbicide in rice planting in Taiwan and Southeast Asia) on the organism *Fejervarya limnocharis* (alpine cricket frog) exposed to concentrations used in the field. In this study no effect on the growth of tadpoles of *F. limnocharis* was observed, but there was a negative action on survival, development and time of metamorphosis. The authors suggested that the herbicide butachlor can cause serious impacts on anurans that reproduce in rice fields, but this

In a study conducted by Ventura et al. [8], it was observed that the herbicide atrazine has a genotoxic and mutagenic effect on the species *Oreochromis niloticus* (Nile tilapia). In this study, the authors observed that the herbicide can interfere in the genetic material of the organisms exposed, even at doses considered residual, which led the authors to suggest that residual doses of atrazine, resulting from leaching of soils of crops near water bodies, can interfere in

Bouilly et al. [44] studied the impact of the herbicide diuron on *Crassostrea gigas* (Pacific oyster) and observed that the herbicide can cause irreversible damages to the genetic material of the organism studied. Moreover, the authors affirm that, due to the persistence of diuron in environments adjacent to its application site and that it is preferably used in spring, the pollution caused by its use causes negative impact in the aquatic organisms during the

In general, when herbicides contaminate the aquatic ecosystem, they can cause deleterious effects on the organisms of this system. Thus, organisms that live in regions impacted by these substances, whose breeding period coincides with the application period of the herbicides, can

Hladik et al. [45] evaluated the presence of two herbicides (chloroacetamide and triazine), as well as their by-products, in drinking water samples of the Midwest region of the United States. The authors detected the presence of neutral chloroacetamide degradates in median concen‐ trations (1 to 50 ng/L) of the water samples. Furthermore, they found that neither the original chloroacetamide herbicides nor their degradation products were efficiently removed by conventional water treatment processes (coagulation/flocculation, filtration, chlorination). According to Bannink [46], about 40% of the drinking water from Netherlands is derived from surface water. The Dutch water companies are facing problems with the water quality due to contamination by herbicides used to eliminate ruderal plants. These data serve as alerts for the presence of herbicides and their degradation products in drinking water, pointing out the need for the development of new treatment systems that could be more efficient to eliminate

According to Ying and Williams [40], organic herbicides, when in aquatic ecosystems, can be distributed in several compartments depending on their solubility in water. These compart‐ ments include water, aquatic organisms, suspended sediment and bottom sediment. The more hydrophilic the organic pesticide, the more it is transported to the aqueous phase, and the more hydrophobic a pesticide is, the more it will be associated to the organic carbon of the

impact varies from species to species.

408 Herbicides - Current Research and Case Studies in Use

breeding season.

this class of contaminants.

a negative form in the stability of aquatic ecosystems.

suffer serious risks of development and survival of their offspring.

A study conducted by Jacomini et al. [48] evaluated the contamination of three matrices (water, sediment and bivalve molluscs) collected in rivers influenced by crops of sugar cane in São Paulo State-Brazil. In this study, the authors observed that the highest concentrations of residues of the herbicide ametrin were present in the sediment, showing the persistence of this compound in the sediments of rivers and its potential to mobilize between the compartments of the aquatic system, such as water and biota.

When the herbicides are dispersed in the water or sediments in suspension of the rivers, they can end up in other ecosystems such as estuaries. Duke et al. [49], when studying the effect of herbicides on mangroves of the Mackay region, found out that diuron, and even other herbicides, are potentially responsible for the mangrove dieback. According to the authors, the consequences for this death would be the impoverishment of the quality of the coastal water with an increase of the turbidity, nutrients and sediment deposition, problems in the fixation of seedlings and consequent erosion of the estuaries.

In a review conducted by Jones [50], the author highlights the contamination of marine environments by herbicides (such as diuron), discussing that the contamination of these environments can occur by transport of these substances of agricultural or non cultivated areas (roadsides, sports fields, train tracks), runoff by storms and tailwater irrigation release), pulverizations and accidental spills. These contaminations mean that the photochemical efficiency of intracellular symbiotic algae of the coral, in long term, may be compromised, leading to a loss in the symbiotic relationship of the coral with the algae and a consequent bleaching of corals. Still considering the marine ecosystem, Lewis et al. [51] verified that the runoff of pesticides from agricultural areas influence the health of the Great Barrier Reef in Australia and can disturb this sensitive ecosystem.

Considering the prior literature, it is likely possible that the effects of herbicides do not occur only at the places that they are applied but also in places distant from their application. Moreover, herbicides can induce alterations in non-target organisms, altering the survival and the equilibrium of the ecosystems, whether they are aquatic or terrestrial. Thus, much care must be taken when introducing these substances into the environment and more studies should be conducted in order to thoroughly understand the environmental consequences that herbicides can cause.

### **2. The effects of herbicides using different bioassays and test-organisms**

Many studies have evaluated the impact of different chemical classes of herbicides using different doses, organisms and bioassays, focusing on toxic, cytotoxic, genotoxic, mutagenic, embryotoxic, teratogenic, carcinogenic and estrogenic effects.

With respect to the toxicity, some herbicides pose major concerns when applied in regions close to water resources due to their highly toxic potential to many aquatic organisms [52].

The toxic, cytotoxic, genotoxic, mutagenic, embryotoxic, teratogenic carcinogenic and estro‐ genic effects caused by herbicides on various organisms could be exemplified by studies as

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

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411

Atrazine is a triazinic herbicide, classified as moderately toxic of pre- and post-emergence, used for the control of weeds in crops of asparagus, corn, sorghum, sugarcane and pineapple [70]. According to Eldridge et al. [71], triazinic herbicides are among the most used pesticides

Triazine herbicides are extensively used in the United States to control grass, sedge and broadleaf weedsduring the cultivation of maize, wheat, sorghum, sugarcane and conifers [72]. In Brazil, these herbicides are widely used on crops of sugarcane and maize. Due to the widespread use of triazine herbicides in the agriculture and, therefore, its high exposure potential for humans, the United States Environmental Protection Agency (USEPA) has conducted a special review on the published and non published data of several triazine herbicides [73]. According to Nwani et al. [22], the herbicide atrazine is widely used in crops worldwide. The dangers, both toxic and genotoxic of this herbicide have been revised; however, there is an urgent need for more detailed studies on the mode of action of this compound. Atrazine has been tested in several systems, but there are shortcomings in relation to certain tests performed and some evidences of the genotoxic effects, *in vivo*, still need to be

Several studies using the test system *Aspergillus* have shown that atrazine is not mutagenic to these organisms [75, 76, 77], although it is considered mutagenic for other test systems such as *Drosophila melanogaster* [78, 79]. According to Ribas et al. [74], atrazine was responsible for a significant frequency of aneuploidies in *Neurospora crassa*, given by the chromosomal nondisjunction in *Aspergillus nidulans*, and by the induction of loss of sexual chromosomes in

Sorghum plants treated with atrazine presented an increase in the number of their chromo‐ somes, multinucleated cells, aneuploidy and polyploidy, and abnormalities in the mother cells of the pollen grain, which suggests that this herbicide interferes in the stability and also in the

Popa et al. [70] observed that atrazine, when applied in high concentrations in maize seedlings, can induce chromosome breaks, visualized by the presence of single and paired chromosome fragments; a high frequency of chromatids and chromosome bridges; lagging chromosomes and presence of heteropolyploid or polyploid cells. Grant and Owens [81] showed that atrazine induced chromosome breaks (in mitosis and meiosis) in the species

Hayes et al. [82] investigated the effect of the herbicide atrazine on wild leopard frogs (*Rana pipens*), in different regions of the United States. The authors observed that a great percentage of males exposed to the herbicide presented abnormalities in the gonads, such as development

in agriculture due to their ability to inhibit the photosynthesis of weeds in crops [16].

described below.

**2.1. Atrazine**

confirmed [74].

*Drosophila melanogaster*.

*Pisum sativum* and *Allium cepa.*

meiosis [80].

Biological tests of toxicity and mutagenicity are, according to Moraes [53], indispensable for the evaluation of the reactions of living organisms to environmental pollution and also for the identification of the potential synergistic effects of several pollutants. The impact that toxic materials can promote in the integrity and function of DNA of several organisms has been investigated [54]. Several biomarkers have been used as tools for the detection of the toxic, genotoxic and mutagenic effects of pollution. Among them we can cite the presence of DNA adducts, chromosome aberrations, breaks in the DNA strands, micronuclei formation and other nuclear abnormalities, besides induction of cell death [55].

Most of the tests used to detect the mutagenic potential of chemical substances are based on the investigation of possible inductions of chromosome damages such as structural alterations, formation of micronuclei, sister chromatid exchanges, assessment of mutant genes or damages in the DNA, using different test organisms, such as bacteria, plants and animals, both *in vitro* and *in vivo* [56].

According to Veiga [57], it is possible to estimate the genotoxic, mutagenic, carcinogenic and teratogenic effects of agrochemicals by relatively simple methods. Several studies have been carried out by several researchers concerned with the harmful effects of pesticides in an attempt to verify their possible physiological [58, 59], mutagenic [7, 8, 60, 61, 62] and carcino‐ genic effects [63].

The interaction between different methods of evaluating the toxic, genotoxic and mutagenic potential provides a more global and comprehensive view of the effect of a chemical agent. For the monitoring of organisms exposed to chemical agents, the chromosome aberration test, micronucleus test and comet assay have been widely used [64]. A few studies also have shown the toxic effects of chemicals, by cell death processes, both necrotic and apoptotic [65].

According to Kristen [66], the dramatic expansion in the production of xenobiotic compounds by anthropogenic activities has compromised the environment by the introduction of millions of chemicals with toxic potential to biological systems.

Cytogenetic tests are adequate to identify the harmful effects of substances, in their several concentrations and different periods of exposure. These tests, generally performed with test organisms, are commonly applied in biomonitoring to the extent of pollution and in the evaluation of the combined effects of toxic and mutagenic substances on the organisms in the natural environment [53]. Micronuclei assays are efficient to assess the mutagenic activity of herbicides both in laboratorial and field assays [67]. The comet assay can be used to evaluate damages in proliferating cells or not, in *in vitro* or *in vivo* tests and can be applied with the purpose of genotoxicological analyses [68]. According to these same authors, these tests are considered one of the best tools to biomonitor several chemical compounds, including herbicides. According to Ribas et al. [69], the simplicity, reproducibility and rapidity of the comet test, associated to the ability of this assay in evaluating damages in the DNA, makes this technique highly applicable to environmental genotoxicology.

The toxic, cytotoxic, genotoxic, mutagenic, embryotoxic, teratogenic carcinogenic and estro‐ genic effects caused by herbicides on various organisms could be exemplified by studies as described below.

#### **2.1. Atrazine**

With respect to the toxicity, some herbicides pose major concerns when applied in regions close to water resources due to their highly toxic potential to many aquatic organisms [52].

Biological tests of toxicity and mutagenicity are, according to Moraes [53], indispensable for the evaluation of the reactions of living organisms to environmental pollution and also for the identification of the potential synergistic effects of several pollutants. The impact that toxic materials can promote in the integrity and function of DNA of several organisms has been investigated [54]. Several biomarkers have been used as tools for the detection of the toxic, genotoxic and mutagenic effects of pollution. Among them we can cite the presence of DNA adducts, chromosome aberrations, breaks in the DNA strands, micronuclei formation and

Most of the tests used to detect the mutagenic potential of chemical substances are based on the investigation of possible inductions of chromosome damages such as structural alterations, formation of micronuclei, sister chromatid exchanges, assessment of mutant genes or damages in the DNA, using different test organisms, such as bacteria, plants and animals, both *in*

According to Veiga [57], it is possible to estimate the genotoxic, mutagenic, carcinogenic and teratogenic effects of agrochemicals by relatively simple methods. Several studies have been carried out by several researchers concerned with the harmful effects of pesticides in an attempt to verify their possible physiological [58, 59], mutagenic [7, 8, 60, 61, 62] and carcino‐

The interaction between different methods of evaluating the toxic, genotoxic and mutagenic potential provides a more global and comprehensive view of the effect of a chemical agent. For the monitoring of organisms exposed to chemical agents, the chromosome aberration test, micronucleus test and comet assay have been widely used [64]. A few studies also have shown the toxic effects of chemicals, by cell death processes, both necrotic and apoptotic [65].

According to Kristen [66], the dramatic expansion in the production of xenobiotic compounds by anthropogenic activities has compromised the environment by the introduction of millions

Cytogenetic tests are adequate to identify the harmful effects of substances, in their several concentrations and different periods of exposure. These tests, generally performed with test organisms, are commonly applied in biomonitoring to the extent of pollution and in the evaluation of the combined effects of toxic and mutagenic substances on the organisms in the natural environment [53]. Micronuclei assays are efficient to assess the mutagenic activity of herbicides both in laboratorial and field assays [67]. The comet assay can be used to evaluate damages in proliferating cells or not, in *in vitro* or *in vivo* tests and can be applied with the purpose of genotoxicological analyses [68]. According to these same authors, these tests are considered one of the best tools to biomonitor several chemical compounds, including herbicides. According to Ribas et al. [69], the simplicity, reproducibility and rapidity of the comet test, associated to the ability of this assay in evaluating damages in the DNA, makes this

other nuclear abnormalities, besides induction of cell death [55].

of chemicals with toxic potential to biological systems.

technique highly applicable to environmental genotoxicology.

*vitro* and *in vivo* [56].

410 Herbicides - Current Research and Case Studies in Use

genic effects [63].

Atrazine is a triazinic herbicide, classified as moderately toxic of pre- and post-emergence, used for the control of weeds in crops of asparagus, corn, sorghum, sugarcane and pineapple [70]. According to Eldridge et al. [71], triazinic herbicides are among the most used pesticides in agriculture due to their ability to inhibit the photosynthesis of weeds in crops [16].

Triazine herbicides are extensively used in the United States to control grass, sedge and broadleaf weedsduring the cultivation of maize, wheat, sorghum, sugarcane and conifers [72]. In Brazil, these herbicides are widely used on crops of sugarcane and maize. Due to the widespread use of triazine herbicides in the agriculture and, therefore, its high exposure potential for humans, the United States Environmental Protection Agency (USEPA) has conducted a special review on the published and non published data of several triazine herbicides [73]. According to Nwani et al. [22], the herbicide atrazine is widely used in crops worldwide. The dangers, both toxic and genotoxic of this herbicide have been revised; however, there is an urgent need for more detailed studies on the mode of action of this compound. Atrazine has been tested in several systems, but there are shortcomings in relation to certain tests performed and some evidences of the genotoxic effects, *in vivo*, still need to be confirmed [74].

Several studies using the test system *Aspergillus* have shown that atrazine is not mutagenic to these organisms [75, 76, 77], although it is considered mutagenic for other test systems such as *Drosophila melanogaster* [78, 79]. According to Ribas et al. [74], atrazine was responsible for a significant frequency of aneuploidies in *Neurospora crassa*, given by the chromosomal nondisjunction in *Aspergillus nidulans*, and by the induction of loss of sexual chromosomes in *Drosophila melanogaster*.

Sorghum plants treated with atrazine presented an increase in the number of their chromo‐ somes, multinucleated cells, aneuploidy and polyploidy, and abnormalities in the mother cells of the pollen grain, which suggests that this herbicide interferes in the stability and also in the meiosis [80].

Popa et al. [70] observed that atrazine, when applied in high concentrations in maize seedlings, can induce chromosome breaks, visualized by the presence of single and paired chromosome fragments; a high frequency of chromatids and chromosome bridges; lagging chromosomes and presence of heteropolyploid or polyploid cells. Grant and Owens [81] showed that atrazine induced chromosome breaks (in mitosis and meiosis) in the species *Pisum sativum* and *Allium cepa.*

Hayes et al. [82] investigated the effect of the herbicide atrazine on wild leopard frogs (*Rana pipens*), in different regions of the United States. The authors observed that a great percentage of males exposed to the herbicide presented abnormalities in the gonads, such as development retardation and hermaphroditism. This effect can, in long term, lead to a decline in the amphibian population of the sites contaminated with this herbicide.

of age, promoted alterations in the levels of several hormones in the serum of these individuals, observed by slight increases in the levels of androstenedione testosterone, estradiol, estrone,

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413

To obtain more concise data on the genotoxicity of triazine herbicides, Tennant et al. [89] used the comet assay methodology, which showed to be highly sensitive for the detection of low rates of damages in the DNA. According to these authors, the comet assay showed that atrazine induced a small increase in the damages in the DNA in leukocytes of rats. Moreover, by the comet assay, Clements et al. [90] reported that atrazine induced a significant increase in the frequencies of damages in the DNA of erythrocytes of bullfrog tadpoles, noting the genotoxic potential of this herbicide for this species of amphibian, from the concentration of 4.8 mg/L. Studies about the cytotoxicity, genotoxicity and mutagenicity of the atrazine herbicide (oral gavage - dose 400 mg/kg/day), carried out by Campos-Pereira et al. [91], have shown the induction of lipid peroxidation and liver damage, death of hepatocytes, and micronucleus formation in exposed Wistar rats. Tests performed by Ventura et al. [8] showed that the same triazine pesticide was able to induce significant DNA fragmentation when using the comet assay, and nuclear alterations and micronuclei using the micronucleus test in *Oreochromis niloticus* (Nile tilapia) erythrocytes exposed to different concentrations of atrazine (6.25, 12.5,

25 μg/L), thus corroborating the studies performed by Campos-Pereira et al. [91].

and in the presence of the S9 fractions, when the strains were exposed to atrazine.

cells exposed to 1 ppm of the herbicide atrazine.

mainly at concentrations of 100 and 200 μg/l.

Ruiz and Marzin [92] assessed the genotoxic and mutagenic effects of the herbicide atrazine by two *in vitro* assays (*Salmonella* assay and SOS Chromotest), one to detect bacterial mutage‐ nicity and the other to verify primary damages in the DNA. The assays were carried out both in the absence and in the presence of S9 fractions from rat liver homogenate (Sprague-Dawley). The authors found that the herbicide atrazine did not present genotoxic potential neither to the *in vitro* test with Salmonella/microsome nor by the SOS Chromotest, both in the absence

*In vitro* studies, performed with human lymphocytes, treated with 0.10 ppm of atrazine, detected a slight increase in the chromosome aberrations rates [85]. However, for concentra‐ tions below 0.001 ppm of this herbicide, chromosome aberrations were not detected [86] Lioi et al. [93] observed a small increase in the number of sister chromatid exchange but a great increase of chromosome aberrations in human lymphocytes exposed to atrazine. Meisner et al. [94] observed a significant increase in the frequency of chromosome breaks in human blood

The genotoxicity of herbicides, such as atrazine, has also been evaluated by the comet assay by the use of human blood lymphocytes. According to Ribas et al. [69], blood cells treated with the herbicide atrazine, at concentrations of 50-200 μg/l, showed an extensive migration of DNA,

In mammalian test systems, submitted to the action of the herbicide atrazine, most of the results seem to be negative, except for the results of Loprieno and Adler [95], who obtained a significant increase in the frequency of chromosome aberrations in bone marrow cells of rats, and the data obtained by Meisner et al. [94], who described an induction of chromosome aberrations in cultured human lymphocytes. While the results from bacteria and mammal test

progesterone and corticosterone, quantified by radioimmunoassay.

According to Gammon et al. [83], some publications have reported a possible feminization of frogs, both in laboratorial assays and field studies. This effect is mainly due to the action of the enzyme aromatase; however, published research not shown the measures of this enzyme. Thus, there are doubts about the feminization theory, except for the studies that presented a great number of frogs with morphological alterations related to very high levels of atrazine.

Nwani et al. [22] evaluated the genotoxic and mutagenic effects of the herbicide Rasayanzine, whose active ingredient is atrazine, using the comet assay and micronucleus test, in erythro‐ cytes and gill cells of the fish *Channa punctatus.* By the data analysis of the two cell types, significant effects for all the concentrations (4.24, 5.30 and 8.48 mg/L) and exposure periods tested (1, 3, 5, 7, 14, 21, 28 and 35 days) were observed. The highest damages were observed for the highest concentrations and exposure times, showing the genotoxic and mutagenic doseresponse potential of atrazine for the aquatic organism. Furthermore, it was found that gills were more sensitive to the action of the herbicide, when compared to erythrocytes. From the results obtained, the authors suggested a careful and judicious use of the herbicide atrazine in order to protect the aquatic ecosystems and human population.

A study carried out by Çavas [84] compared the genotoxic effects of the active ingredient atrazine and its commercial formulation Gesaprim, in the concentrations of 5, 10 and 15 μg/L, by the comet assay and micronucleus test, in erythrocytes of the fish gibel carp (*Carassius auratus*). The results showed that there was a significant increase in the frequencies of the micronuclei and DNA strand breaks in the erythrocytes treated with all the concentrations of the commercial formulation of atrazine, showing the genotoxic and mutagenic potential of Gesaprim for this species of fish. While the commercial formulation presented a high genotoxic potential, the assays showed that the active ingredient atrazine is not genotoxic, suggesting that the adjuvants present in Gesaprim must be the responsible for the genotoxic effects observed in this species of fish. Despite the comparative analysis of the genotoxicity between the active ingredient and the commercial product has showed to be a very effective tool for the discovery of genotoxic environmental risks, it is not easy to determine the exact identity of the products used as adjuvants and of the agents of surface action of pesticides due to the existence of the patent protection system.

Atrazine has also been tested to evaluate the ability to induce cytogenetic damages in rodents. Meisner et al. [85] submitted rats to 20 ppm of atrazine (by water ingestion) and did not observe, after exposure to the herbicide, an increase in the number of chromosome aberrations. In a similar study, Roloff et al. [86] reported that there was no significant increase of chromo‐ some aberrations in cells of rat bone marrow, when they were fed with 20 ppm of atrazine.

Wu et al. [87] assessed the embryotoxic and teratogenic effects of atrazine, at the doses of 25, 100 and 200 mg/Kg/day, in Sprague-Dawley rats. Prenatal exposure to the highest dose of the herbicide tested caused hypospadias in 10.23% of male newborn rats, and the lowest dose induced diverse embryotoxic damages in some individuals. According to Modic et al. [88], high doses of atrazine (50 or 200 mg/kg/day), administered daily in male Wistar rats at 60 days of age, promoted alterations in the levels of several hormones in the serum of these individuals, observed by slight increases in the levels of androstenedione testosterone, estradiol, estrone, progesterone and corticosterone, quantified by radioimmunoassay.

retardation and hermaphroditism. This effect can, in long term, lead to a decline in the

According to Gammon et al. [83], some publications have reported a possible feminization of frogs, both in laboratorial assays and field studies. This effect is mainly due to the action of the enzyme aromatase; however, published research not shown the measures of this enzyme. Thus, there are doubts about the feminization theory, except for the studies that presented a great number of frogs with morphological alterations related to very high levels of atrazine.

Nwani et al. [22] evaluated the genotoxic and mutagenic effects of the herbicide Rasayanzine, whose active ingredient is atrazine, using the comet assay and micronucleus test, in erythro‐ cytes and gill cells of the fish *Channa punctatus.* By the data analysis of the two cell types, significant effects for all the concentrations (4.24, 5.30 and 8.48 mg/L) and exposure periods tested (1, 3, 5, 7, 14, 21, 28 and 35 days) were observed. The highest damages were observed for the highest concentrations and exposure times, showing the genotoxic and mutagenic doseresponse potential of atrazine for the aquatic organism. Furthermore, it was found that gills were more sensitive to the action of the herbicide, when compared to erythrocytes. From the results obtained, the authors suggested a careful and judicious use of the herbicide atrazine in

A study carried out by Çavas [84] compared the genotoxic effects of the active ingredient atrazine and its commercial formulation Gesaprim, in the concentrations of 5, 10 and 15 μg/L, by the comet assay and micronucleus test, in erythrocytes of the fish gibel carp (*Carassius auratus*). The results showed that there was a significant increase in the frequencies of the micronuclei and DNA strand breaks in the erythrocytes treated with all the concentrations of the commercial formulation of atrazine, showing the genotoxic and mutagenic potential of Gesaprim for this species of fish. While the commercial formulation presented a high genotoxic potential, the assays showed that the active ingredient atrazine is not genotoxic, suggesting that the adjuvants present in Gesaprim must be the responsible for the genotoxic effects observed in this species of fish. Despite the comparative analysis of the genotoxicity between the active ingredient and the commercial product has showed to be a very effective tool for the discovery of genotoxic environmental risks, it is not easy to determine the exact identity of the products used as adjuvants and of the agents of surface action of pesticides due to the

Atrazine has also been tested to evaluate the ability to induce cytogenetic damages in rodents. Meisner et al. [85] submitted rats to 20 ppm of atrazine (by water ingestion) and did not observe, after exposure to the herbicide, an increase in the number of chromosome aberrations. In a similar study, Roloff et al. [86] reported that there was no significant increase of chromo‐ some aberrations in cells of rat bone marrow, when they were fed with 20 ppm of atrazine.

Wu et al. [87] assessed the embryotoxic and teratogenic effects of atrazine, at the doses of 25, 100 and 200 mg/Kg/day, in Sprague-Dawley rats. Prenatal exposure to the highest dose of the herbicide tested caused hypospadias in 10.23% of male newborn rats, and the lowest dose induced diverse embryotoxic damages in some individuals. According to Modic et al. [88], high doses of atrazine (50 or 200 mg/kg/day), administered daily in male Wistar rats at 60 days

amphibian population of the sites contaminated with this herbicide.

412 Herbicides - Current Research and Case Studies in Use

order to protect the aquatic ecosystems and human population.

existence of the patent protection system.

To obtain more concise data on the genotoxicity of triazine herbicides, Tennant et al. [89] used the comet assay methodology, which showed to be highly sensitive for the detection of low rates of damages in the DNA. According to these authors, the comet assay showed that atrazine induced a small increase in the damages in the DNA in leukocytes of rats. Moreover, by the comet assay, Clements et al. [90] reported that atrazine induced a significant increase in the frequencies of damages in the DNA of erythrocytes of bullfrog tadpoles, noting the genotoxic potential of this herbicide for this species of amphibian, from the concentration of 4.8 mg/L.

Studies about the cytotoxicity, genotoxicity and mutagenicity of the atrazine herbicide (oral gavage - dose 400 mg/kg/day), carried out by Campos-Pereira et al. [91], have shown the induction of lipid peroxidation and liver damage, death of hepatocytes, and micronucleus formation in exposed Wistar rats. Tests performed by Ventura et al. [8] showed that the same triazine pesticide was able to induce significant DNA fragmentation when using the comet assay, and nuclear alterations and micronuclei using the micronucleus test in *Oreochromis niloticus* (Nile tilapia) erythrocytes exposed to different concentrations of atrazine (6.25, 12.5, 25 μg/L), thus corroborating the studies performed by Campos-Pereira et al. [91].

Ruiz and Marzin [92] assessed the genotoxic and mutagenic effects of the herbicide atrazine by two *in vitro* assays (*Salmonella* assay and SOS Chromotest), one to detect bacterial mutage‐ nicity and the other to verify primary damages in the DNA. The assays were carried out both in the absence and in the presence of S9 fractions from rat liver homogenate (Sprague-Dawley). The authors found that the herbicide atrazine did not present genotoxic potential neither to the *in vitro* test with Salmonella/microsome nor by the SOS Chromotest, both in the absence and in the presence of the S9 fractions, when the strains were exposed to atrazine.

*In vitro* studies, performed with human lymphocytes, treated with 0.10 ppm of atrazine, detected a slight increase in the chromosome aberrations rates [85]. However, for concentra‐ tions below 0.001 ppm of this herbicide, chromosome aberrations were not detected [86] Lioi et al. [93] observed a small increase in the number of sister chromatid exchange but a great increase of chromosome aberrations in human lymphocytes exposed to atrazine. Meisner et al. [94] observed a significant increase in the frequency of chromosome breaks in human blood cells exposed to 1 ppm of the herbicide atrazine.

The genotoxicity of herbicides, such as atrazine, has also been evaluated by the comet assay by the use of human blood lymphocytes. According to Ribas et al. [69], blood cells treated with the herbicide atrazine, at concentrations of 50-200 μg/l, showed an extensive migration of DNA, mainly at concentrations of 100 and 200 μg/l.

In mammalian test systems, submitted to the action of the herbicide atrazine, most of the results seem to be negative, except for the results of Loprieno and Adler [95], who obtained a significant increase in the frequency of chromosome aberrations in bone marrow cells of rats, and the data obtained by Meisner et al. [94], who described an induction of chromosome aberrations in cultured human lymphocytes. While the results from bacteria and mammal test systems are almost all negative, atrazine exhibits clear mutagenic effects in different plant test systems, by inducing chromosome aberrations in *Hordeum vulgare* and *Vicia faba* [96, 97], in *Zea mays* [98], in *Sorghum vulgare* [99] and in *Allium cepa* [62] ; sister chromatid exchanges in maize [100] ; and point mutation in maize [98].

On the other hand, Hrelia et al. [106] showed that males and females of Sprague-Dawley rats exposed by oral gavage to doses of 56, 112 and 224 mg/kg of cyanazine, did not present

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415

Taets et al. [107] evaluated the clastogenic potential of environmental concentrations of the triazine herbicides simazine (0.001 to 0.004 μg/mL), cyanazine (0.003 to 0.012 μg/mL) and atrazine (0.003 to 0.018 μg/mL), in Chinese Hamster Ovary (CHO) cells, using flow cytometry assay. The authors proved the clastogenic action for the herbicides atrazine and cyanazine, proven by the high indices of damages in the cells exposed to atrazine and by the significant

The herbicide terbutryn is an s-triazine herbicide used pre- and post-emergence and widely used worldwide as an agent to control grass, sedge, and broadleaf weeds in vegetables, cereals and fruit trees. It is an herbicide persistent in the environment, which tends to dislocate by the

An *in vitro* study performed by Moretti et al. [108] investigated the genotoxicity of the herbicide terbutryn, by analyzing the relationship between the cytogenetic damage, evaluated by the assays of SCE (sister chromatid exchanges) and MN (micronucleus), and the primary damage in the DNA, assessed by the comet assay, in leukocytes newly-isolated from peripheral human blood. The results showed that terbutryn did not produce significant increases of SCE or MN, both in the absence and in the presence of the metabolic activation system from rat liver (S9 fraction), although terbutryn has induced primary damages in the DNA in a more pronounced form in the absence of S9. The apparent lack of sensitivity of the assays of SCE and MN test for the genotoxicity of terbutryn, in comparison to the comet assay, can be attributed to the generation of specific types of damages, since the SCE and MN are determined in proliferative cells and are sensitive indicators of lesions that survive for, at least, one mitotic cycle, while the comet assay identifies repairable lesions in the DNA of on resting (G0) cells. According to these results, the authors suggest that terbutryn must be considered a genotoxic compound.

The 2,4-D (2,4-dichlorophenoxyacetic acid) is an herbicide from the group of the polychlori‐ nated aromatic hydrocarbons that has been widely used throughout the world [109] since 1944, to control broadleaf weeds and woody plants [110]. Its action mimics the auxin of plants [111]. According to Martínez-Tabche et al. [112], this herbicide mimics the action of the hormone indole acetic acid, when used in small quantities but it is highly cytotoxic in

According to Ateeq et al. [113], the increase in the frequency of micronuclei and altered cells was significant, when erythrocytes of catfish (*Clarias batrachus*) were analyzed, after exposure to the herbicides 2,4-D and butachlor. There was a positive dose-response relationship in all

exposures to the two herbicides and in all exposure periods tested.

significant increases in chromosome aberrations.

**2.4. Terbutryn**

flow of water and leachate [108].

**2.5. 2,4-D (2,4-dichlorophenoxyacetic acid)**

high concentrations.

frequencies of damages observed in the cells exposed to cyanazine.

Studies performed by Zeljezic et al. [101] had already reported that atrazine does not present genotoxicity or capacity to induce apoptosis or necrosis in human lymphocytes, while the treatment of these cells with the commercial formulation, Gesaprim, significantly increased the rates of damages in DNA, observed by the comet assay. Srivastava and Mishra [102] observed results that are in agreement with the findings of Zeljezic et al. [101] and Çavas [84], in which the exposure to different concentrations of Gesaprim inhibited the mitotic index and increased the frequencies of micronuclei and chromosome aberrations in somatic cells of *Allium cepa* and *Vicia faba*.

#### **2.2. Atrazine and butachlor**

Toxic effects of atrazine, alone or associated with the herbicide butachlor, for the freshwater species such as the green alga *Scenedesmus obliquus* and the cladoceran *Daphnia carinata*, were evaluated, showing values of 96 h-EC50 for *S. obliquus* (atrazine= 0.0147 mg/L and butachlor= 2.31 mg/L, and of 48h-LC50 for *D. carinata* (atrazine= 60.6 mg/L and butachlor= 3.40 mg/L) [20]. These results suggest that atrazine has a highly toxic potential for *S. obliquus* and slightly toxic for *D. carinata*, while butachlor exhibits a moderate toxic potential for both organisms. Now, the analysis of the mixture atrazine-butachlor allowed the authors to verify that the toxic effects were significantly antagonistic for *S. obliquus*, and that there was no significant synergism for *D. carinata* [20].

#### **2.3. Atrazine, simazine ande cyanazine**

Simazine and cyanazine, as well as atrazine, are widely used as triazine herbicides of pre- and post-emergence weed control, whose residues have been carried to the source of drinking water of several agricultural communities. These compounds also present a potential risk to humans, mainly due to their presence in food [103]. Studies on the effect of atrazine, simazine and cyanazine performed by Kligerman et al. [104], found that there was not a significant increase in the sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to these herbicides, up to the solubility limit in aqueous solution using 0.5% of dimethyl sulfoxide. However, Adler [105] observed that doses of 1500 and 2000 mg/Kg of atrazine, administered by oral gavage in rats, induced dominant lethal mutations and chromatin breaks in the bone marrow of these organisms.

Kligerman et al. [103] observed that the association of the herbicides atrazine, simazine and cyanazine did not induce micronuclei in polychromatic erythrocytes of bone marrow of female rats (C57B1/6) exposed by intraperitoneal injection, even when very high doses of these herbicides were administered (125, 250 and 500 mg/Kg of atrazine; 500, 1000 and 2000 mg/Kg of simazine; 100, 200 and 400 mg/kg of cyanazine), showing an absence of genotoxic potential of these compounds for the organism tested.

On the other hand, Hrelia et al. [106] showed that males and females of Sprague-Dawley rats exposed by oral gavage to doses of 56, 112 and 224 mg/kg of cyanazine, did not present significant increases in chromosome aberrations.

Taets et al. [107] evaluated the clastogenic potential of environmental concentrations of the triazine herbicides simazine (0.001 to 0.004 μg/mL), cyanazine (0.003 to 0.012 μg/mL) and atrazine (0.003 to 0.018 μg/mL), in Chinese Hamster Ovary (CHO) cells, using flow cytometry assay. The authors proved the clastogenic action for the herbicides atrazine and cyanazine, proven by the high indices of damages in the cells exposed to atrazine and by the significant frequencies of damages observed in the cells exposed to cyanazine.

### **2.4. Terbutryn**

systems are almost all negative, atrazine exhibits clear mutagenic effects in different plant test systems, by inducing chromosome aberrations in *Hordeum vulgare* and *Vicia faba* [96, 97], in *Zea mays* [98], in *Sorghum vulgare* [99] and in *Allium cepa* [62] ; sister chromatid exchanges in

Studies performed by Zeljezic et al. [101] had already reported that atrazine does not present genotoxicity or capacity to induce apoptosis or necrosis in human lymphocytes, while the treatment of these cells with the commercial formulation, Gesaprim, significantly increased the rates of damages in DNA, observed by the comet assay. Srivastava and Mishra [102] observed results that are in agreement with the findings of Zeljezic et al. [101] and Çavas [84], in which the exposure to different concentrations of Gesaprim inhibited the mitotic index and increased the frequencies of micronuclei and chromosome aberrations in somatic cells of

Toxic effects of atrazine, alone or associated with the herbicide butachlor, for the freshwater species such as the green alga *Scenedesmus obliquus* and the cladoceran *Daphnia carinata*, were evaluated, showing values of 96 h-EC50 for *S. obliquus* (atrazine= 0.0147 mg/L and butachlor= 2.31 mg/L, and of 48h-LC50 for *D. carinata* (atrazine= 60.6 mg/L and butachlor= 3.40 mg/L) [20]. These results suggest that atrazine has a highly toxic potential for *S. obliquus* and slightly toxic for *D. carinata*, while butachlor exhibits a moderate toxic potential for both organisms. Now, the analysis of the mixture atrazine-butachlor allowed the authors to verify that the toxic effects were significantly antagonistic for *S. obliquus*, and that there was no significant synergism for

Simazine and cyanazine, as well as atrazine, are widely used as triazine herbicides of pre- and post-emergence weed control, whose residues have been carried to the source of drinking water of several agricultural communities. These compounds also present a potential risk to humans, mainly due to their presence in food [103]. Studies on the effect of atrazine, simazine and cyanazine performed by Kligerman et al. [104], found that there was not a significant increase in the sister chromatid exchanges and chromosome aberrations in cultured human lymphocytes exposed to these herbicides, up to the solubility limit in aqueous solution using 0.5% of dimethyl sulfoxide. However, Adler [105] observed that doses of 1500 and 2000 mg/Kg of atrazine, administered by oral gavage in rats, induced dominant lethal mutations

Kligerman et al. [103] observed that the association of the herbicides atrazine, simazine and cyanazine did not induce micronuclei in polychromatic erythrocytes of bone marrow of female rats (C57B1/6) exposed by intraperitoneal injection, even when very high doses of these herbicides were administered (125, 250 and 500 mg/Kg of atrazine; 500, 1000 and 2000 mg/Kg of simazine; 100, 200 and 400 mg/kg of cyanazine), showing an absence of genotoxic potential

maize [100] ; and point mutation in maize [98].

414 Herbicides - Current Research and Case Studies in Use

*Allium cepa* and *Vicia faba*.

**2.2. Atrazine and butachlor**

**2.3. Atrazine, simazine ande cyanazine**

and chromatin breaks in the bone marrow of these organisms.

of these compounds for the organism tested.

*D. carinata* [20].

The herbicide terbutryn is an s-triazine herbicide used pre- and post-emergence and widely used worldwide as an agent to control grass, sedge, and broadleaf weeds in vegetables, cereals and fruit trees. It is an herbicide persistent in the environment, which tends to dislocate by the flow of water and leachate [108].

An *in vitro* study performed by Moretti et al. [108] investigated the genotoxicity of the herbicide terbutryn, by analyzing the relationship between the cytogenetic damage, evaluated by the assays of SCE (sister chromatid exchanges) and MN (micronucleus), and the primary damage in the DNA, assessed by the comet assay, in leukocytes newly-isolated from peripheral human blood. The results showed that terbutryn did not produce significant increases of SCE or MN, both in the absence and in the presence of the metabolic activation system from rat liver (S9 fraction), although terbutryn has induced primary damages in the DNA in a more pronounced form in the absence of S9. The apparent lack of sensitivity of the assays of SCE and MN test for the genotoxicity of terbutryn, in comparison to the comet assay, can be attributed to the generation of specific types of damages, since the SCE and MN are determined in proliferative cells and are sensitive indicators of lesions that survive for, at least, one mitotic cycle, while the comet assay identifies repairable lesions in the DNA of on resting (G0) cells. According to these results, the authors suggest that terbutryn must be considered a genotoxic compound.

#### **2.5. 2,4-D (2,4-dichlorophenoxyacetic acid)**

The 2,4-D (2,4-dichlorophenoxyacetic acid) is an herbicide from the group of the polychlori‐ nated aromatic hydrocarbons that has been widely used throughout the world [109] since 1944, to control broadleaf weeds and woody plants [110]. Its action mimics the auxin of plants [111]. According to Martínez-Tabche et al. [112], this herbicide mimics the action of the hormone indole acetic acid, when used in small quantities but it is highly cytotoxic in high concentrations.

According to Ateeq et al. [113], the increase in the frequency of micronuclei and altered cells was significant, when erythrocytes of catfish (*Clarias batrachus*) were analyzed, after exposure to the herbicides 2,4-D and butachlor. There was a positive dose-response relationship in all exposures to the two herbicides and in all exposure periods tested.

Studies carried out by Suwalsky et al. [114] in nerve cells of *Caudiverbera caudiverbera* demon‐ strated the toxicity of the herbicide 2,4-D. The authors observed a reduction in the dosedependent response to nerve stimulation in the simpact junction of the frog when they were exposed to this herbicide. This reduction is probably due to a mechanism of lipid perturbation and interference in the properties of the plasma membrane, such as protein conformation and/ or interaction with protein receptors, which leads to an inhibition of the glandular chloride channel from the mucosal skin of this test organism.

herbicide atrazine in relation to 2,4-D, showing that atrazine is potentially more teratogenic

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The estrogenic potential of 4 herbicides (triclopyr; 2,4-D; diquat dibromide and glyphosate), was evaluated by the *in vivo* de vitellogenin assay with rainbow trout. A significant estrogenic potential was shown for 2,4-D, since it induced a 93 fold increase in the levels of plasma

Glyphosate is a non-selective organophosphorus, broad spectrum, post-emergence herbicide, widely used in agriculture, mainly to control grasses, sedges, and broadleaf weeds [121]. Its action occurs by the inhibition of the biosynthesis of aromatic amino acids [122]. Its main mode of action is by the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is essential in plants for the synthesis of the referred amino acids. Since this enzyme is absent in animals, this herbicide should be relatively non toxic for these organisms [123]. There are many conflicting data on the toxicity of glyphosate and its commercial

According to Solomon and Thompson [124], environmental toxicology of glyphosate has been extensively reviewed by a series of international regulatory agencies. According to the authors, as glyphosate binds strongly with organic matter, it is considered immobile in soils and sediments. This binding also removes glyphosate from water, reducing efficiently, the exposure of aquatic organisms. As the acute exposures are most likely to occur, the measures of effect are the most adequate for the purpose of risk assessment. However, in general, the authors affirm that glyphosate presents a low potential of acute toxicity for wild animals,

Williams et al. [125] carried out a critical review on the toxicity of the herbicide RoundUp™ and of its active ingredient glyphosate. The analysis of the toxicity data, carried out by pattern tests and evaluation criteria, indicated that there is no evidence that glyphosate causes direct damages in the DNA, both in assays performed *in vitro* and *in vivo*. The authors concluded that Roundup™ and its components do not represent a risk for the induction of inheritable/ somatic mutations in humans. Furthermore, the authors assert that, by the studies performed, glyphosate is not carcinogenic or teratogenic, nor does it cause significant adverse effects in the reproduction, development or in the endocrine system of humans and other mammals and,

A study on the impact of the herbicide glyphosate and its commercial formulation Roundup™, in three microorganisms of food interest (*Geotrichum candidum*, *Lactococcus lactis subsp. cremoris* and *Lactobacillus delbrueckii subsp. bulgaricus*), showed that Roundup™ has an inhibi‐ tory effect on the microbial growth and a microbiocide effect at concentrations lower than the recommended for agricultural use. It was also observed in this study that glyphosate did not induce significant toxic effects for the three microorganisms studied. These differences between the toxic actions resulted from Roundup™ and glyphosate could be explained by a possible amplified effect of the commercial formulation due to the presence, according to Cox

vitellogenin of the fish treated with this herbicide during 7 days [120].

including mammals, birds, fish and aquatic invertebrates.

therefore, does not represent a risk for the health of human beings.

than 2,4-D, for frog embryos.

**2.6. Glyphosate**

formulations.

According to Gómez et al. [115], the main and most common entrance route of 2,4-D in fish is through gills. This herbicide can cause several adverse symptoms to these organisms, such as bleeding, increased damage to the kidneys and renal functions, as well as hepatic degeneration.

Martínez-Tabche et al. [112] evaluated the toxicity of different concentrations of the herbicides 2,4-D and paraquat (0, 5, 75 and 150 mg/L), using several assays (acute lethality test, lipid peroxidation assay by quantification of MDA – Malondialdehyde – and comet assay) in rainbow trout (*Oncorhynchus mykiss*). For the acute lethality tests, it was observed a more evident toxic action for the organisms exposed to the treatment of 24 h with the herbicide paraquat, which presented high indices of mortality, analyzed by the values of LC50 (LC50 of paraquat = 0.084 mg/L; LC50 of 2,4-D = 362.38 mg/L). The authors also showed that individuals exposed to the two higher concentrations of both herbicides had apnoea and white spots in their scales. All concentrations of 2,4-D and paraquat induced a significant increase in the DNA damages and the amount of MDA in the gills exposed.

González et al. [116] proved the genotoxicity of 2,4-D due to a significant increase of SCE in CHO cells treated with the concentrations of 2 to 4 ug/mL of this herbicide. Madrigal-Bujaidar et al. [117] also showed the genotoxic potential of 2,4-D, due to a clastogenc effect of this herbicide at the doses of 100 and 200 mg/Kg, detected by a significant increase of SCE in bone marrow cells and germ cells of rats. Soloneski et al. [118] studied the genotoxic effects of different concentrations (0, 10, 25, 50 and 100 mg/mL) of the herbicide 2,4-D (2,4-dichlorophe‐ noxyacetic) and its commercial derivative 2,4-D DMA (Dimethylamine 2,4-D salt), by the SCE assay and analyses of cell cycle progression and mitotic index human lymphocytes maintained in culture, in the presence (human whole blood - WBC) and absence (plasma leukocyte cultures - PLC) of erythrocytes. These compounds did not induce significant frequencies of SCE and only the concentration of 100 mg/mL of 2,4-D caused alterations in the progression of the cell cycle in PLC, while the different concentrations of 2,4-D and 2,4-D DMA induced a significant increase in the frequency of SCE and a significant delay in the cell proliferation rates in WBC. Moreover, both 2,4-D and 2,4-D DMA presented a dose-response inhibition of the mitotic activity in PLC and WBC. Based on these results, the authors concluded that the herbicide and its commercial derivative presented genotoxic potential, which was higher in the presence of human erythrocytes.

Morgan et al. [119] showed, by embryotoxicity and teratogenicity assays carried out with *Xenopus* (FETAX - frog embryo teratogenic assay – *Xenopus*), that high concentrations of 2,4- D, induce potentially more embryotoxic effects than teratogenic in frog embryos, demonstrat‐ ed by the values of EC50 and LC50 of 245 mg/L and 254 mg/L, respectively, and by the Teratogenic Index of 1.04. Moreover, the same authors compared the teratogenic action of the herbicide atrazine in relation to 2,4-D, showing that atrazine is potentially more teratogenic than 2,4-D, for frog embryos.

The estrogenic potential of 4 herbicides (triclopyr; 2,4-D; diquat dibromide and glyphosate), was evaluated by the *in vivo* de vitellogenin assay with rainbow trout. A significant estrogenic potential was shown for 2,4-D, since it induced a 93 fold increase in the levels of plasma vitellogenin of the fish treated with this herbicide during 7 days [120].

### **2.6. Glyphosate**

Studies carried out by Suwalsky et al. [114] in nerve cells of *Caudiverbera caudiverbera* demon‐ strated the toxicity of the herbicide 2,4-D. The authors observed a reduction in the dosedependent response to nerve stimulation in the simpact junction of the frog when they were exposed to this herbicide. This reduction is probably due to a mechanism of lipid perturbation and interference in the properties of the plasma membrane, such as protein conformation and/ or interaction with protein receptors, which leads to an inhibition of the glandular chloride

According to Gómez et al. [115], the main and most common entrance route of 2,4-D in fish is through gills. This herbicide can cause several adverse symptoms to these organisms, such as bleeding, increased damage to the kidneys and renal functions, as well as hepatic degeneration. Martínez-Tabche et al. [112] evaluated the toxicity of different concentrations of the herbicides 2,4-D and paraquat (0, 5, 75 and 150 mg/L), using several assays (acute lethality test, lipid peroxidation assay by quantification of MDA – Malondialdehyde – and comet assay) in rainbow trout (*Oncorhynchus mykiss*). For the acute lethality tests, it was observed a more evident toxic action for the organisms exposed to the treatment of 24 h with the herbicide paraquat, which presented high indices of mortality, analyzed by the values of LC50 (LC50 of paraquat = 0.084 mg/L; LC50 of 2,4-D = 362.38 mg/L). The authors also showed that individuals exposed to the two higher concentrations of both herbicides had apnoea and white spots in their scales. All concentrations of 2,4-D and paraquat induced a significant increase in the DNA

González et al. [116] proved the genotoxicity of 2,4-D due to a significant increase of SCE in CHO cells treated with the concentrations of 2 to 4 ug/mL of this herbicide. Madrigal-Bujaidar et al. [117] also showed the genotoxic potential of 2,4-D, due to a clastogenc effect of this herbicide at the doses of 100 and 200 mg/Kg, detected by a significant increase of SCE in bone marrow cells and germ cells of rats. Soloneski et al. [118] studied the genotoxic effects of different concentrations (0, 10, 25, 50 and 100 mg/mL) of the herbicide 2,4-D (2,4-dichlorophe‐ noxyacetic) and its commercial derivative 2,4-D DMA (Dimethylamine 2,4-D salt), by the SCE assay and analyses of cell cycle progression and mitotic index human lymphocytes maintained in culture, in the presence (human whole blood - WBC) and absence (plasma leukocyte cultures - PLC) of erythrocytes. These compounds did not induce significant frequencies of SCE and only the concentration of 100 mg/mL of 2,4-D caused alterations in the progression of the cell cycle in PLC, while the different concentrations of 2,4-D and 2,4-D DMA induced a significant increase in the frequency of SCE and a significant delay in the cell proliferation rates in WBC. Moreover, both 2,4-D and 2,4-D DMA presented a dose-response inhibition of the mitotic activity in PLC and WBC. Based on these results, the authors concluded that the herbicide and its commercial derivative presented genotoxic potential, which was higher in the presence of

Morgan et al. [119] showed, by embryotoxicity and teratogenicity assays carried out with *Xenopus* (FETAX - frog embryo teratogenic assay – *Xenopus*), that high concentrations of 2,4- D, induce potentially more embryotoxic effects than teratogenic in frog embryos, demonstrat‐ ed by the values of EC50 and LC50 of 245 mg/L and 254 mg/L, respectively, and by the Teratogenic Index of 1.04. Moreover, the same authors compared the teratogenic action of the

channel from the mucosal skin of this test organism.

416 Herbicides - Current Research and Case Studies in Use

damages and the amount of MDA in the gills exposed.

human erythrocytes.

Glyphosate is a non-selective organophosphorus, broad spectrum, post-emergence herbicide, widely used in agriculture, mainly to control grasses, sedges, and broadleaf weeds [121]. Its action occurs by the inhibition of the biosynthesis of aromatic amino acids [122]. Its main mode of action is by the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is essential in plants for the synthesis of the referred amino acids. Since this enzyme is absent in animals, this herbicide should be relatively non toxic for these organisms [123]. There are many conflicting data on the toxicity of glyphosate and its commercial formulations.

According to Solomon and Thompson [124], environmental toxicology of glyphosate has been extensively reviewed by a series of international regulatory agencies. According to the authors, as glyphosate binds strongly with organic matter, it is considered immobile in soils and sediments. This binding also removes glyphosate from water, reducing efficiently, the exposure of aquatic organisms. As the acute exposures are most likely to occur, the measures of effect are the most adequate for the purpose of risk assessment. However, in general, the authors affirm that glyphosate presents a low potential of acute toxicity for wild animals, including mammals, birds, fish and aquatic invertebrates.

Williams et al. [125] carried out a critical review on the toxicity of the herbicide RoundUp™ and of its active ingredient glyphosate. The analysis of the toxicity data, carried out by pattern tests and evaluation criteria, indicated that there is no evidence that glyphosate causes direct damages in the DNA, both in assays performed *in vitro* and *in vivo*. The authors concluded that Roundup™ and its components do not represent a risk for the induction of inheritable/ somatic mutations in humans. Furthermore, the authors assert that, by the studies performed, glyphosate is not carcinogenic or teratogenic, nor does it cause significant adverse effects in the reproduction, development or in the endocrine system of humans and other mammals and, therefore, does not represent a risk for the health of human beings.

A study on the impact of the herbicide glyphosate and its commercial formulation Roundup™, in three microorganisms of food interest (*Geotrichum candidum*, *Lactococcus lactis subsp. cremoris* and *Lactobacillus delbrueckii subsp. bulgaricus*), showed that Roundup™ has an inhibi‐ tory effect on the microbial growth and a microbiocide effect at concentrations lower than the recommended for agricultural use. It was also observed in this study that glyphosate did not induce significant toxic effects for the three microorganisms studied. These differences between the toxic actions resulted from Roundup™ and glyphosate could be explained by a possible amplified effect of the commercial formulation due to the presence, according to Cox [126] of adjuvants, such as polyethoxylated tallowamine (POEA), used for a better stability and penetration of the chemical compound [127].

The oral administration of high doses of glyphosate (3500 mg/Kg) in Charles River COBS CD rats, between the 6th to the 19th day of pregnancy, and in rabbits, between the 6th to the 27th day of pregnancy, showed significant indices of maternal mortality for both species, as well as increase in the number of foetuses with reduced ossification of sternebrae [131], proving the toxicity and teratogenicity of this concentration of the herbicide for the organisms tested.

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Relyea [132] performed a study to observe the impact of two herbicides (glyphosate and 2,4- D) in the biodiversity of aquatic communities containing algae and more 25 species of animals. In this study the author observed that 2,4-D did not cause great impacts in the community and this is in agreement with previous studies that showed that this substance presents high LC-50 for several species. However, glyphosate had great impact in the community, causing a decrease of 22% of the species richness, while 2,4-D did not cause effects on this diversity. The authors also observed that neither of the two herbicides caused reduction in the periphyton

Reglone is a bypiridylium herbicide, whose active ingredient is diquat (1,1'-ethylene -2,2' ipyridyl dibromide), and of foliar application, used to eliminate weeds of different crops [133]. Reglone, in the concentrations tested (0.005, 0.01, 0.05 and 0.1% of the active ingredient for *Crepis capillaris* L.; 34.17 and 8.5 mg/Kg for mouse bone marrow polychromatic erythrocytes - PCEs), did not induce chromosome aberrations in any test system but promoted an increase in the frequency of micronuclei in both plant cells and PCEs [129], and thus is considered a

The herbicide Stomp 330, belongs to the dinitroanilines class, whose active ingredient is pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine], it is applied as a systematic selective herbicide of the soil [133]. The responses of the two test systems for Stomp were very different: the concentrations tested (0.005, 0.1, 0.2 and 0.4% of the active ingredient for *Crepis capillaris* L.; 122.2, 244.5 and 489 mg/Kg for rats - PCEs) did not cause significant increases in the frequencies of chromosome aberrations in plant cells, but increased its incidence in rat cells, moreover, it induced an increase in the frequency of micronuclei in both test systems. This could be explained by the proven aneugenic effect of this herbicide, since all the concentrations

Paraquat (1,1'-dimethyl-4-4'-bipyridium dichloride) is a non-selective herbicide with fast action, widely used worldwide, mainly in the pre-harvest of cotton and potato and also to control a broad spectrum of weeds [134, 135, 136]. According to Tortorelli et al. [134], paraquat is able to modify the activity of several enzymes of fish, affecting the cardiac contraction and

potential mutagenic herbicide for these test organisms.

tested produced C-mitoses in the assays with PCEs [129].

**2.7. 2,4-D and glyphosate**

biomass.

**2.8. Diquat**

**2.9. Pendimethalin**

**2.10. Paraquat**

Relyea [128] assessed the toxic potential of environmentally relevant concentrations of glyphosate on three species of tadpoles (wood frog [*Rana sylvatica* or *sylvaticus Lithobates*], leopard frog [*Rana pipiens pipiens* or L.], and American toad [*Bufo americanus* or *Anaxyrus americanus*] ), by morphological analysis of individuals, before and after the application of the herbicide, showing that there is a significant induction of morphological alterations in the tadpoles of the three species. Specifically in the case of the wood frog and leopard frog, the exposure to the chemical compound has led to an evident alteration of the size of the tadpole tail, suggesting that the herbicide could be activating physiological mechanisms of develop‐ ment that are normally used as defence responses against predators. These results showed that glyphosate can have widespread and relevant effects on non target species, contradicting other studies, such as the one performed by Solomon and Thompson [124], who affirmed the inexistence or irrelevance of the toxicity of this compound on organisms and the environment.

Studies on the genotoxic potential of the active ingredient glyphosate, present in the commer‐ cial formulation Roundup, were performed on the roots of smooth hawksbeard (*Crepis capillaris* L.), in the concentrations of 0.05, 0.1, 0.5 and 1.0% of the active ingredient and for polychromatic erythrocytes (PCEs) of the bone marrow of C57BL rat, at doses inferior to half the LD50 (1080 mg/Kg). In these studies the chromosome aberrations assay and micronucleus test were used, which showed that this chemical compound did not induce significant responses for any of the biological systems tested [129].

Martini et al. [123] studied the effects of the commercial formulation of glyphosate in the proliferation, survival and differentiation of the 3T3-L1 fibroblasts (a mammal cell line), by the cell viability test with Trypan, MTT test, enzymatic activity assay of caspase-3 and staining assay with annexin-V and propidium iodide. The results showed that glyphosate inhibits the cell proliferation and induces apoptosis in a dose-dependent way, besides decreasing signifi‐ cantly the ability of the fibroblasts to differentiate to adipocytes. These data suggest the occurrence of important cell damages mediated by the action of this herbicide, indicating that glyphosate presents a potential risk factor for human health and the environment.

Dallegrave et al. [130] evaluated the teratogenicity of the herbicide glyphosate, marketed in Brazil as Roundup (36% of glyphosate and 18% of the surfactant polyoxyethyleneamine), to females of Wistar rats. The females treated orally with three different doses of glyphosate (500, 750, 1000 mg/Kg) from the 6th to the 15th day of gestation. After performing caesarean sections on day 21 of gestation, the number of corpora lutea, implantations, live and dead foetuses and reabsorptions, as well as the external malformations and skeletal malformation were recorded and analyzed. It was observed a mortality rate of 50% of the females treated with the highest concentration of glyphosate; the authors verified that there was a dose-response relationship directly proportional to the increase in the number of skeletal alterations found. These results led the authors to conclude that the commercial formulation of glyphosate (Roundup) is toxic for females of Wistar rats and is able to induce a delay in the fetal skeletal development of this species. It is important to consider that the toxicity and teratogenicity observed can result from both the action of glyphosate as well asthe surfactant present in the commercial formulation.

The oral administration of high doses of glyphosate (3500 mg/Kg) in Charles River COBS CD rats, between the 6th to the 19th day of pregnancy, and in rabbits, between the 6th to the 27th day of pregnancy, showed significant indices of maternal mortality for both species, as well as increase in the number of foetuses with reduced ossification of sternebrae [131], proving the toxicity and teratogenicity of this concentration of the herbicide for the organisms tested.

#### **2.7. 2,4-D and glyphosate**

Relyea [132] performed a study to observe the impact of two herbicides (glyphosate and 2,4- D) in the biodiversity of aquatic communities containing algae and more 25 species of animals. In this study the author observed that 2,4-D did not cause great impacts in the community and this is in agreement with previous studies that showed that this substance presents high LC-50 for several species. However, glyphosate had great impact in the community, causing a decrease of 22% of the species richness, while 2,4-D did not cause effects on this diversity. The authors also observed that neither of the two herbicides caused reduction in the periphyton biomass.

### **2.8. Diquat**

[126] of adjuvants, such as polyethoxylated tallowamine (POEA), used for a better stability

Relyea [128] assessed the toxic potential of environmentally relevant concentrations of glyphosate on three species of tadpoles (wood frog [*Rana sylvatica* or *sylvaticus Lithobates*], leopard frog [*Rana pipiens pipiens* or L.], and American toad [*Bufo americanus* or *Anaxyrus americanus*] ), by morphological analysis of individuals, before and after the application of the herbicide, showing that there is a significant induction of morphological alterations in the tadpoles of the three species. Specifically in the case of the wood frog and leopard frog, the exposure to the chemical compound has led to an evident alteration of the size of the tadpole tail, suggesting that the herbicide could be activating physiological mechanisms of develop‐ ment that are normally used as defence responses against predators. These results showed that glyphosate can have widespread and relevant effects on non target species, contradicting other studies, such as the one performed by Solomon and Thompson [124], who affirmed the inexistence or irrelevance of the toxicity of this compound on organisms and the environment. Studies on the genotoxic potential of the active ingredient glyphosate, present in the commer‐ cial formulation Roundup, were performed on the roots of smooth hawksbeard (*Crepis capillaris* L.), in the concentrations of 0.05, 0.1, 0.5 and 1.0% of the active ingredient and for polychromatic erythrocytes (PCEs) of the bone marrow of C57BL rat, at doses inferior to half the LD50 (1080 mg/Kg). In these studies the chromosome aberrations assay and micronucleus test were used, which showed that this chemical compound did not induce significant

Martini et al. [123] studied the effects of the commercial formulation of glyphosate in the proliferation, survival and differentiation of the 3T3-L1 fibroblasts (a mammal cell line), by the cell viability test with Trypan, MTT test, enzymatic activity assay of caspase-3 and staining assay with annexin-V and propidium iodide. The results showed that glyphosate inhibits the cell proliferation and induces apoptosis in a dose-dependent way, besides decreasing signifi‐ cantly the ability of the fibroblasts to differentiate to adipocytes. These data suggest the occurrence of important cell damages mediated by the action of this herbicide, indicating that

Dallegrave et al. [130] evaluated the teratogenicity of the herbicide glyphosate, marketed in Brazil as Roundup (36% of glyphosate and 18% of the surfactant polyoxyethyleneamine), to females of Wistar rats. The females treated orally with three different doses of glyphosate (500, 750, 1000 mg/Kg) from the 6th to the 15th day of gestation. After performing caesarean sections on day 21 of gestation, the number of corpora lutea, implantations, live and dead foetuses and reabsorptions, as well as the external malformations and skeletal malformation were recorded and analyzed. It was observed a mortality rate of 50% of the females treated with the highest concentration of glyphosate; the authors verified that there was a dose-response relationship directly proportional to the increase in the number of skeletal alterations found. These results led the authors to conclude that the commercial formulation of glyphosate (Roundup) is toxic for females of Wistar rats and is able to induce a delay in the fetal skeletal development of this species. It is important to consider that the toxicity and teratogenicity observed can result from both the action of glyphosate as well asthe surfactant present in the commercial formulation.

glyphosate presents a potential risk factor for human health and the environment.

and penetration of the chemical compound [127].

418 Herbicides - Current Research and Case Studies in Use

responses for any of the biological systems tested [129].

Reglone is a bypiridylium herbicide, whose active ingredient is diquat (1,1'-ethylene -2,2' ipyridyl dibromide), and of foliar application, used to eliminate weeds of different crops [133]. Reglone, in the concentrations tested (0.005, 0.01, 0.05 and 0.1% of the active ingredient for *Crepis capillaris* L.; 34.17 and 8.5 mg/Kg for mouse bone marrow polychromatic erythrocytes - PCEs), did not induce chromosome aberrations in any test system but promoted an increase in the frequency of micronuclei in both plant cells and PCEs [129], and thus is considered a potential mutagenic herbicide for these test organisms.

### **2.9. Pendimethalin**

The herbicide Stomp 330, belongs to the dinitroanilines class, whose active ingredient is pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine], it is applied as a systematic selective herbicide of the soil [133]. The responses of the two test systems for Stomp were very different: the concentrations tested (0.005, 0.1, 0.2 and 0.4% of the active ingredient for *Crepis capillaris* L.; 122.2, 244.5 and 489 mg/Kg for rats - PCEs) did not cause significant increases in the frequencies of chromosome aberrations in plant cells, but increased its incidence in rat cells, moreover, it induced an increase in the frequency of micronuclei in both test systems. This could be explained by the proven aneugenic effect of this herbicide, since all the concentrations tested produced C-mitoses in the assays with PCEs [129].

#### **2.10. Paraquat**

Paraquat (1,1'-dimethyl-4-4'-bipyridium dichloride) is a non-selective herbicide with fast action, widely used worldwide, mainly in the pre-harvest of cotton and potato and also to control a broad spectrum of weeds [134, 135, 136]. According to Tortorelli et al. [134], paraquat is able to modify the activity of several enzymes of fish, affecting the cardiac contraction and opercular ventilation, effects that can alter the initial development of these organisms. According to Tomita et al. [137], paraquat causes oxidative stress in different species of fish by generating elevated levels of superoxide ion.

rat liver), but the data on the chromosome aberrations and micronuclei assays were not significant, which led the authors to conclude that paraquat is an inductor of primary damages

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A study performed by Hoffman and Eastin [143] evaluated the embryotoxic and teratogenic effects of two insecticides (lindane and toxaphene) and two herbicides (paraquat and 2,4,5-T), by external treatment of eggs of mallard duck (*Anas platyrhynchos*), using concentrations of field application. The authors showed that paraquat was the most significantly embryotoxic compound for this organism, independent of the type of vehicle in which the herbicide was associated, besides proving that paraquat impaired the growth of the organisms and was slightly teratogenic. The LC50 for this species was 1.5 Kg of the active ingredient/hectare in aqueous emulsion and 1 lb/acre in oil vehicles. When the organisms treated with paraquat were compared to the ones exposed to the herbicide 2,4,5-T, they presented little damages and

The harmful effects of herbicides on human health are determined by several factors, such as the chemical class of those compounds, dose, time, and exposure route. Herbicides can be toxic to humans at high and lower doses [144]. The prolonged exposure can lead to a number of health effects, including the induction of diseases such as cancer and neurodegenerative [145,

Doll and Peto [149] estimated that 35% of all cases of cancer in the U.S. population originate from diet, and the herbicides present in foods are responsible. Estrogenicity assays made by Hernández et al. [150] show that organochlorine pesticides may act as endocrine disruption through more than one mechanism, including agonist or antagonist effects of different receptors. Chloro-s-triazize herbicides, pre-emergent pesticides used worldwide, have been generally considered as chemical compounds of low toxic potential for humans; however, there are many controversies on this issue. According to several international agencies, including the Environmental Protection Agency (EPA), Development for Environmental Assessment Center of the United States and IARC Monographs (International Agency for Research on Cancer), the herbicide atrazine, for example, was classified as a chemical agent probably carcinogenic to humans, although the basis for this conclusion is only evidenced in other animals [151, 152]. Due to the fact that atrazine induce mammary tumours in female Sprague-Dawley rats, the Peer Review Committee of the EPA Office of Pesticide Program (OPP) also concluded that atrazine should be considered in the Possibly Carcinogenic to Humans Group [153]. However, EPA [154] has classified this herbicide as a compound

Some experimental studies have shown that exposure of humans to high doses of atrazine can result in an increased loss of body weight. However, a great number of epidemiological studies carried out with workers occupationally exposed to triazine herbicides indicate that these compounds do not have carcinogenic potential for these individuals. By analyses of different

146], reproductive and developmental changes [147] and respiratory effects [148].

in the DNA, although they have not shown that it has a clastogenic action.

it was observed few individuals bearing severe defects.

probably non carcinogenic to humans.

**3. Harmful effects of herbicides on human health**

A study conducted by D'Souza et al. [138] evaluated the toxicity of the herbicide paraquat for germ cells of male Sprague-Dawley rats by dermal exposure to this chemical. The authors verified that paraquat, even at low doses, significantly reduced the amount of spermatozoa, increased the frequency of spermatozoa bearing abnormalities and the mortality rate of these germ cells, as well as affected the mobility of the spermatozoa of the individuals studied, showing that the herbicide is a cytotoxic and genotoxic agent for the germ cells of this organism.

Hanada [136], analyzing the karyotype of species of *Rana ornativentris*, after exposure for 6 hours to the herbicide paraquat at the concentrations of 10-8 to 10-6 M, showed that this compound is able to induce genotoxic effects in this organism. The author observed that paraquat promoted, in a dose-dependent manner, a significant increase in the quantity of chromosome breaks in leukocytes of this test organism, suggesting that this species of anuran is highly sensitive to the genotoxic action of the herbicide.

According to Bus et al. [139], the genotoxic action of paraquat may be associated with the transference of a single electron of reduced oxygen to paraquat, forming superoxide ions. The singlet oxygen can be formed from the superoxide ion and subsequently react with lipids to form hydroperoxides and fatty acids. According to Tanaka and Amano [140], lipid peroxida‐ tion is responsible for the origin of several chromosome aberrations. Bauer Dial and Dial [141] still affirm that the oxidative stress induced by paraquat may be related to the teratogenic action of this compound to embryos and tadpoles of anurans.

Speit et al. [142] evaluated the genotoxic potential of the herbicide paraquat in Chinese hamster V79 cells, by chromosome aberrations and comet assays. Using a modified protocol of the comet assay with the modified protein FPG (formamidopyrimidine-DNA glycosylase), a repair enzyme that specifically nicks the DNA at sites of 8-oxo-guanines and formamidopyr‐ imidines, it was not possible to detect oxidative damages in the bases of DNA after treatment with paraquat. Now, when the cells were treated directly on the slides, after lysis (i.e., after the cell membrane barrier has been eliminated), a significant increase in the migration of DNA was observed, only after treatment with high concentrations of the herbicide. Thus, the authors verified that the herbicide induced chromosome aberrations but was not able to induce relevant DNA lesions to promote mutations in the gene HPRT in cultured V79 cells.

Ribas et al. [135] assessed the cytotoxic, genotoxic and mutagenic potentials of different concentrations of the herbicide paraquat (0, 1, 5, 25, 50, 100, 250, 500, 1000, 2000 and 4000 μg/ mL), by the assays of SCE, chromosome aberrations and micronuclei, in lymphocytes main‐ tained in culture. The results showed that paraquat is an agent that induces cytotoxicity for lymphocytes, since it promoted the reduction in the nuclear division rate in all the concentra‐ tions tested and a significant decrease in the cell proliferation rates, when the cells were exposed to the highest concentration of the herbicide. In relation to the genotoxicity, the herbicide induced a significant increase in the frequencies of SCE of the lymphocytes treated, whose damage was not modified by co-treatment with the metabolic activation (S9 fraction of rat liver), but the data on the chromosome aberrations and micronuclei assays were not significant, which led the authors to conclude that paraquat is an inductor of primary damages in the DNA, although they have not shown that it has a clastogenic action.

A study performed by Hoffman and Eastin [143] evaluated the embryotoxic and teratogenic effects of two insecticides (lindane and toxaphene) and two herbicides (paraquat and 2,4,5-T), by external treatment of eggs of mallard duck (*Anas platyrhynchos*), using concentrations of field application. The authors showed that paraquat was the most significantly embryotoxic compound for this organism, independent of the type of vehicle in which the herbicide was associated, besides proving that paraquat impaired the growth of the organisms and was slightly teratogenic. The LC50 for this species was 1.5 Kg of the active ingredient/hectare in aqueous emulsion and 1 lb/acre in oil vehicles. When the organisms treated with paraquat were compared to the ones exposed to the herbicide 2,4,5-T, they presented little damages and it was observed few individuals bearing severe defects.

### **3. Harmful effects of herbicides on human health**

opercular ventilation, effects that can alter the initial development of these organisms. According to Tomita et al. [137], paraquat causes oxidative stress in different species of fish by

A study conducted by D'Souza et al. [138] evaluated the toxicity of the herbicide paraquat for germ cells of male Sprague-Dawley rats by dermal exposure to this chemical. The authors verified that paraquat, even at low doses, significantly reduced the amount of spermatozoa, increased the frequency of spermatozoa bearing abnormalities and the mortality rate of these germ cells, as well as affected the mobility of the spermatozoa of the individuals studied, showing that the herbicide is a cytotoxic and genotoxic agent for the germ cells of this organism.

Hanada [136], analyzing the karyotype of species of *Rana ornativentris*, after exposure for 6 hours to the herbicide paraquat at the concentrations of 10-8 to 10-6 M, showed that this compound is able to induce genotoxic effects in this organism. The author observed that paraquat promoted, in a dose-dependent manner, a significant increase in the quantity of chromosome breaks in leukocytes of this test organism, suggesting that this species of anuran

According to Bus et al. [139], the genotoxic action of paraquat may be associated with the transference of a single electron of reduced oxygen to paraquat, forming superoxide ions. The singlet oxygen can be formed from the superoxide ion and subsequently react with lipids to form hydroperoxides and fatty acids. According to Tanaka and Amano [140], lipid peroxida‐ tion is responsible for the origin of several chromosome aberrations. Bauer Dial and Dial [141] still affirm that the oxidative stress induced by paraquat may be related to the teratogenic

Speit et al. [142] evaluated the genotoxic potential of the herbicide paraquat in Chinese hamster V79 cells, by chromosome aberrations and comet assays. Using a modified protocol of the comet assay with the modified protein FPG (formamidopyrimidine-DNA glycosylase), a repair enzyme that specifically nicks the DNA at sites of 8-oxo-guanines and formamidopyr‐ imidines, it was not possible to detect oxidative damages in the bases of DNA after treatment with paraquat. Now, when the cells were treated directly on the slides, after lysis (i.e., after the cell membrane barrier has been eliminated), a significant increase in the migration of DNA was observed, only after treatment with high concentrations of the herbicide. Thus, the authors verified that the herbicide induced chromosome aberrations but was not able to induce

relevant DNA lesions to promote mutations in the gene HPRT in cultured V79 cells.

Ribas et al. [135] assessed the cytotoxic, genotoxic and mutagenic potentials of different concentrations of the herbicide paraquat (0, 1, 5, 25, 50, 100, 250, 500, 1000, 2000 and 4000 μg/ mL), by the assays of SCE, chromosome aberrations and micronuclei, in lymphocytes main‐ tained in culture. The results showed that paraquat is an agent that induces cytotoxicity for lymphocytes, since it promoted the reduction in the nuclear division rate in all the concentra‐ tions tested and a significant decrease in the cell proliferation rates, when the cells were exposed to the highest concentration of the herbicide. In relation to the genotoxicity, the herbicide induced a significant increase in the frequencies of SCE of the lymphocytes treated, whose damage was not modified by co-treatment with the metabolic activation (S9 fraction of

generating elevated levels of superoxide ion.

420 Herbicides - Current Research and Case Studies in Use

is highly sensitive to the genotoxic action of the herbicide.

action of this compound to embryos and tadpoles of anurans.

The harmful effects of herbicides on human health are determined by several factors, such as the chemical class of those compounds, dose, time, and exposure route. Herbicides can be toxic to humans at high and lower doses [144]. The prolonged exposure can lead to a number of health effects, including the induction of diseases such as cancer and neurodegenerative [145, 146], reproductive and developmental changes [147] and respiratory effects [148].

Doll and Peto [149] estimated that 35% of all cases of cancer in the U.S. population originate from diet, and the herbicides present in foods are responsible. Estrogenicity assays made by Hernández et al. [150] show that organochlorine pesticides may act as endocrine disruption through more than one mechanism, including agonist or antagonist effects of different receptors. Chloro-s-triazize herbicides, pre-emergent pesticides used worldwide, have been generally considered as chemical compounds of low toxic potential for humans; however, there are many controversies on this issue. According to several international agencies, including the Environmental Protection Agency (EPA), Development for Environmental Assessment Center of the United States and IARC Monographs (International Agency for Research on Cancer), the herbicide atrazine, for example, was classified as a chemical agent probably carcinogenic to humans, although the basis for this conclusion is only evidenced in other animals [151, 152]. Due to the fact that atrazine induce mammary tumours in female Sprague-Dawley rats, the Peer Review Committee of the EPA Office of Pesticide Program (OPP) also concluded that atrazine should be considered in the Possibly Carcinogenic to Humans Group [153]. However, EPA [154] has classified this herbicide as a compound probably non carcinogenic to humans.

Some experimental studies have shown that exposure of humans to high doses of atrazine can result in an increased loss of body weight. However, a great number of epidemiological studies carried out with workers occupationally exposed to triazine herbicides indicate that these compounds do not have carcinogenic potential for these individuals. By analyses of different studies, it was observed that, although the chloro-s-triazine herbicides interfere in the endo‐ crine responses of different species of mammals, their potential impact on humans seem to be mainly related to reproduction and development and not with human carcinogenesis [155].

Mladinic et al. [122] evaluated the genotoxic potential, by the comet assay and FISH, and oxidative damages, by the TBARS lipid peroxidation, of different concentrations of glyphosate (three similar to those observed in residential and occupational exposures and two related to LC50) in human lymphocytes. The comet assay showed that the concentration of 580 μg/mL promoted a significant increase in the tail length, while the concentration of 92.8 μg/mL caused an increase in the tail intensity, both in relation to the control test. With the addition of the S9 fraction, the tail length was significantly increased for all the concentrations tested. When the lymphocytes were exposed to the three highest concentrations without S9, there was an increase in the frequency of micronuclei, nuclear buds and nucleoplasmic bridges. The addition of a metabolic activation system only promoted a significant increase of the nuclear instabilities for the highest concentration tested. The values of TBARS significantly increased with the increase of the concentrations tested, regardless the presence or absence of the S9 fraction. Due to the fact that dose-dependent effects for all the assays used were not observed, the authors concluded that these concentrations of glyphosate are not relevant for human

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

423

According to Mladinic et al. [122], the increase in the number of crops genetically modified used in assays and diagnosis of resistance to glyphosate, may be related to the fact that these crops tolerate increasingly higher concentrations of the active ingredient necessary for an effective control of weeds, which results from the introduction of increasing amounts of glyphosate into the environment. Thus, some epidemiological studies have shown that human exposure to glyphosate present in the environment is correlated to the development of diseases

According to He et al. [161], paraquat, the second most widely used herbicide in the world, is able to selectively accumulate in human lungs by causing oxidative injury and fibrosis, leading several individuals to death. Chronic exposure to this herbicide is also associated with hepatic

Studies carried out by He et al. [161] evaluated the toxicity of paraquat on BEAS-2B normal cells (human bronchial epithelial cells), showing that it is dose-dependent and results in mitochondrial damages, oxidative stress, death of lung cells exposed, as well as production of cytokines, pro-fibrogenic growth factors and transformation of myofibroblasts. The authors also proved that administration of resveratrol, a polyphenolic phytoalexin naturally produced by several plants, to control bacteria and fungi, was able to inhibit the production of reactive oxygen species, inflammations and fibrotic reactions induced by paraquat, by the activation of the Nrf2 signaling (Nuclear Factor Erythroid-2), revealing a new molecular mechanism for the intervention against oxidative damages and pulmonary fibrosis resulted from the action

The study on the influence of a complex mixture of herbicides (atrazine, 2,4-D, alachlor, ciazine and malathion) in workers occupationally exposed to them, was carried out using cytogenetic methods standardly established (chromosome aberrations and micronucleus assay) and the comet assay technique. This assay showed a significant increase in the DNA migration (P<0.001), suggesting that long-term exposure to the pesticides could cause damages in the genome of somatic cells and, therefore, would represent a potential risk to human health [164].

exposure, since they did not present a significant risk for human health.

such as the non-Hodgkin lymphoma [159, 160].

of toxic chemical compounds.

lesions, kidney failure and Parkinson´s disease [162, 163].

Gammon et al. [83] discussed the extensive list of epidemiological studies with the herbicide atrazine, which describes that the carcinogenic potential of this compound to humans is not conclusive, although some studies have indicated a relationship between a high risk of prostate cancer and exposure to the herbicide.

Mladinic et al. [156] evaluated the genotoxic and mutagenic effects of low concentrations of the herbicides glyphosate and terbuthylazine, considered safe and, therefore, considered possible to occur in occupational and residential exposures (ADI – Acceptable Daily Intake, REL – Residential Exposure Level, OEL – Occupational Exposure Level, and 1/100 and 1/16 LD50 – Lethal Dose 50% - oral, rat), in human lymphocytes, with and without the use of metabolic activation (S9 fraction), by the FSH cytome assay, using pan-centromeric DNA probes to assess the content of micronuclei and other chromatinic instabilities. The authors verified that the frequencies of micronuclei, nuclear buds and nucleoplasmic bridges of cells treated with glyphosate slightly increased after the concentration of OEL 3.5 μg/mL, but no concentration induced an increase of the centromeric signals (C+) or DAPI (DAPI+). Now, the treatment with the herbicide terbuthylazine without metabolic activation showed a doseresponse increase in the frequency of micronuclei of the lymphocytes exposed, and the significant data were from the concentration of 0.0008 μg/mL (REL) tested. The concentrations ADI (0.00058 μg/mL), REL (0.0008 μg/mL) and OEL (0.008 μg/mL) of terbuthylazine induced a significant occurrence of micronuclei hybridized with the centromeric probe (C+), regardless the presence or absence of S9, and of nuclear buds containing centromeric signals, only in the presence of S9. By the results obtained, it was suggested that the lowest concentrations of glyphosate do not have relevant harmful effects for the DNA molecule, while terbuthylazine presents a predominant aneugenic potential for the genetic material of human lymphocytes.

Terbuthylazine belongs to the chloro-s-triazine herbicides class, which inhibits the photosyn‐ thesis of weeds, by reaching the photosystem II. It is a chemical used for a variety of crops, such as maize, sugarcane, olive and pineapple [157]. Since the banishment of atrazine in European countries in 2006, terbuthylazine was recommended as its substitute. Due to the fact that the herbicide terbuthylazine is suspect of causing diseases in humans, such as non-Hodgkin lymphoma and lung cancer, Mladinic et al. [158] evaluated the effects of prolonged exposure (14 days) to low concentrations of this compound (0.58 ng/ml and 8 ng/ml) in human lymphocytes, using the comet assay and the comet-FISH assay (with the c-Myc and TP 53 genes). Treatment with terbuthylazine induced the migration of fragments of DNA in a significant manner, only for the highest concentration treated. The results showed an impair‐ ment of the structural integrity of c-Myc and TP 53, due to the prolonged exposure of human lymphocytes to terbuthylazine. The fact that several copies of TP53 were affected by the herbicide can indicate its ability to negatively interfere in the control of the cell cycle. However, the authors concluded that, for a more detailed assessment of the risk of cancer associated with exposure to terbuthylazine, it should be evaluated the impact of this pesticide on other housekeeping genes and markers.

Mladinic et al. [122] evaluated the genotoxic potential, by the comet assay and FISH, and oxidative damages, by the TBARS lipid peroxidation, of different concentrations of glyphosate (three similar to those observed in residential and occupational exposures and two related to LC50) in human lymphocytes. The comet assay showed that the concentration of 580 μg/mL promoted a significant increase in the tail length, while the concentration of 92.8 μg/mL caused an increase in the tail intensity, both in relation to the control test. With the addition of the S9 fraction, the tail length was significantly increased for all the concentrations tested. When the lymphocytes were exposed to the three highest concentrations without S9, there was an increase in the frequency of micronuclei, nuclear buds and nucleoplasmic bridges. The addition of a metabolic activation system only promoted a significant increase of the nuclear instabilities for the highest concentration tested. The values of TBARS significantly increased with the increase of the concentrations tested, regardless the presence or absence of the S9 fraction. Due to the fact that dose-dependent effects for all the assays used were not observed, the authors concluded that these concentrations of glyphosate are not relevant for human exposure, since they did not present a significant risk for human health.

studies, it was observed that, although the chloro-s-triazine herbicides interfere in the endo‐ crine responses of different species of mammals, their potential impact on humans seem to be mainly related to reproduction and development and not with human carcinogenesis [155].

Gammon et al. [83] discussed the extensive list of epidemiological studies with the herbicide atrazine, which describes that the carcinogenic potential of this compound to humans is not conclusive, although some studies have indicated a relationship between a high risk of prostate

Mladinic et al. [156] evaluated the genotoxic and mutagenic effects of low concentrations of the herbicides glyphosate and terbuthylazine, considered safe and, therefore, considered possible to occur in occupational and residential exposures (ADI – Acceptable Daily Intake, REL – Residential Exposure Level, OEL – Occupational Exposure Level, and 1/100 and 1/16 LD50 – Lethal Dose 50% - oral, rat), in human lymphocytes, with and without the use of metabolic activation (S9 fraction), by the FSH cytome assay, using pan-centromeric DNA probes to assess the content of micronuclei and other chromatinic instabilities. The authors verified that the frequencies of micronuclei, nuclear buds and nucleoplasmic bridges of cells treated with glyphosate slightly increased after the concentration of OEL 3.5 μg/mL, but no concentration induced an increase of the centromeric signals (C+) or DAPI (DAPI+). Now, the treatment with the herbicide terbuthylazine without metabolic activation showed a doseresponse increase in the frequency of micronuclei of the lymphocytes exposed, and the significant data were from the concentration of 0.0008 μg/mL (REL) tested. The concentrations ADI (0.00058 μg/mL), REL (0.0008 μg/mL) and OEL (0.008 μg/mL) of terbuthylazine induced a significant occurrence of micronuclei hybridized with the centromeric probe (C+), regardless the presence or absence of S9, and of nuclear buds containing centromeric signals, only in the presence of S9. By the results obtained, it was suggested that the lowest concentrations of glyphosate do not have relevant harmful effects for the DNA molecule, while terbuthylazine presents a predominant aneugenic potential for the genetic material of human lymphocytes.

Terbuthylazine belongs to the chloro-s-triazine herbicides class, which inhibits the photosyn‐ thesis of weeds, by reaching the photosystem II. It is a chemical used for a variety of crops, such as maize, sugarcane, olive and pineapple [157]. Since the banishment of atrazine in European countries in 2006, terbuthylazine was recommended as its substitute. Due to the fact that the herbicide terbuthylazine is suspect of causing diseases in humans, such as non-Hodgkin lymphoma and lung cancer, Mladinic et al. [158] evaluated the effects of prolonged exposure (14 days) to low concentrations of this compound (0.58 ng/ml and 8 ng/ml) in human lymphocytes, using the comet assay and the comet-FISH assay (with the c-Myc and TP 53 genes). Treatment with terbuthylazine induced the migration of fragments of DNA in a significant manner, only for the highest concentration treated. The results showed an impair‐ ment of the structural integrity of c-Myc and TP 53, due to the prolonged exposure of human lymphocytes to terbuthylazine. The fact that several copies of TP53 were affected by the herbicide can indicate its ability to negatively interfere in the control of the cell cycle. However, the authors concluded that, for a more detailed assessment of the risk of cancer associated with exposure to terbuthylazine, it should be evaluated the impact of this pesticide on other

cancer and exposure to the herbicide.

422 Herbicides - Current Research and Case Studies in Use

housekeeping genes and markers.

According to Mladinic et al. [122], the increase in the number of crops genetically modified used in assays and diagnosis of resistance to glyphosate, may be related to the fact that these crops tolerate increasingly higher concentrations of the active ingredient necessary for an effective control of weeds, which results from the introduction of increasing amounts of glyphosate into the environment. Thus, some epidemiological studies have shown that human exposure to glyphosate present in the environment is correlated to the development of diseases such as the non-Hodgkin lymphoma [159, 160].

According to He et al. [161], paraquat, the second most widely used herbicide in the world, is able to selectively accumulate in human lungs by causing oxidative injury and fibrosis, leading several individuals to death. Chronic exposure to this herbicide is also associated with hepatic lesions, kidney failure and Parkinson´s disease [162, 163].

Studies carried out by He et al. [161] evaluated the toxicity of paraquat on BEAS-2B normal cells (human bronchial epithelial cells), showing that it is dose-dependent and results in mitochondrial damages, oxidative stress, death of lung cells exposed, as well as production of cytokines, pro-fibrogenic growth factors and transformation of myofibroblasts. The authors also proved that administration of resveratrol, a polyphenolic phytoalexin naturally produced by several plants, to control bacteria and fungi, was able to inhibit the production of reactive oxygen species, inflammations and fibrotic reactions induced by paraquat, by the activation of the Nrf2 signaling (Nuclear Factor Erythroid-2), revealing a new molecular mechanism for the intervention against oxidative damages and pulmonary fibrosis resulted from the action of toxic chemical compounds.

The study on the influence of a complex mixture of herbicides (atrazine, 2,4-D, alachlor, ciazine and malathion) in workers occupationally exposed to them, was carried out using cytogenetic methods standardly established (chromosome aberrations and micronucleus assay) and the comet assay technique. This assay showed a significant increase in the DNA migration (P<0.001), suggesting that long-term exposure to the pesticides could cause damages in the genome of somatic cells and, therefore, would represent a potential risk to human health [164].

### **4. Conclusion**

The authors present in this manuscript the bioassays and the test-systems most commonly used to evaluate the effects of herbicides and the test-organisms to best suit the assessments of herbicide effects. In these considerations, the authors attempted to present the most sensitive and efficient organisms capable of detecting environmental contamination resulting from the action of these chemical agents. Additionally, we present in this paper the need to carry out research aimed at more effective methods to prevent and/or reduce the deleterious effects of such compounds on the environment, the biota potentially exposed, and especially to human health.

**Herbicide Test-organism Endpoint Results Tested**

for 500 mg/Kg

damages, mainly at the

increase in the rates of lipid peroxidation, hepatic damages, death of hepatocytes and induction of

micronuclei, in the tested concentrations, in all the exposure periods (from 1 to 35 days), with more significant effects in the highest concentrations and exposure periods; higher sensitivity for gill cells

damages, from the concentration of

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

concentrations of 100 and 200 µg/L

induction of damages in the DNA and

significant induction of DNA strand breaks and micronuclei, in all tested concentrations of the commercial product (Gesaprim), but there was not a induction of these genotoxic and mutagenic effects for the active

DNA exposed to the commercial product Gesaprim, but there was no induction of genotoxicity for the active ingredient atrazine, for all tested concentrations.

significant inhibition of the mitotic index, significant increase in the frequencies of micronuclei and chromosome aberrations of both test organisms, when exposed to the commercial product Gesaprim, but there was no induction of any significant effects when cells were exposed to the active ingredient atrazine, for all tested concentrations.

there was no significant induction of genotoxic damages nor mutagenic

comet assay significant increase in the DNA

4.8 mg/L

micronuclei.

ingredient.

Atrazine Rat leukocytes comet assay increase in the damages in the DNA

Atrazine Human lymphocytes comet assay significant increase in the DNA

test

lipid peroxidation assay; micronucleus

micronucleus test; comet assay

micronucleus test; comet assay

Atrazine Human lymphocytes comet assay significant increase of damage in the

chromosome aberration assay; micronucleus test

*Salmonella* assay and SOS Chromotest

Atrazine Erythrocytes of bullfrog tadpoles

Atrazine Hepatocytes of Wistar rats

Atrazine Erythrocytes and gill cels

Atrazine Erythrocytes of the gibel

Atrazine Somatic cells of *Allium*

Atrazine *Salmonella* and hepatic

rats

*cepa* and *Vicia faba*

cells of Sprague-Dawley

of the fish *Channa punctatus*

carp fish (*Carassius auratus*)

**concentrations**

125, 250, 500 mg/Kg

4.8, 19.75, 77, 308 mg/L

50, 100, 200 µg/L

4.24, 5.30. 8.48 mg/L

0.047, 0.47, 4.7 ug/L

*A. cepa:* 15, 30, 60 mg/L; *V. faba:* 17,5, 35, 70 mg/L

1 – 1000 µg/ plate

400 ppm [91] Campos-

5, 10, 15 µg/L [84] Çavas, 2011

**References**

425

http://dx.doi.org/10.5772/55851

[89] Tennant et al., 2001

[90] Clements et al., 1997

[69] Ribas et al., 1995

Pereira et al., 2012

[22] Nwani et al., 2011

[101] Zeljezic et al., 2006

[102] Srivastava and Mishra, 2009

[92] Ruiz and Marzin, 1997

In this study it was addressed several studies that used different methodologies, which evaluated the toxicity and action of herbicides on different non-target organisms, including human species. The table below summarizes the main researches addressed in the text.


#### Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health http://dx.doi.org/10.5772/55851 425


**4. Conclusion**

424 Herbicides - Current Research and Case Studies in Use

health.

Atrazine Erythrocytes of Nile

Atrazine Wild leopard frogs

tilapia (*Oreochromis niloticus*)

(*Rana pipiens*)

Atrazine *Sorghum vulgare* chromosome

Atrazine Human lymphocytes chromosome

Atrazine Human lymphocytes chromosome

Atrazine Human blood cells chromosome

Atrazine Rat chromosome

Atrazine Human lymphocytes chromosome

Atrazine Bone marrow cells of rats

The authors present in this manuscript the bioassays and the test-systems most commonly used to evaluate the effects of herbicides and the test-organisms to best suit the assessments of herbicide effects. In these considerations, the authors attempted to present the most sensitive and efficient organisms capable of detecting environmental contamination resulting from the action of these chemical agents. Additionally, we present in this paper the need to carry out research aimed at more effective methods to prevent and/or reduce the deleterious effects of such compounds on the environment, the biota potentially exposed, and especially to human

In this study it was addressed several studies that used different methodologies, which evaluated the toxicity and action of herbicides on different non-target organisms, including human species. The table below summarizes the main researches addressed in the text.

toxicity assay induction of abnormalities in the

breaks

aberrations

increase in the DNA fragmentation; induction of micronuclei and nuclear abnormalities in all tested concentrations

gonads; developmental delay and hermaphroditism (≥ 0.1ppb)

induction of multinucleated, aneuploid and polyploid cells; abnormalities in the mother cells of pollen grains; meiotic instability

increase in the chromosome aberrations frequency at 0.10 ppm

increase in the frequency of chromosome aberrations; increase in the frequency of sister chromatid exchange in all tested concentrations

Significant increase of chromosome

there was no significant increase in the frequency of chromosome aberrations at 20 ppm

there was no significant increase in the frequency of chromosome

induction of chromosome aberrations 0.0001 µg/mL [94] Meisner et

**concentrations**

6.25, 12.5, 25 µg/L

0.01, 0.1, 0.4, 0.8, 1, 10, 25, 200 ppb

0.01, 1, 0.10 mg/ml

**References**

[8] Ventura et al., 2008

[82] Hayes et al., 2002

[85] Meisner et al., 1992

2.7 Kg a.i./ha [80] Liang et al., 1967

5, 8.5, 17, 51 µM [93] Lioi et al., 1998

1 ppm [94] Meisner et al., 1993

20 ppm [85] Meisner et al., 1992

20 ppm [86] Roloff et al., 1992

al., 1993

**Herbicide Test-organism Endpoint Results Tested**

micronucleus test; comet assay

aberration assay

aberration assay

aberration assay

aberration assay

chromosome aberration assay

aberration assay

aberration assay; SCE


**Herbicide Test-organism Endpoint Results Tested**

teratogenic assay

acute lethality test, lipid peroxidation assay by quantification of MDA; comet assay

SCE; analysis of the cell cycle progression and mitotic index

Vitellogenin estrogenic assay

microbial growth assay

chromosome aberration assay; micronucleus test

2,4-D Gills of different species of fishes

2,4-D Chinese Hamster Ovary – CHO – cells

2,4-D Bone marrow and germ cells of rats

2,4-D and Butachlor Erythrocytes of the

2,4-D and Paraquat Rainbow trout

2,4-D and 2,4-D DMA

2,4-D; Triclopyr; Diquat dibromide; glyphosate

2,4-D Frog *Xenopus* FETAX - frog embryo

(*Oncorhynchus mykiss*)

Humanh lymphocytes and erythrocytes

Rainbow trout (*Oncorhynchus mykiss*)

*Lactococcus lactis* subsp. *Cremoris; Lactobacillus delbrueckii* subsp. *bulgaricus*

Glyphosate *Geotrichum candidum,*

Glyphosate Tadpoles of wood frog

(*Rana sylvatica* or *Sylvaticus lithobates*),

catfish (*Clarias batrachus*)

**concentrations**

2, 4, 6, 10 µg/mL

50,100, 200 mg/kg

2,4-D: 25, 50, 75ppm; Butachlor: 1, 2, 2.5ppm

2,4-D: 316, 346, 389, 436, 489 mg/L; Paraquat: 0.055, 0.066, 0.083, 0.116, 0.133 mg/L

10, 25, 50, 100 µg/mL

0.11, 1.64, 2.07, 1.25 mg/L

0.1, 1, 10, 100, 1000, 10000 ppm

0, 1, 2, or 3 mg acid equivalents [a.e.] /L of

nerve stimulation due to inhibition of the glandular chloride channel in

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

of the renal functions and hepatic

chromatid exchange at 2 and 4 µg/ml

chromatid exchange at 100 and 200

significant induction of embryotoxic

significant increase in the frequency of micronuclei and altered cells in a dose-response manner for both

toxic action more evident for paraquat (high indices of mortality); apnea and white spots in the scales of individuals exposed to the 2 herbicides; increase in the rates of MDA and damages in the DNA after exposure to all concentrations of the

ppm, for both cell types

and teratogenic effects

mucosa skin

toxicity assay bleeding, renal increase, impairment

degeneration

SCE significant increase in the sister

SCE significant increase in the sister

herbicides

tested herbicides

erythrocytes

exposed to 2,4-D

acute toxicity assay significant induction of morphological

alterations in the cell cycle and induction of SCE for some concentrations only with more significant genotoxic effects for

significant increase in the levels of vitellogenin of the plasma of fishes

inhibition of microbial growth by the commercial product Roundup; microbiocide effect at concentrations lower than the recommended by agricultural use for the commercial product Roundup; non induction of significant toxic effects for the three microorganisms by the active ingredient glyphosate

alterations of tadpoles of the three species; for the wood frogs and

**References**

427

[116] González et al., 2005

[117] Madrigal-Bujaidar et al., 2001

[113] Ateeq et al.,

[112] Martínez-Tabche et al., 2004

[118] Soloneski et al., 2007

[120] Xie et al., 2005

[127] Clair et al., 2012

[128] Relyea, 2012

400 mg/L [115] Gómez et al., 1998

http://dx.doi.org/10.5772/55851

245 mg/L [119] Morgan et al., 1996

2002


**Herbicide Test-organism Endpoint Results Tested**

induction of hypospadias in male newborns at 200 ppm and diverse embryotoxic damages at 25 ppm.

testosterone, androstenedione, estradiol, estrone, progesterone and corticosterone to 50 or 200 ppm for

there was no significant increase of chromosome aberrations and sister

damages by atrazine for the tested concentrations, proven clastogenic

and slightly toxic for *D. carinata* and butachlor is moderately toxic for both; the toxic effects of the mixture of the herbicides were significantly antagonistic for *S. obliquus* and there was no significative synergism for *D.*

affected the survival, development and metamorphosis time of tadpoles in different concentrations; DNA damage (0.4-0.8 mg/L)

there was no significant induction of micronuclei and SCE; significant induction of DNA damages for all tested concentrations

response of the simpatic junction to

chromatid exchanges

micronucleus test there was no significant induction of micronuclei

flow cytometry assay significant induction of chromosome

acute toxicity assay atrazine is highly toxic for *S. obliquus*

*carinata*

toxicity assay dose-dependent reduction in the

potential of cyanazine

60 days

teratogenic tests

aberration assay and

Atrazine Wistar rats Radioimmunoassay alterations in the levels of

SCE

Human lymphocytes chromosome

Atrazine Sprague-Dawley rats embryotoxic and

426 Herbicides - Current Research and Case Studies in Use

Polychromatic erythrocytes of the bone marrow of female C57B1/6 rats

Green alga *Scenedesmus obliquus* and cladoceran *Daphnia*

*carinata*

Butachlor Alpine cricket frog

2,4-D *Caudiverbera*

(*Fejervarya limnocharis*)

Terbutryn Human leukocytes micronucleus test;

*caudiverbera* frog

chromosome aberration assay

comet assay; SCE

Chinese Hamster Ovary – CHO – cells

Atrazine, Simazine and Cyanazine

Atrazine, Simazine and Cyanazine

Atrazine, Simazine and Cyanazine

Atrazine and Butachlor

**concentrations**

25, 100, 200 mg/kg/d

50, 200 mg / kg / day

0, 125, 250, 500 mg/kg

0.003 µg/mL, 0.018 µg/ mL(atrazine); 0.003 µg/mL, 0.012 µg/mL (cyanazine)

*S. obliquus*: 0, 0.5, 1, 2, 4, 8 mg/L (butachlor) and 0, 0.008, 0.016, 0.032, 0.064, 0.128 mg/L (atrazine) / *D. carinata*: 0, 1, 1.8, 3, 5, 8 mg/L (butachlor) and 0, 7.5, 15, 30, 60, 120 mg/L (atrazine)

ranging from 0.025 to 3.2 mg/l

0, 5, 10, 50, 100, 150 µg/mL

**References**

[87] Wu et al., 2007

[88] Modic et al., 2004

0.5, 5, 50 ppb [104] Kligerman

et al., 1993

[103] Kligerman et al., 2000

[107] Taets et al., 1998

[20] He et al., 2012

[43] Liu et al., 2011

[108] Moretti et al., 2002

0.01, 0.1, 1 mM [114] Suwalsky et al., 1999


**Herbicide Test-organism Endpoint Results Tested**

assay-FISH

enzyme activity assay

Paraquat Several species of fishes enzyme activity assay induction of oxidative stress; increase

conventional cytogenetics assay

aberration assay; micronucleus test;

cytotoxicity assay, oxidative stress assay

chromosome aberration test; micronucleus test

SCE

Terbuthylazine Human lymphocytes comet assay; comet

Paraquat Several species of fishes acute toxicity assay;

Paraquat Germ cells of Sprague-Dawley rats

Paraquat Leukocytes of *Rana*

Paraquat BEAS 2B normal cells

Diuron Pacific oyster

Diquat Roots of smooth

rat

*ornativentris*

Paraquat Human lymphocytes chromosome

(human bronchial epithelial cells)

(*Crassostrea* gigas)

hawksbeard (*Crepis capillaris* L.); polychromatic erythrocytes of the bone marrow of C57BL **concentrations**

Terbuthylazine: 0.58 ng/ml, 8 ng/ml; carbofuran: 8 ng/ml, 21.6 ng/ml

0, 6, 15, 30 mg/kg

0, 1, 5, 25, 50, 250, 500, 1000, 2000, 4000 µg/mL

300 ng/L, 3 µg/L

*Crepis capillaris*: 0.005, 0.01, 0.05, 0.1%; erythrocytes: 8.5, 34.17 mg/Kg

centromeric probe and nuclear buds with centromeric signals in the presence of S9 (0.008 ug/mL onward)

Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

alteration in the activity of different enzymes; negative effects on cardiac contraction and opercular ventilation

in the levels of SOD

concentrations

µg/mL)

toxicity assay irreversible damages to the genetic

systems

spermatozoa; increase in the mortality rates and abnormalities in spermatozoa for the higher

genotoxic effects, such as chromosome breaks

reduction in the cell division index; decrease in the cell proliferation rates; significant increase in the frequencies of SCE (50 µg/mL for 24h treatment; 4000 µg/mL for 2h treatment), significant increase in the MN frequencies (concentrations ≥ 25

mitochondrial damage; oxidative stress; cell death; production of cytokines, pro-fibrogenic growth facts and transformation of myofibroblast

material, negative impacts in the reproduction of aquatic organisms

there was no induction of chromosome aberrations for any test system; significant increase of the frequency of micronuclei for both test

cytotoxicity assay reduction in the quantity of

induction of the migration of fragments of DNA, significant only at the highest concentration; impairment of the structural integrity of c-Myc and TP 53 due to prolonged exposure to terbuthylazine

**References**

429

http://dx.doi.org/10.5772/55851

[158] Mladinic et al., 2012

[138] D'Souza et al., 2006

[135] Ribas et al., 1998

[44] Bouilly et al., 2007

[129] Dimitrov et al., 2006

0.1-2.0 mg/L [134] Tortorelli et al., 1990

0.2-50 mM [137] Tomita et al., 2007

10-6 M [136] Hanada, 2011

10 uM He et al., 2012

#### Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health http://dx.doi.org/10.5772/55851 429


**Herbicide Test-organism Endpoint Results Tested**

chromosome aberration assay; micronucleus assay

teratogenicity assay

lipid peroxidation assay – TBARS

leopard frog (*Rana pipiens pipiens* or L.), and American toad (*Bufo americanus* or *Anaxyrus americanus*)

428 Herbicides - Current Research and Case Studies in Use

hawksbeard (*Crepis capillaris* L.); polychromatic erythrocytes of the bone marrow of C57BL

Glyphosate Female Wistar rats acute toxicity assay;

Glyphosate Human lymphocytes comet assay; FISH;

Algae and 25 species of aquatic animals

Glyphosate Roots from the smooth

rat

Glyphosate adn 2,4-D

Glyphosate and Terbuthylazine

**concentrations**

*Crepis capillaris*: 0.05, 0.1, 0.5, 1 %; erythrocytes: doses inferior to half the LD50 (1080 mg/Kg)

500, 750, 1000 mg/kg

0.5, 2.91, 3.5, 92.8, 580 µg/mL

0, 1, 2, or 3 mg acid equivalents [a.e.] /L of Roundup Original MAX

0.5, 2.91, 3.50, 92.8, 580 µg/mL (glyphosate); 0,00058, 0,0008, 0,008, 25, 156,5 µg/mL (terbuthylazine)

Roundup Original MAX

leopard frogs, exposure to glyphosate affected the size of the tail of tadpoles, for all tested concentrations

there was no induction of genotoxic and/or mutagenic effects for any of

high mortality index of females treated with the highest concentration of the commercial product Roundup; increase in the dose-response of fetal skeletal

significant increase in the DNA migration at 580 µg/mL; significant increase of the comet tail intensity at 92.8 µg/mL; greater lesion in the DNA in the presence of S9; increase in the frequency micronuclei, nuclear buds and nucleoplasmic bridges, without S9; significant increase of nuclear instabilities in the highest concentration tested with S9; significant dose-response increase of

the species

alterations

the levels of TBARS

of periphyton by the 2 herbicides; there was no great impacts to the aquatic community by 2,4-D; high impact to the aquatic community by glyphosate by the significative decrease in the species richness

frequencies of micronuclei, nuclear buds and nucleoplasmic bridges of clells treated (3.5 µg/mL onward), but without induction of centromeric signals; terbuthylazine induced an increase in the frequency of micronuclei hybridized with

acute toxicity assay there was no reduction in the biomass

Human lymphocytes cytome FISH glyphosate caused an increase in the

**References**

[129] Dimitrov et al., 2006

[130] Dallegrave et al., 2003

[122] Mladinic et al., 2009

[132] Relyea, 2005

[156] Mladinic et al., 2009


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Toxicity of Herbicides: Impact on Aquatic and Soil Biota and Human Health

http://dx.doi.org/10.5772/55851

431

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ticide Biochemistry and Physiology, v. 88, n. 3, p. 252-259, 2007.

**Table 2.** List o the main researches carried out with several bioindicators to evaluate the toxicity of herbicides.

### **Author details**

Maria Aparecida Marin-Morales\* , Bruna de Campos Ventura-Camargo and Márcia Miyuki Hoshina

\*Address all correspondence to: mamm@rc.unesp.br

Department of Biology, Institute of Biosciences, São Paulo State University (UNESP), SP, Brazil

### **References**


[5] Spacie, A.; Hamelink, J.L. Bioaccumulation, in: RAND, G.M.; PETROCELLI, S.R. (Eds.), Fundamentals of Aquatic Toxicology: Methods and Applications, Hemi‐ sphere, New York, 1985, pp. 495-525.

**Herbicide Test-organism Endpoint Results Tested**

*Silurana tropicalis* toxicity assay toxic effects for tadpoles, more

aberration assay; micronucleus test; comet assay

there was no significant increase in the frequencies of chromosome aberrations in plant cells, but an increase of their incidence in cells of rats; significant increase in the frequency of micronuclei for both test

systems.

of the DNA

**Table 2.** List o the main researches carried out with several bioindicators to evaluate the toxicity of herbicides.

Department of Biology, Institute of Biosciences, São Paulo State University (UNESP), SP,

[1] Guzzella, L.; Pozzoni, F.; Giuliano, G. Herbicide contamination of surficial ground‐

[3] Bolognesi, C.; Merlo, F.D. Pesticides: Human Health Effects. Encyclopedia of Envi‐

[4] Nehls, S.; Segner, H. Detection of DNA damage in two cell lines from rainbow trout, RTG-W1, using the comet assay. Environmental Toxicology, v. 16, p. 321-329, 2001.

water in Northern Italy. Environmental Pollution, v. 142, p. 344-353, 2006.

[2] Kortekamp, A. Herbicides and Environment. Kroatia, 2011, 760 p.

significant for thiobencarb

significant increase in the migration

, Bruna de Campos Ventura-Camargo and

chromosome aberration test; micronucleus test

Pendimethalin Roots of smooth

rat

Simetryn, mefenacet and thiobencarb

Complex mixture of pesticides (atrazine, 2,4-D, alachlor, ciazine and malathion)

**Author details**

Brazil

**References**

Márcia Miyuki Hoshina

Maria Aparecida Marin-Morales\*

hawksbeard (*Crepis capillaris* L.); polychromatic erythrocytes of the bone marrow of C57BL

430 Herbicides - Current Research and Case Studies in Use

Workers exposed chromosome

\*Address all correspondence to: mamm@rc.unesp.br

ronmental Health, p. 438-453, 2011.

**concentrations**

*Crepis capillaris*: 0.005, 0.1, 0.2, 0.4%; erythrocytes: 122.2, 244.5, 489 mg/Kg

Thiobencarb: 6.85-2.92 mM

Mixture of various concentrations of pesticides

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[163] Garaj-Vrhovac and Zeljezic, 2002


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**Chapter 17**

**Herbicide Resistant Weeds:**

Additional information is available at the end of the chapter

Jamal R. Qasem

**1. Introduction**

http://dx.doi.org/10.5772/56036

**The Technology and Weed Management**

Pest resistance to control methods in general is not an isolated phenomenon but usually expected and well demonstrated when any method is repeatedly applied over a long period of time without being changed or modified in nature, structure, principals of application or formulation. All pests that growers must control in agricultural land have the capacity to become resistant to whatever tactic is used to control them [11]. It is usually expressed as a gradual adaptation or "fitness" of some individuals or populations of the targeted pest or organism to the frequently applied control methods and available conditions. This adaptation may be physical, morphological or phenological, physiological, anatomical or biochemical or could result from the interaction between any two or more of these. It may also be due to some genetic changes as mutations occur on the key site at which a specific method operates. These mutations are at least partially dominant and inherited. Traits are conferred by modifications to single nuclear genes. This indicates that the rate of resistance evolution will be driven by mutation, the intensity of selection, the dominance and relative fitness of mutations in presence or absence of the herbicide and by dispersal of resistance alleles within and between weed populations [28]. However, no proof that the herbicides cause the mutations leads to resistance [37]. However, most often resistance is controlled by a single, dominant or semi-dominant gene [38] although recessive genes control of herbicide resistant trait in natural weed popula‐ tions has been also implicated in resistance to dintroanaline, while wild populations exposed to herbicide stresses for the first time may efficiently express herbicide-resistant genes.

Most weed modifications and adaptations, if not all, are advantageous to the pest, since allow its escape on time and/or place and thus avoid external hazard or threat to its existence and genetic line. Resistance therefore should not be confused with natural tolerance or low

> © 2013 Qasem; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Qasem; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.
