**Herbicide Resistant Weeds: The Technology and Weed Management**

Jamal R. Qasem

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56036

### **1. Introduction**

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

susceptibility due to a normal physiological or behavioristic property of an unselected population [23].

resistance will be monogenic, resulting in a large change in the resistance phenotype. However, when doses are lower and selection acts within the range of standing genetic variation, polygenic responses will be possible and resistance will evolve by a gradual change in the mean susceptibility of the population [28]. On the other hand, population of not or less adapted individuals, decline in growth and number until greatly suppressed, limited and may become extinct. Therefore, with continuous dependence on a single method of weed control, a weed population is usually shifting toward better adapted species or individuals that cope well with existing control measures and new conditions. Self-thinning of a weed population is continued toward complete tolerance to employed control measures. Therefore, weeds adapted to mowing tend to grow short, in a rosette form, creeping above the soil surface or show high plasticity and softness of aerial parts and stems and become difficult to mow and also escape hand weeding. Deep rooted weed species are difficult to pull out even by soil tillers. Seasonal dormancy and shifts in the weed population in the growing season is well recognized for certain weed species such as *Senecio vulgaris* [29; 37], while physiological adaptation of *Echinochloa crusi-galli* and *Cyperus rotundus* to flooding conditions and the role of Alcohol dehydroginase enzyme (Adh) in *E. crusi-galli* is well documented [5; 14]. Similar adaptations of *Cirsium arvense* ecotypes to temperature variations [43] and *Typha anguistifolia* and *Typha latifolia* genetic and clonal variations [27; 40] have also been reported. In this regard, it is important to differentiate between tolerance and resistance of weeds to herbicides. Tolerance is the inherited ability of a species to survive and reproduce after herbicide treatment; it refers to the natural variability to herbicides and exists within individuals of a species and quickly evolves. It usually refers to relatively minor or gradual differences in intraspecific variability. Resistance is the inherited ability of a plant or a biotype to survive and reproduce following exposure to a dose of herbicide that is normally lethal to wild type [16; 23; 30; 37]. Therefore, it is a decreased response of a population of weed to herbicides as a result of their application.

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However, both terms sometimes are misused or used interchangeably.

resistant individuals to utilize more resources [9; 22].

Tolerant weed species are less harmed by herbicides; they exhibit a certain degree of avoidance or adaptation strategy that allows recovery and thus escape control measures. They may respond by timing stomata closure or having sunken pores or stomata, thick waxy cutical on upper leaf surface, encased growing points or some biochemical, physiological or anatomical properties better developed by time until they become best fit and adapted to applied herbicides and become thereafter resistant. This, however, leads to gradual but radical changes in the weed population composition and distribution spectrum at which resistant individuals or certain weed species increased and dominate and susceptible ones are reduced and replaced. Adaptation or exclusion of the less tolerant species depends on performance of these by time. Generally a weed population becomes rich in individuals and poor in species with the continuous use of the same herbicide or different herbicides of similar mode/mechanism of action. This shift does not however, reflect better competitiveness or higher regenerative ability but most likely due to absence of sensitive highly competing species or forms that allow

In cultivated fields, associating weeds bear more resemblance to crop plants in morphology, physiology and responses to control measures and other agricultural practices in general. They

Organisms are varied in sensitivity, responses and thus adaptability to such conditions and in responses to any treatment or imposed external factors. Tolerance and then gradual resistance of agricultural pests to any control method or environmental stress is thus a strategy through which organisms/ or pests encounter hazards and maintain life and therefore may be applied to any method of pest or weed control including prevention, mechanical, cultural, physical, biological and chemical [30]. For example, weeds resisting soil mulch cover show some morphological and/or physical characteristics that allow penetration of the mulch layer; also, flooding of resistant species possess water impermeable seed coat or generate O2 and reduce CO2 penetration. Firing or flaming is resisted through presence of a hard seed coat or deeply buried regenerative propagules; certain weed species show feedback mechanisms or luxury accumulation of mineral nutrients and thus avoid toxicity; high temperature and low soil moisture harmful effects are avoided by adoption of secondary or enforced seed dormancy, while harmful effects of excessive light is avoided by some morpho-physiological alterations. Soil acidity may be encountered in the microhabitat by root exudates or selective mineral absorption and salinity by excretion of salt through different mechanisms and formation of salt glands or vacuoles or shedding salt saturated organs; microbes attack is avoided by production of repellent allelochemicals, and pests through some morpho-chemical adapta‐ tions. However, the mechanism behind tolerance or resistance is different and based on the type of target pest or the hazard imposed.

Herbicides represent one of the external factors and form a group of synthetic- plus some biochemicals used to suppress or kill unwanted vegetation and are a major component of pesticides. They assist in management and restoration of areas invaded by invasive species. Herbicides are a major technological tool and responsible, in part, for an agricultural revolution and increase in food production in the last few decades. However, at present this technology faces radical changes in effectiveness under field conditions that lead in different cases to failure of weed control operation due to continued development of weed tolerance/resistance and evolution and limitations in the herbicide industry and development.

### **2. Agriculture practices and weed evolution**

General weed control methods (tillage, hoeing, hand weeding, flooding, cuttings or mowing, flaming, use of general herbicides) are all nonselective and usually applied to a composite weed species or vegetation of inter and intra-specific variations in richness, morphology, growth habit and responses. Each species may adapt, or not, to any of these methods. Since weeds are widely different in mechanisms by which they encounter hazards they are exposed to, they are different in plasticity and responses. With continued use of a single control method for a long period of time, species migrate, flourish or die. Flourishing species gradually became better fit and adapted, and increase in number and population size in absence of others. The only surviving individuals are those possessing rare single gene mutations and evolved resistance will be monogenic, resulting in a large change in the resistance phenotype. However, when doses are lower and selection acts within the range of standing genetic variation, polygenic responses will be possible and resistance will evolve by a gradual change in the mean susceptibility of the population [28]. On the other hand, population of not or less adapted individuals, decline in growth and number until greatly suppressed, limited and may become extinct. Therefore, with continuous dependence on a single method of weed control, a weed population is usually shifting toward better adapted species or individuals that cope well with existing control measures and new conditions. Self-thinning of a weed population is continued toward complete tolerance to employed control measures. Therefore, weeds adapted to mowing tend to grow short, in a rosette form, creeping above the soil surface or show high plasticity and softness of aerial parts and stems and become difficult to mow and also escape hand weeding. Deep rooted weed species are difficult to pull out even by soil tillers. Seasonal dormancy and shifts in the weed population in the growing season is well recognized for certain weed species such as *Senecio vulgaris* [29; 37], while physiological adaptation of *Echinochloa crusi-galli* and *Cyperus rotundus* to flooding conditions and the role of Alcohol dehydroginase enzyme (Adh) in *E. crusi-galli* is well documented [5; 14]. Similar adaptations of *Cirsium arvense* ecotypes to temperature variations [43] and *Typha anguistifolia* and *Typha latifolia* genetic and clonal variations [27; 40] have also been reported. In this regard, it is important to differentiate between tolerance and resistance of weeds to herbicides. Tolerance is the inherited ability of a species to survive and reproduce after herbicide treatment; it refers to the natural variability to herbicides and exists within individuals of a species and quickly evolves. It usually refers to relatively minor or gradual differences in intraspecific variability. Resistance is the inherited ability of a plant or a biotype to survive and reproduce following exposure to a dose of herbicide that is normally lethal to wild type [16; 23; 30; 37]. Therefore, it is a decreased response of a population of weed to herbicides as a result of their application. However, both terms sometimes are misused or used interchangeably.

susceptibility due to a normal physiological or behavioristic property of an unselected

Organisms are varied in sensitivity, responses and thus adaptability to such conditions and in responses to any treatment or imposed external factors. Tolerance and then gradual resistance of agricultural pests to any control method or environmental stress is thus a strategy through which organisms/ or pests encounter hazards and maintain life and therefore may be applied to any method of pest or weed control including prevention, mechanical, cultural, physical, biological and chemical [30]. For example, weeds resisting soil mulch cover show some morphological and/or physical characteristics that allow penetration of the mulch layer; also, flooding of resistant species possess water impermeable seed coat or generate O2 and reduce CO2 penetration. Firing or flaming is resisted through presence of a hard seed coat or deeply buried regenerative propagules; certain weed species show feedback mechanisms or luxury accumulation of mineral nutrients and thus avoid toxicity; high temperature and low soil moisture harmful effects are avoided by adoption of secondary or enforced seed dormancy, while harmful effects of excessive light is avoided by some morpho-physiological alterations. Soil acidity may be encountered in the microhabitat by root exudates or selective mineral absorption and salinity by excretion of salt through different mechanisms and formation of salt glands or vacuoles or shedding salt saturated organs; microbes attack is avoided by production of repellent allelochemicals, and pests through some morpho-chemical adapta‐ tions. However, the mechanism behind tolerance or resistance is different and based on the

Herbicides represent one of the external factors and form a group of synthetic- plus some biochemicals used to suppress or kill unwanted vegetation and are a major component of pesticides. They assist in management and restoration of areas invaded by invasive species. Herbicides are a major technological tool and responsible, in part, for an agricultural revolution and increase in food production in the last few decades. However, at present this technology faces radical changes in effectiveness under field conditions that lead in different cases to failure of weed control operation due to continued development of weed tolerance/resistance

General weed control methods (tillage, hoeing, hand weeding, flooding, cuttings or mowing, flaming, use of general herbicides) are all nonselective and usually applied to a composite weed species or vegetation of inter and intra-specific variations in richness, morphology, growth habit and responses. Each species may adapt, or not, to any of these methods. Since weeds are widely different in mechanisms by which they encounter hazards they are exposed to, they are different in plasticity and responses. With continued use of a single control method for a long period of time, species migrate, flourish or die. Flourishing species gradually became better fit and adapted, and increase in number and population size in absence of others. The only surviving individuals are those possessing rare single gene mutations and evolved

and evolution and limitations in the herbicide industry and development.

**2. Agriculture practices and weed evolution**

population [23].

446 Herbicides - Current Research and Case Studies in Use

type of target pest or the hazard imposed.

Tolerant weed species are less harmed by herbicides; they exhibit a certain degree of avoidance or adaptation strategy that allows recovery and thus escape control measures. They may respond by timing stomata closure or having sunken pores or stomata, thick waxy cutical on upper leaf surface, encased growing points or some biochemical, physiological or anatomical properties better developed by time until they become best fit and adapted to applied herbicides and become thereafter resistant. This, however, leads to gradual but radical changes in the weed population composition and distribution spectrum at which resistant individuals or certain weed species increased and dominate and susceptible ones are reduced and replaced. Adaptation or exclusion of the less tolerant species depends on performance of these by time. Generally a weed population becomes rich in individuals and poor in species with the continuous use of the same herbicide or different herbicides of similar mode/mechanism of action. This shift does not however, reflect better competitiveness or higher regenerative ability but most likely due to absence of sensitive highly competing species or forms that allow resistant individuals to utilize more resources [9; 22].

In cultivated fields, associating weeds bear more resemblance to crop plants in morphology, physiology and responses to control measures and other agricultural practices in general. They mimic crops from sowing and germination until harvest. Since herbicides used on crop plants are selective, weeds respond by exhibiting similar morphology, physiology and biochemistry as crop plants to avoid hazards. However, weeds derived from crop plants as hybrids, crop relatives or wild-weedy forms are better fit to such conditions than others. Weed-crop associations also exist between weed species of different taxa from crop plants. In this case, the longer the use of the same herbicide/s, the greater the close association between crops and certain well performed weed species that later transfer into adapted weed races. Crop relative weeds however, are of great potential to intra- and inter- gene exchange and efficient mating system among themselves and with crops, thus become best adapted and more difficult to control.

becomes a real trouble. Its residual negative effects may not possible to overcome for a long

Herbicide Resistant Weeds: The Technology and Weed Management

http://dx.doi.org/10.5772/56036

449

**4. Field evidence of weed resistance and herbicide resistance protocol**

In the field all growth patterns and distribution of weed species may be observed. Some species grow in colonies, in certain growth patterns, forming an ecological niche, sporadically distributed, or randomly scattered within crop plants. Certain species are dominant while others show moderate growth or are suppressed while some grow vigorous or have limited growth and short stature. This however, depends on the microhabitat and place they occupy in the field and their performance. Under intense cultivation and thick crop stands, individuals of certain weed species express phenotypic plasticity (phenotypes) at which they change/ modify their appearance, reduce or drop lower branches and thus lateral growth, elongate and increase cell divisions, overtopping crop plants and trapping light, although some shade tolerant species perform better under such conditions. Phenotypic plasticity modifying the mode of growth and energy allocation in response to environmental changes is considered to be important adaptive mechanism. These phenological variations can be easily observed among different weed species. Uniform application of herbicides in the field should equally affect all individuals of a single weed species. When herbicides are best timed and properly applied they should yield similar mode of action on species individuals. While differences in influence of a herbicide on different weed species is expected, hence differences in taxonomy, morphology, physiology and biochemistry, but such differences among individuals of a single species should have resulted from some morphogenetic or other variations within the same or different populations of that species. Certain individuals are totally killed, others less injured and some escape control unharmed. When the same herbicide or herbicides of the same mechanism of action are used, it becomes clearer that previously less or unaffected individuals should exhibit similar responses as were first shown. Gradually these individuals increase in number and growth until they dominate the site with continuous use of the same herbicide or its analogues while sensitive individuals are suppressed or removed. This however, takes a relatively long time for the population to shift from susceptible to complete resistant and depends on herbicide, environment and plant factors. These are positive signs on possible herbicide-resistance development in the field. If less affected or unharmed individuals in the first herbicide application are killed or severely injured in repeated treatments then there should be another cause of escape or partial control at first application and herbicide resistance should be then excluded. On the other hand, unharmed individuals may also tolerate higher application rates. Therefore, farmers should keep observing changes in the weed population as long as the herbicides are in use. They must get familiarized with weed species, populations and densities at pre- and post- herbicide treatments, comparing weed growth, performance and densities and recording any changes in populations thereafter. Less or unharmed indi‐ viduals of any species should be followed up throughout subsequent applications of the same

period after abandonment.

herbicide or herbicides of similar mode of action.

### **3. Selection pressure and weed races**

With continuous use of the same agricultural practice/s, interspecies selection occurs and plant species are gradually purified (intraspecific selection) by time until they become best adapted. Since all control measures including herbicides aim to eliminate weeds without causing injury to crop plants, weeds respond by developing mechanism/s allowing escape of chemical hazards. Under such conditions, sensitive individuals are first limited or disappear. Tolerant individuals increase in number and accumulate tolerance until they become resistant. There‐ fore, a resistant population of any weed species is exposed to long-term selection pressure through which it is purified and performs well under prevailing conditions in absence of sensitive weed species. With continuous exposure to herbicide pressure, a population of resistance is usually developed.

Weeds tend to avoid herbicide toxicity by changing normal growth habits, or exhibiting some phenological (such as changes in germination patterns), physical and/or physiological changes through which they adjust emergence time, external appearance or physiology. These however, are inherited traits that allow plants to survive herbicide treatments. One best adaptation is that of weeds similar to crop plants in most or all growth aspects. These form weed races similar to crop plants and well adapted to their habitats. Among reported weed races are *Camelina sativa* to flax crop, *Echinochloa crus-galli var. Oryzicola* that associate with rice and the weedy wild rice or red rice in India and east-south Africa [8; 20]. All are genetically irrelevant to crop plants. However, in some cases weed races are of the same botanical family or belong to the same crop species. This kind of association leads to development of "cropraces" that possess weedy characters very well adapted to cultural practices; they are similar to crop plants in most growth aspects and difficult to control by herbicides or other control methods including hand weeding. They take an advantage from conditions under which crop plants are growing until they become difficult to leave their habitats or even become dependent on crop plants in their growth and environment. These weeds are specialized to certain crop plants or cultivars. Moreover, many genetically related species can exchange genes with crop individuals and mimic crops. It can be concluded that any agricultural practice exerts selection pressure and may become troublesome to farmers when repeatedly applied for a long period. Its positive impact on crop growth and productivity is usually negated with time until it becomes a real trouble. Its residual negative effects may not possible to overcome for a long period after abandonment.

### **4. Field evidence of weed resistance and herbicide resistance protocol**

mimic crops from sowing and germination until harvest. Since herbicides used on crop plants are selective, weeds respond by exhibiting similar morphology, physiology and biochemistry as crop plants to avoid hazards. However, weeds derived from crop plants as hybrids, crop relatives or wild-weedy forms are better fit to such conditions than others. Weed-crop associations also exist between weed species of different taxa from crop plants. In this case, the longer the use of the same herbicide/s, the greater the close association between crops and certain well performed weed species that later transfer into adapted weed races. Crop relative weeds however, are of great potential to intra- and inter- gene exchange and efficient mating system among themselves and with crops, thus become best adapted and more difficult to

With continuous use of the same agricultural practice/s, interspecies selection occurs and plant species are gradually purified (intraspecific selection) by time until they become best adapted. Since all control measures including herbicides aim to eliminate weeds without causing injury to crop plants, weeds respond by developing mechanism/s allowing escape of chemical hazards. Under such conditions, sensitive individuals are first limited or disappear. Tolerant individuals increase in number and accumulate tolerance until they become resistant. There‐ fore, a resistant population of any weed species is exposed to long-term selection pressure through which it is purified and performs well under prevailing conditions in absence of sensitive weed species. With continuous exposure to herbicide pressure, a population of

Weeds tend to avoid herbicide toxicity by changing normal growth habits, or exhibiting some phenological (such as changes in germination patterns), physical and/or physiological changes through which they adjust emergence time, external appearance or physiology. These however, are inherited traits that allow plants to survive herbicide treatments. One best adaptation is that of weeds similar to crop plants in most or all growth aspects. These form weed races similar to crop plants and well adapted to their habitats. Among reported weed races are *Camelina sativa* to flax crop, *Echinochloa crus-galli var. Oryzicola* that associate with rice and the weedy wild rice or red rice in India and east-south Africa [8; 20]. All are genetically irrelevant to crop plants. However, in some cases weed races are of the same botanical family or belong to the same crop species. This kind of association leads to development of "cropraces" that possess weedy characters very well adapted to cultural practices; they are similar to crop plants in most growth aspects and difficult to control by herbicides or other control methods including hand weeding. They take an advantage from conditions under which crop plants are growing until they become difficult to leave their habitats or even become dependent on crop plants in their growth and environment. These weeds are specialized to certain crop plants or cultivars. Moreover, many genetically related species can exchange genes with crop individuals and mimic crops. It can be concluded that any agricultural practice exerts selection pressure and may become troublesome to farmers when repeatedly applied for a long period. Its positive impact on crop growth and productivity is usually negated with time until it

control.

**3. Selection pressure and weed races**

448 Herbicides - Current Research and Case Studies in Use

resistance is usually developed.

In the field all growth patterns and distribution of weed species may be observed. Some species grow in colonies, in certain growth patterns, forming an ecological niche, sporadically distributed, or randomly scattered within crop plants. Certain species are dominant while others show moderate growth or are suppressed while some grow vigorous or have limited growth and short stature. This however, depends on the microhabitat and place they occupy in the field and their performance. Under intense cultivation and thick crop stands, individuals of certain weed species express phenotypic plasticity (phenotypes) at which they change/ modify their appearance, reduce or drop lower branches and thus lateral growth, elongate and increase cell divisions, overtopping crop plants and trapping light, although some shade tolerant species perform better under such conditions. Phenotypic plasticity modifying the mode of growth and energy allocation in response to environmental changes is considered to be important adaptive mechanism. These phenological variations can be easily observed among different weed species. Uniform application of herbicides in the field should equally affect all individuals of a single weed species. When herbicides are best timed and properly applied they should yield similar mode of action on species individuals. While differences in influence of a herbicide on different weed species is expected, hence differences in taxonomy, morphology, physiology and biochemistry, but such differences among individuals of a single species should have resulted from some morphogenetic or other variations within the same or different populations of that species. Certain individuals are totally killed, others less injured and some escape control unharmed. When the same herbicide or herbicides of the same mechanism of action are used, it becomes clearer that previously less or unaffected individuals should exhibit similar responses as were first shown. Gradually these individuals increase in number and growth until they dominate the site with continuous use of the same herbicide or its analogues while sensitive individuals are suppressed or removed. This however, takes a relatively long time for the population to shift from susceptible to complete resistant and depends on herbicide, environment and plant factors. These are positive signs on possible herbicide-resistance development in the field. If less affected or unharmed individuals in the first herbicide application are killed or severely injured in repeated treatments then there should be another cause of escape or partial control at first application and herbicide resistance should be then excluded. On the other hand, unharmed individuals may also tolerate higher application rates. Therefore, farmers should keep observing changes in the weed population as long as the herbicides are in use. They must get familiarized with weed species, populations and densities at pre- and post- herbicide treatments, comparing weed growth, performance and densities and recording any changes in populations thereafter. Less or unharmed indi‐ viduals of any species should be followed up throughout subsequent applications of the same herbicide or herbicides of similar mode of action.

Sometimes partial effect or failure of the applied herbicide to control certain weed species or individual weeds in the first application may be thought as due to wrong calibration, misap‐ plication, incomplete coverage treatment by a general herbicide or unsprayed gaps resulting from low sprayer boom during spray, unfavorable weather conditions, improper timing of herbicide application, and weed flushes after application of a non-resisted herbicide [16]. This could be easily judged in the repeated application to these species or individuals. When the herbicide failed to control these for the second time or at higher rates then resistance may be underway. With continued use of the same herbicide for different times, resistant individuals aggregate forming irregular patches while other weeds are controlled. A patch of uncontrolled weeds starts spreading and healthy weeds are mixed with uncontrolled weeds of the same species (Fig. 1).

ecotypes, genotypes, biotypes or phenotypes. Some of the basic differences in the definitions of pest resistance depend on these terms. The basic unit of plant classifications is the "species" that is defined as a group of individuals displaying common characteristics and having the ability to mate and produce fully viable progeny. A species usually consists of several to many populations. A population is a group of organisms within a species that co-exist in time and space [35; 36] and share a distinct range of genetic variations. While a genotype is the sum of the genetic coding or the genome of an individual, a biotype may not be coincident with genotype asanindividualhasmanygenes.Certaingenesmaybeexpressedorunexpressedandnotpertain tothephenotypeassociatedwiththebiotype.Abiotypeisaphenotypethatconsistentlyexpresses or exhibits a specific trait or set of traits; it represents a group of individuals or a population within a species with a distinctive genetic variation of biochemical or morphological traits. Phenotype refers to the physiological and morphological profile of the expressed gene in an individual[42].Asinglegenotypecanproducedifferentphenotypes inresponsetoenvironmen‐ tal conditions and the fundamental properties of organisms are known as phenotypic plastici‐ ty. The epigenetic change is thus reflecting the alteration of phenotype (morphological or biochemical) without change in either the coding sequence of a gene or the upstream promot‐ er region. Therefore biotypes within the same species may be developed due to this interac‐ tion. On the other hand, ecotype is a population within a species that has developed distinctive morphological or physiological characters (herbicide resistance) in response to a specific environment andpersistswhenindividuals aremovedto adifferent environment.Ecotypes are of different germination and growth optima for the same environmental factor and pheno‐ types may be emerged and observed in weed populations. These alter their morphological features in response to certain prevailing environmental conditions which aim at protection of theirindividualsagainstunfavorableecologicalstresses.Somaticpolymorphismofcertainweed species is well recognized and expressed as seed polymorphism of different morphological or physiological requirements for germination on different parts of the same weed individual.

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These however, are somatic rather than genetically based differences.

Crop relative weeds are usually derived from the same species of crop plants and thus are genetically related. Most crop species have wild relatives and can interact with them under field conditions. Examples are radish, carrots, vetch, celery, lettuce, fennel, eggplants, wheat, barley, oat, etc. In addition, crop plants which are domesticated from wild forms possess a high degree of compatibility with crops. These are referred to as wild and weedy relatives, in spite of the fact that all species are related because their cells can read a common genetic code [15]. Crop weedy relatives are genetically compatible with crop plants and easily exchange genes. The emerged hybrids may become noxious weeds with certain weedy characteristics derived from both crop plants and wild forms. They could exhibit a certain degree of dormancy that is usually weak or absent in its parents and possess other weed traits making them difficult to control. These new generations have the ability to resist environmental hazards much better than parents and can exist and dominate in both productive and unproductive habitats. These

**6. Herbicide resistance and crop relative weeds**

Therefore irregularly shaped patches of a single weed species in the field are an indicator of herbicide resistance, especially when:


However, the rate at which a resistant weed population is selected depends on the number and frequency of herbicide applications it receives, the size of the population and its genetic diversity, and characteristics of the herbicide target site. Resistance buildup is accelerated when the management of crops does not include different weed control methods that limit herbicide use. In addition, this may be greatly enhanced in conservation or zero tillage because weeds are not killed by mechanical disturbance and general herbicides.

### **5. Interaction between environment and genetics**

Growth and productivity of any plant species are mainly influenced by genetics, ecology and their interactions. Weeds are different from crops in their responses to both factors. They are more flexible and thus better responsive and adapted to extremes in environmental condi‐ tions such as high temperature, freezing, excessive light, salinity, drought, etc. Tolerance of weeds and better responses are mainly due to better and rapid interaction between environ‐ ment and genetics compared to crop plants. In addition, the long term breeding and selection pressure imposed on crop plants has lead to selection of less adapted species or cultivars that are highly sensitive to ecological stresses and deficient in certain characteristics that offer protection or defense mechanisms against unfavorable environment. Weed fitness in natural habitats and their rapid responses to the changing environment allow evolution of weed

ecotypes, genotypes, biotypes or phenotypes. Some of the basic differences in the definitions of pest resistance depend on these terms. The basic unit of plant classifications is the "species" that is defined as a group of individuals displaying common characteristics and having the ability to mate and produce fully viable progeny. A species usually consists of several to many populations. A population is a group of organisms within a species that co-exist in time and space [35; 36] and share a distinct range of genetic variations. While a genotype is the sum of the genetic coding or the genome of an individual, a biotype may not be coincident with genotype asanindividualhasmanygenes.Certaingenesmaybeexpressedorunexpressedandnotpertain tothephenotypeassociatedwiththebiotype.Abiotypeisaphenotypethatconsistentlyexpresses or exhibits a specific trait or set of traits; it represents a group of individuals or a population within a species with a distinctive genetic variation of biochemical or morphological traits. Phenotype refers to the physiological and morphological profile of the expressed gene in an individual[42].Asinglegenotypecanproducedifferentphenotypes inresponsetoenvironmen‐ tal conditions and the fundamental properties of organisms are known as phenotypic plastici‐ ty. The epigenetic change is thus reflecting the alteration of phenotype (morphological or biochemical) without change in either the coding sequence of a gene or the upstream promot‐ er region. Therefore biotypes within the same species may be developed due to this interac‐ tion. On the other hand, ecotype is a population within a species that has developed distinctive morphological or physiological characters (herbicide resistance) in response to a specific environment andpersistswhenindividuals aremovedto adifferent environment.Ecotypes are of different germination and growth optima for the same environmental factor and pheno‐ types may be emerged and observed in weed populations. These alter their morphological features in response to certain prevailing environmental conditions which aim at protection of theirindividualsagainstunfavorableecologicalstresses.Somaticpolymorphismofcertainweed species is well recognized and expressed as seed polymorphism of different morphological or physiological requirements for germination on different parts of the same weed individual. These however, are somatic rather than genetically based differences.

### **6. Herbicide resistance and crop relative weeds**

Sometimes partial effect or failure of the applied herbicide to control certain weed species or individual weeds in the first application may be thought as due to wrong calibration, misap‐ plication, incomplete coverage treatment by a general herbicide or unsprayed gaps resulting from low sprayer boom during spray, unfavorable weather conditions, improper timing of herbicide application, and weed flushes after application of a non-resisted herbicide [16]. This could be easily judged in the repeated application to these species or individuals. When the herbicide failed to control these for the second time or at higher rates then resistance may be underway. With continued use of the same herbicide for different times, resistant individuals aggregate forming irregular patches while other weeds are controlled. A patch of uncontrolled weeds starts spreading and healthy weeds are mixed with uncontrolled weeds of the same

Therefore irregularly shaped patches of a single weed species in the field are an indicator of

**•** Field history indicates extensive use of the same herbicide or herbicides of the same

**•** There has been a previous failure to control the same species or population in the same field

However, the rate at which a resistant weed population is selected depends on the number and frequency of herbicide applications it receives, the size of the population and its genetic diversity, and characteristics of the herbicide target site. Resistance buildup is accelerated when the management of crops does not include different weed control methods that limit herbicide use. In addition, this may be greatly enhanced in conservation or zero tillage because

Growth and productivity of any plant species are mainly influenced by genetics, ecology and their interactions. Weeds are different from crops in their responses to both factors. They are more flexible and thus better responsive and adapted to extremes in environmental condi‐ tions such as high temperature, freezing, excessive light, salinity, drought, etc. Tolerance of weeds and better responses are mainly due to better and rapid interaction between environ‐ ment and genetics compared to crop plants. In addition, the long term breeding and selection pressure imposed on crop plants has lead to selection of less adapted species or cultivars that are highly sensitive to ecological stresses and deficient in certain characteristics that offer protection or defense mechanisms against unfavorable environment. Weed fitness in natural habitats and their rapid responses to the changing environment allow evolution of weed

**•** No or minimal herbicide symptoms appear on the single uncontrolled weed species.

species (Fig. 1).

herbicide resistance, especially when:

450 Herbicides - Current Research and Case Studies in Use

mechanism of action.

**•** There are no other apparent application problems.

**•** Other weed species on the herbicide label are effectively controlled.

with the same herbicide or with herbicides of the same site of action.

weeds are not killed by mechanical disturbance and general herbicides.

**5. Interaction between environment and genetics**

Crop relative weeds are usually derived from the same species of crop plants and thus are genetically related. Most crop species have wild relatives and can interact with them under field conditions. Examples are radish, carrots, vetch, celery, lettuce, fennel, eggplants, wheat, barley, oat, etc. In addition, crop plants which are domesticated from wild forms possess a high degree of compatibility with crops. These are referred to as wild and weedy relatives, in spite of the fact that all species are related because their cells can read a common genetic code [15]. Crop weedy relatives are genetically compatible with crop plants and easily exchange genes. The emerged hybrids may become noxious weeds with certain weedy characteristics derived from both crop plants and wild forms. They could exhibit a certain degree of dormancy that is usually weak or absent in its parents and possess other weed traits making them difficult to control. These new generations have the ability to resist environmental hazards much better than parents and can exist and dominate in both productive and unproductive habitats. These

gene flow, serves as a mechanism to maintain the biological diversity that helps to ensure long-

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Gene flow is a critical determinant of population genetic structure, playing an important role in both evolutionary and applied plant population genetics [12]. It is also known as 'migration' [13] or admixture [1] and can be defined as the movement of genes between populations of a species and between these populations and inter-fertile relatives [39; 41], conferring new traits, the biophysical characteristics of the organism to individuals of the recipient population [34].

Gene flow could occur through dispersal of pollen (via outcrossing between sexually compat‐ ible individuals within or among populations) or seeds (via seed dispersal), or vegetative parts capable of clonal propagation [34; 41]. Pollen dispersal is the typical method for such exchange of genetic information [15] and pollinating visitors or other agents including wind, animal, water current and other factors could play a significant role in this issue. This happens by crosspollination (hybridization), that is, the pollination of members of one population or genetic pool with that of another [34]. These are natural and ordinary phenomena that occur in

Movement of pollen away from its site of production can result in true gene flow only if (1) the pollen first effects fertilization to form seeds, and (2) seeds germinate, produce plants that express the gene (i.e., are not silen8ced), and are able to reproduce [15]. Gene flow can be from crop to crop or landrace, from crop to wild relative, and even from wild relative to crop plant [34]. Spread of this phenomenon would lead to radical changes in vegetation composition and

However, two types of gene flow are known; horizontal and vertical. Stewart [39] showed that 'horizontal' gene flow is the movement of genes between disparate, unrelated species, such as

Among the world's 180 most damaging weeds, however, cause 90% of all crop losses, only five groups (related weeds of rice, sorghum, rape seed, sugarcane, and oats) are sexually compatible with the most important crops (Table 1). This fact emphasizes that the number of weed-crop crosses likely to lead to extremely troublesome or unmanageable problems is small.

Weed crosses with herbicide-tolerant biotech crops are likely to be favored in some agricultural fields where the herbicide is used. In areas where little or no herbicide is applied (e.g., native lands), the weed–biotech crop crosses will not be favored [15]. Self-pollinating crops are considered of low risk in terms of gene flow to weeds. Roundup Ready, Clearfield, or Liberty Link canola, in contrast, could pollinate nearby herbicide-susceptible canola as well as weedy canola relatives, resulting in volunteer canola plants and weeds that may be resistant to several herbicide families [38]. However, several pieces of evidence clearly show an escape of weedy transgene from fields via seed flow and this escape occurs via man-mediated long-distance dispersal events [4]. Other results revealed that development of weed resistance via selection pressure from repeated herbicide applications in herbicide resistant crops (in the absence of gene flow), often poses greater risks than that from gene flow to related weed species [15].

term survival of populations and species in various environments.

conventional as well as genetically modified crops.

weed ecological distribution and their economic significance.

between plants and microbes while horizontal gene flow is more theoretic.

**Figure 1.** Three resistant weed species (a, b, c) to glyphosate herbicide at different growth stages and spray times. (a). *Conyza canadensis* resistant to glyphosate until harvest stage of wheat. Source http://www.sciencephoto.com/ media/ courtesy of the Montana State University (b). A field infested by suspected glyphosate- resistant *Kochia*, after the field was sprayed with three applications of glyphosate. Photo181407/enlarge Southern Agricultural Research Center. By Dillon Tabish, 08-11-12.Available at: http://www.flatheadbeacon.com/articles/article/scientists\_discov‐ er\_possible\_herbicide\_resistant\_weed\_in\_montana/29184 (c). Palmir Amaranth (*Amaranthus palmeri*) resistance to glyphosate in corn at early growth. Source: E. Larson, April 21st, 2011.Availableat:http:// www.mississippi-crops.com/ 2011/04/21/how -to-deal-with-glyphosate-resistance-and- weed-issues-in-corn/.

are of a high genetic plasticity allowing their individuals to adapt to extensive herbicide applications and thus resist chemical treatments. Crop-weed crossed forms can easily ex‐ change genes with crop plants as well as with weedy relatives and therefore are becoming troublesome weeds in fields with genetically modified crops.

### **7. Gene flow potential with wild/weedy relatives of world crops**

In nature, genetic information is transferred between different individuals, populations, and generations (to progeny) and across spatial dimensions [2; 15]. This phenomenon, known as gene flow, serves as a mechanism to maintain the biological diversity that helps to ensure longterm survival of populations and species in various environments.

Gene flow is a critical determinant of population genetic structure, playing an important role in both evolutionary and applied plant population genetics [12]. It is also known as 'migration' [13] or admixture [1] and can be defined as the movement of genes between populations of a species and between these populations and inter-fertile relatives [39; 41], conferring new traits, the biophysical characteristics of the organism to individuals of the recipient population [34].

Gene flow could occur through dispersal of pollen (via outcrossing between sexually compat‐ ible individuals within or among populations) or seeds (via seed dispersal), or vegetative parts capable of clonal propagation [34; 41]. Pollen dispersal is the typical method for such exchange of genetic information [15] and pollinating visitors or other agents including wind, animal, water current and other factors could play a significant role in this issue. This happens by crosspollination (hybridization), that is, the pollination of members of one population or genetic pool with that of another [34]. These are natural and ordinary phenomena that occur in conventional as well as genetically modified crops.

Movement of pollen away from its site of production can result in true gene flow only if (1) the pollen first effects fertilization to form seeds, and (2) seeds germinate, produce plants that express the gene (i.e., are not silen8ced), and are able to reproduce [15]. Gene flow can be from crop to crop or landrace, from crop to wild relative, and even from wild relative to crop plant [34]. Spread of this phenomenon would lead to radical changes in vegetation composition and weed ecological distribution and their economic significance.

However, two types of gene flow are known; horizontal and vertical. Stewart [39] showed that 'horizontal' gene flow is the movement of genes between disparate, unrelated species, such as between plants and microbes while horizontal gene flow is more theoretic.

Among the world's 180 most damaging weeds, however, cause 90% of all crop losses, only five groups (related weeds of rice, sorghum, rape seed, sugarcane, and oats) are sexually compatible with the most important crops (Table 1). This fact emphasizes that the number of weed-crop crosses likely to lead to extremely troublesome or unmanageable problems is small.

are of a high genetic plasticity allowing their individuals to adapt to extensive herbicide applications and thus resist chemical treatments. Crop-weed crossed forms can easily ex‐ change genes with crop plants as well as with weedy relatives and therefore are becoming

**Figure 1.** Three resistant weed species (a, b, c) to glyphosate herbicide at different growth stages and spray times. (a). *Conyza canadensis* resistant to glyphosate until harvest stage of wheat. Source http://www.sciencephoto.com/ media/ courtesy of the Montana State University (b). A field infested by suspected glyphosate- resistant *Kochia*, after the field was sprayed with three applications of glyphosate. Photo181407/enlarge Southern Agricultural Research Center. By Dillon Tabish, 08-11-12.Available at: http://www.flatheadbeacon.com/articles/article/scientists\_discov‐ er\_possible\_herbicide\_resistant\_weed\_in\_montana/29184 (c). Palmir Amaranth (*Amaranthus palmeri*) resistance to glyphosate in corn at early growth. Source: E. Larson, April 21st, 2011.Availableat:http:// www.mississippi-crops.com/

)

In nature, genetic information is transferred between different individuals, populations, and generations (to progeny) and across spatial dimensions [2; 15]. This phenomenon, known as

**7. Gene flow potential with wild/weedy relatives of world crops**

troublesome weeds in fields with genetically modified crops.

2011/04/21/how -to-deal-with-glyphosate-resistance-and- weed-issues-in-corn/.

(a) (b)

452 Herbicides - Current Research and Case Studies in Use

(c)

Weed crosses with herbicide-tolerant biotech crops are likely to be favored in some agricultural fields where the herbicide is used. In areas where little or no herbicide is applied (e.g., native lands), the weed–biotech crop crosses will not be favored [15]. Self-pollinating crops are considered of low risk in terms of gene flow to weeds. Roundup Ready, Clearfield, or Liberty Link canola, in contrast, could pollinate nearby herbicide-susceptible canola as well as weedy canola relatives, resulting in volunteer canola plants and weeds that may be resistant to several herbicide families [38]. However, several pieces of evidence clearly show an escape of weedy transgene from fields via seed flow and this escape occurs via man-mediated long-distance dispersal events [4]. Other results revealed that development of weed resistance via selection pressure from repeated herbicide applications in herbicide resistant crops (in the absence of gene flow), often poses greater risks than that from gene flow to related weed species [15].


depends mainly on presence of wild or weedy relatives [11]. Transgene (s) transfer may have unpredictable and out of control ecological impacts under intensive cultivation of biotech crops [25]. While different crops can exchange genes with wild relatives, gene escape to wild or weedy relatives and its ecological impacts are outrated. The ecological consequences of gene flow however, depends on the amount of transgenes moved out to a wild population and the genetically modified traits and whether they have an evolutionary advantage under natural selection pressure or not and if enhanced fitness of wild and weedy relatives then the transgene followed by gene flow would persist and spread rapidly in the population of wild relatives through introgression, invade a new area and outcompete other individuals under natural conditions [24]. Weeds receiving transgenes will continue to evolve when exposed to selection pressure and it becomes nearly impossible to move them out from the environments if they

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The development of crops that are resistant to herbicides is a relatively new technology aimed to improve weed control in agricultural land. Herbicide-resistant crops can be created by standard methods of plant breeding, but the use of genetic engineering techniques is more usual. Herbicide-resistant crops are made resistant by either transgene technology or by selection in cell or tissue culture for mutations that confer herbicide resistance [10]. Glyphosate and glufosinate are herbicides most used in this regard. For example, soybean, corn, cotton, sugar beet, and canola are available as glyphosate- resistant cultivars and some are now widely

**•** Develop crops more tolerant/resistant to herbicides and thus increase herbicides uses and

**•** Increase options for weed control when the number of herbicides is limited, such as in minor

**•** Effective control of certain difficult weed species and widening of weed control spectrum

**•** Increase bio-safety and enhance better eco-friendly use of new and less toxic herbicides

However, public concern about the impact of genetically modified crops on the natural environment encouraged more studies on this aspect in the last few years. Among the possible impacts, the 'escape' of the transgene, either through dispersal of the crop plant outside the agricultural area or through hybridization with wild relatives and thus increase the possibility

In the majority of instances, there is a very low probability that an approved biotech crop introduction could create an environmental risk different from that of a nonbiotech version of

planted in different countries. Importance of genetically engineered crops is to:

**•** Eliminate possible injury effects of soil persistent herbicides to crop plants.

can persist and spread in the populations.

**•** Achieve more effective weed control

**•** May be more cost- effective weed control method

selectivity.

crops.

of "weediness" [41].

**8. Transgenic crops and weed evolution**

**Table 1.** Examples of some important food crops and their sexually compatible weed species

In this regard, biotech crops conferring stress tolerance (e.g., to water deficits, diseases, insects, salt stress, or nutritional deficiencies) may need more scrutiny because their crosses with weedy relatives may impart selective advantages in both agricultural and nonagricultural areas. Thus, some traits obtained from biotech crops could theoretically facilitate development into problematic weedy or wild species [15].

The economic consequences due to gene flow from biotech crops will primarily impact the agricultural fields in which those crops are grown, but potentially could impact natural areas given the proper rare combination of sexually compatible relatives, favorable environment, and reproductive/fitness advantages. As an example, rice grown in tropical countries may be relatively more prone to such processes because of the substantial populations of its wild/ weedy relatives that grow naturally in or adjacent to the rice-producing areas [8; 26].

Crop-wild hybridization may also create genotypes with the potential to displace parental taxa in new environments [7]. However, the most important variable affecting gene flow is the degree of relatedness and distance between the crop and the weed, because gene flow is only possible if close relatives are growing near the crop. As a result the possibility of gene flow depends mainly on presence of wild or weedy relatives [11]. Transgene (s) transfer may have unpredictable and out of control ecological impacts under intensive cultivation of biotech crops [25]. While different crops can exchange genes with wild relatives, gene escape to wild or weedy relatives and its ecological impacts are outrated. The ecological consequences of gene flow however, depends on the amount of transgenes moved out to a wild population and the genetically modified traits and whether they have an evolutionary advantage under natural selection pressure or not and if enhanced fitness of wild and weedy relatives then the transgene followed by gene flow would persist and spread rapidly in the population of wild relatives through introgression, invade a new area and outcompete other individuals under natural conditions [24]. Weeds receiving transgenes will continue to evolve when exposed to selection pressure and it becomes nearly impossible to move them out from the environments if they can persist and spread in the populations.

### **8. Transgenic crops and weed evolution**

**Rank Crop Scientific Name Related weeds: sexually compatible with**

1 Wheat Triticum aestivum

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2 Rice Oryza sativa

Source: Different references

into problematic weedy or wild species [15].

Triticum durum

Oryza glaberrima

4 Soybean *Glycine max G. soya* 5 Barley *Hordeum vulgare H. spontaneum* 6 Sorghum *Sorghum bicolor* S. bicolor

3 Maize *Zea mays Z. mays ssp Mexicana*

7 Canola *Brassica napus, B. rapa, B. juncea B. napus, B. rapa, B. nigra* 8 Sunflower *Helianthus annus Helianthus annus*

**Table 1.** Examples of some important food crops and their sexually compatible weed species

In this regard, biotech crops conferring stress tolerance (e.g., to water deficits, diseases, insects, salt stress, or nutritional deficiencies) may need more scrutiny because their crosses with weedy relatives may impart selective advantages in both agricultural and nonagricultural areas. Thus, some traits obtained from biotech crops could theoretically facilitate development

The economic consequences due to gene flow from biotech crops will primarily impact the agricultural fields in which those crops are grown, but potentially could impact natural areas given the proper rare combination of sexually compatible relatives, favorable environment, and reproductive/fitness advantages. As an example, rice grown in tropical countries may be relatively more prone to such processes because of the substantial populations of its wild/

Crop-wild hybridization may also create genotypes with the potential to displace parental taxa in new environments [7]. However, the most important variable affecting gene flow is the degree of relatedness and distance between the crop and the weed, because gene flow is only possible if close relatives are growing near the crop. As a result the possibility of gene flow

weedy relatives that grow naturally in or adjacent to the rice-producing areas [8; 26].

**crops**

T.aestivum Aegilops cylindrical A. tauschii A. triumcialis Agropyron spp

O. sativa O. glaberrima O. barthii O. longistaminata O. rufipogon O. punctata

S. almum S. halepense S. propinguum S. sudanense

The development of crops that are resistant to herbicides is a relatively new technology aimed to improve weed control in agricultural land. Herbicide-resistant crops can be created by standard methods of plant breeding, but the use of genetic engineering techniques is more usual. Herbicide-resistant crops are made resistant by either transgene technology or by selection in cell or tissue culture for mutations that confer herbicide resistance [10]. Glyphosate and glufosinate are herbicides most used in this regard. For example, soybean, corn, cotton, sugar beet, and canola are available as glyphosate- resistant cultivars and some are now widely planted in different countries. Importance of genetically engineered crops is to:


However, public concern about the impact of genetically modified crops on the natural environment encouraged more studies on this aspect in the last few years. Among the possible impacts, the 'escape' of the transgene, either through dispersal of the crop plant outside the agricultural area or through hybridization with wild relatives and thus increase the possibility of "weediness" [41].

In the majority of instances, there is a very low probability that an approved biotech crop introduction could create an environmental risk different from that of a nonbiotech version of the same crop. This however, does not lessen the serious concerns about possible consequences of the escape of transgenes into the environment [41]. Examples of the risks mentioned in the context of gene flow from genetically modified plants are: i) new emerged weeds resulting from an escape by the crop itself; ii) super weeds resulted by hybridization of a (wild/weedy) species with the transgenic crop; iii) genetic erosion (loss of original diversity of wild relatives). To date, all instances of weeds becoming resistant have resulted from the weed evolving its own biochemical mechanism and not by acquiring genes for resistance from the crop. How‐ ever, in some cases it would be possible for the herbicide resistance gene to flow from the crop to the weed [11].

**•** Physiological or biochemical tolerance to the herbicide

**•** Growth stage of weed and crop or other plant development

giving a resistant value for crop plants. Some of these are listed below:

factors when selecting an herbicide or application method.

**9.1. Plant factors and herbicide selectivity**

**9.2. Plant age and growth rate**

or unshaded.

**9.3. Morphology**

Many of the principles and practices of how herbicides used or applied to attain selective chemical and effective weed control are important. These involve the role of plant morphology and physiology, chemical properties, and environmental factors [31]. Herbicide selectivity in one way or another is in direct link with herbicide resistance. Crops are resistant to herbicides selectively used to kill weeds. Even with repeated treatment, crop plants can resist or tolerate higher rates of selective applied herbicide or repeated treatments. This depends on some level of tolerance/resistance higher in crop plants compared with weeds for that specific herbicide or herbicide group. For example, Syrian marjoram (*Origanum syriacum*) was found to with‐ stand up to 4 times higher rates of oxadiazon and oxyfluorfen herbicides either applied on foliage parts or through the soil [32; 33]. Certainly many factors have an important role in

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Plant factors that influence the way weeds and crops respond to herbicides are genetic inheritance, age, growth rate, morphology, growth form and anatomy, and physiological and biochemical processes. The most effective use of herbicides results from considering these

Weed seedlings or young plants are usually killed more easily than large or mature vegetation. In addition, some preemergence herbicides that suppress seed germination are often not effective when used to control larger, better established plants. Plants that are growing rapidly or in shaded places generally are more susceptible to herbicides than are plants of slow growth

The morphology or growth habit of plants can determine the degree of sensitivity to some herbicides. Morphological differences in root structure, location of growing points, and leaf

**•** Weather patterns (temperature, light, wind, rain, etc.)

**•** Variation in microenvironment or micro- topography

**•** Herbicide formulations and surfactants used.

**•** Herbicide application rate

**•** Variation in resource level

**•** Soil type and pH

**•** Time of application

Possible consequences of hybridization and introgression depend on the plant, gene, trait, and ecological factors [39]. In the case where transgenes might be introgressed into "weedy wild relatives", there are concerns about exacerbating "weediness" traits or even the disruption of natural ecosystems. Therefore, to assess the risk of gene flow it needs to be examined not only the probability of genes moving between plants, but how possible is it for the new plants to survive [39].

In general, people ideally would like to minimize or prevent gene flow from transgenic organisms to weedy wild relatives or to places where extensive crop breeding takes place [39]. Three approaches to gene flow mitigation are possible [3].The first is by keeping the genetic modification out of the pollen, preventing the formation of pollen, and keeping the pollen inside the flower. It requires transplastomic plants hence the modified DNA is not situated in the cell's nucleus but is present in plastids, which are cellular compartments outside the nucleus. The second approach relies on male sterile plants unable to produce functioning flowers and therefore cannot release viable pollen. Cytoplasmic male sterile plants are known to produce higher yields. The third approach works by preventing the flowers from opening "cleistogamy" that occurs naturally in some plants. Cleistogamous plants produce flowers which either open only partly or not at all.

However, herbicide-resistant genes have no ecological significance in places where the corresponding herbicide is not used. When paired with a gene that might have an effect in a natural ecosystem, there is a potential problem with gene flow. Repeated application of the herbicide (especially general herbicides) would select for and protect crosses and backcrosses, increasing the possibility of successful gene flow to wild, related species [10].

### **9. Weed control spectrum of selective herbicides and population shifts**

Some plants are genetically tolerant to certain herbicides while others have evolved resistance after repeated exposure to an herbicide. Tolerant and resistant plants usually degrade or metabolize the chemical to nonphytotoxic substances. In some cases of resistance, such as with triazine herbicides, the herbicide does not reach the key site in treated plants. Although tolerance and resistance are common, herbicide selectivity among plants is often conditional; thus it depends on plant, herbicide and environment factors.

Some of the factors that influence herbicide selectivity are as follows:


the same crop. This however, does not lessen the serious concerns about possible consequences of the escape of transgenes into the environment [41]. Examples of the risks mentioned in the context of gene flow from genetically modified plants are: i) new emerged weeds resulting from an escape by the crop itself; ii) super weeds resulted by hybridization of a (wild/weedy) species with the transgenic crop; iii) genetic erosion (loss of original diversity of wild relatives). To date, all instances of weeds becoming resistant have resulted from the weed evolving its own biochemical mechanism and not by acquiring genes for resistance from the crop. How‐ ever, in some cases it would be possible for the herbicide resistance gene to flow from the crop

Possible consequences of hybridization and introgression depend on the plant, gene, trait, and ecological factors [39]. In the case where transgenes might be introgressed into "weedy wild relatives", there are concerns about exacerbating "weediness" traits or even the disruption of natural ecosystems. Therefore, to assess the risk of gene flow it needs to be examined not only the probability of genes moving between plants, but how possible is it for the new plants to

In general, people ideally would like to minimize or prevent gene flow from transgenic organisms to weedy wild relatives or to places where extensive crop breeding takes place [39]. Three approaches to gene flow mitigation are possible [3].The first is by keeping the genetic modification out of the pollen, preventing the formation of pollen, and keeping the pollen inside the flower. It requires transplastomic plants hence the modified DNA is not situated in the cell's nucleus but is present in plastids, which are cellular compartments outside the nucleus. The second approach relies on male sterile plants unable to produce functioning flowers and therefore cannot release viable pollen. Cytoplasmic male sterile plants are known to produce higher yields. The third approach works by preventing the flowers from opening "cleistogamy" that occurs naturally in some plants. Cleistogamous plants produce flowers

However, herbicide-resistant genes have no ecological significance in places where the corresponding herbicide is not used. When paired with a gene that might have an effect in a natural ecosystem, there is a potential problem with gene flow. Repeated application of the herbicide (especially general herbicides) would select for and protect crosses and backcrosses,

**9. Weed control spectrum of selective herbicides and population shifts**

Some plants are genetically tolerant to certain herbicides while others have evolved resistance after repeated exposure to an herbicide. Tolerant and resistant plants usually degrade or metabolize the chemical to nonphytotoxic substances. In some cases of resistance, such as with triazine herbicides, the herbicide does not reach the key site in treated plants. Although tolerance and resistance are common, herbicide selectivity among plants is often conditional;

increasing the possibility of successful gene flow to wild, related species [10].

thus it depends on plant, herbicide and environment factors.

Some of the factors that influence herbicide selectivity are as follows:

to the weed [11].

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survive [39].

which either open only partly or not at all.


Many of the principles and practices of how herbicides used or applied to attain selective chemical and effective weed control are important. These involve the role of plant morphology and physiology, chemical properties, and environmental factors [31]. Herbicide selectivity in one way or another is in direct link with herbicide resistance. Crops are resistant to herbicides selectively used to kill weeds. Even with repeated treatment, crop plants can resist or tolerate higher rates of selective applied herbicide or repeated treatments. This depends on some level of tolerance/resistance higher in crop plants compared with weeds for that specific herbicide or herbicide group. For example, Syrian marjoram (*Origanum syriacum*) was found to with‐ stand up to 4 times higher rates of oxadiazon and oxyfluorfen herbicides either applied on foliage parts or through the soil [32; 33]. Certainly many factors have an important role in giving a resistant value for crop plants. Some of these are listed below:

#### **9.1. Plant factors and herbicide selectivity**

Plant factors that influence the way weeds and crops respond to herbicides are genetic inheritance, age, growth rate, morphology, growth form and anatomy, and physiological and biochemical processes. The most effective use of herbicides results from considering these factors when selecting an herbicide or application method.

#### **9.2. Plant age and growth rate**

Weed seedlings or young plants are usually killed more easily than large or mature vegetation. In addition, some preemergence herbicides that suppress seed germination are often not effective when used to control larger, better established plants. Plants that are growing rapidly or in shaded places generally are more susceptible to herbicides than are plants of slow growth or unshaded.

#### **9.3. Morphology**

The morphology or growth habit of plants can determine the degree of sensitivity to some herbicides. Morphological differences in root structure, location of growing points, and leaf properties between crops or other desirable plants and weeds can determine the selectivi‐ ty pattern of some herbicides. Annual weeds in a perennial crop, meadow, or pasture usually can be controlled by herbicides because of their different root distribution and structure compared to those of perennial plants. For example, perennial crops such as alfalfa can recover from moderate contact herbicide injury to foliage whereas annual weeds, because of their small size and shallow root system, will be killed by the same herbicide application.

**9.5. Genetic inheritance**

weed control duration.

Plant species within a genus usually respond to herbicides in a similar manner, while responses to herbicides by plants in different genera often vary. The reason is that plants with similar taxonomic traits often have similar morphogenetic and enzymatic components. Thus, crops and weeds that belong to the same genera are usually susceptible to the same herbicides and are similarly affected since they have similar biochemistry. This rule is not absolute, however, because varieties of many crops are known to respond differently to the same herbicide and weeds usually adopt different mechanisms of herbicide resistance while crop plants have lost

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Soil factors affect herbicide performance and their effectiveness. These including soilorganic matter content, microorganism populations, soil water table and moisture con‐ tent and soil pH. Organic matter acts through adsorption and release of chemical molecules. Certain herbicides are tightly adsorbed on soil particles and thus become unavailable to weeds. These molecules may be totally inactivated upon their release. Therefore weed control may be complete or not based on the amount of the herbicide adsorbed and whether the held amount on soil colloids is compensated or not before applied. The higher the percentage of organic matter and clay particles, the greater the adsorption in amount and time of herbicide molecules and the lower the herbicide activity and *vice versa*. This requires that some operations should be well managed when soil applied herbicides are used including their incorporation or placement in/on the soil.

Activity of soil microorganisms is another factor affecting activity of soil- applied herbi‐ cides and persistence. Microorganisms may degrade herbicide molecules and feed on organic herbicides. In general, favorable soil factors to microorganism populations stimulate their activity and thus rapid herbicide degradation. Therefore, soil-microbe population is an important factor in increasing or decreasing herbicide persistence and

Soil water also affects herbicide activity and performance. When high amounts of soil water are available or at high soil water levels, herbicide molecules may by hydrated. On the other hand, moisture is necessary to transfer herbicide molecules into the root system and then

Soil pH affects cation exchange capacity of soil particles. Salt or mineral forms of certain herbicides may interact with soil particles under these conditions by exchanging cations or

All above soil factors and others such as soil- root temperature and soil mechanical properties can affect herbicide activity and performance and their effectiveness in control‐ ling weed species and herbicide selectivity. Weeds may become adapted to certain soil

translocate these upward to vegetative parts through the xylem.

anions and thus lead to breakdown of herbicide molecules and inactivation.

many of their traits in breeding programs that present in wild relatives.

**10. Herbicides and edaphic factors**

The meristematic regions of most grasses, such as cereal crops and grassy weeds, are located at the base of the plant or even below the soil surface. The growing points are protected from herbicide exposure by the foliage or soil that surrounds them. Thus, herbicide that contacts only foliage may injure some leaves but will not typically impair the ability of the plant to grow. In contrast, most dicot plants have their meristems exposed at shoot tips and leaf axils. For this reason, these plants are more susceptible than grasses to foliage-applied herbicides, especially of contact action.

Leaf properties of some plants can impart selectivity to certain herbicides, while other plants are effectively controlled. Spray droplets do not adhere well to the surfaces of narrow, upright, waxy leaves that characterize many monocot plants like cereals, onion, and most grasses. Thus, spray droplets do not adequately cover such leaves following herbicide application and the effect of the herbicide is reduced. In contrast, dicot plants have relatively wide leaves that are usually horizontal to the main stem. Leaves of dicot plants, therefore, intercept more spray solution than leaves of grasses and spray droplets spread more evenly over dicot foliage. Herbicide effectiveness is best when spray interception and coverage are greatest and with use of surfactants. However, ecological factors and geographical regions under which weeds are growing have significant influence on herbicide selectivity and rates of applications since they affect or modify weeds morphology and internal anatomy.

#### **9.4. Physiological and biochemical processes**

Plant physiology influences herbicide passage after its application. This process is called "absorption". The extent of herbicide movement in a plant- "translocation"- after it has been absorbed is also a physiological process. Both absorption and translocation are important processes governing herbicide activity and vary markedly among plant species. Generally, plant species that readily absorb and translocate herbicides are most easily killed.

Biochemical and biophysical processes are also important plant factors determining herbicide selectivity. Herbicide adsorption can be responsible for differential herbicide susceptibility among plant species. During this process an herbicide is bound so tightly by cellular constit‐ uents (usually cell walls) that it cannot be translocated readily and thus is inactivated. Membrane stability is another biochemical/biophysical process that results in herbicide selectivity among plants. In this case, the cell membranes of tolerant plants can withstand the disruptive action of the herbicide. The ability of carrot to withstand the toxicity of certain oils is an example of this form of herbicide selectivity.

#### **9.5. Genetic inheritance**

properties between crops or other desirable plants and weeds can determine the selectivi‐ ty pattern of some herbicides. Annual weeds in a perennial crop, meadow, or pasture usually can be controlled by herbicides because of their different root distribution and structure compared to those of perennial plants. For example, perennial crops such as alfalfa can recover from moderate contact herbicide injury to foliage whereas annual weeds, because of their small size and shallow root system, will be killed by the same herbicide

The meristematic regions of most grasses, such as cereal crops and grassy weeds, are located at the base of the plant or even below the soil surface. The growing points are protected from herbicide exposure by the foliage or soil that surrounds them. Thus, herbicide that contacts only foliage may injure some leaves but will not typically impair the ability of the plant to grow. In contrast, most dicot plants have their meristems exposed at shoot tips and leaf axils. For this reason, these plants are more susceptible than grasses to foliage-applied herbicides,

Leaf properties of some plants can impart selectivity to certain herbicides, while other plants are effectively controlled. Spray droplets do not adhere well to the surfaces of narrow, upright, waxy leaves that characterize many monocot plants like cereals, onion, and most grasses. Thus, spray droplets do not adequately cover such leaves following herbicide application and the effect of the herbicide is reduced. In contrast, dicot plants have relatively wide leaves that are usually horizontal to the main stem. Leaves of dicot plants, therefore, intercept more spray solution than leaves of grasses and spray droplets spread more evenly over dicot foliage. Herbicide effectiveness is best when spray interception and coverage are greatest and with use of surfactants. However, ecological factors and geographical regions under which weeds are growing have significant influence on herbicide selectivity and rates of applications since they

Plant physiology influences herbicide passage after its application. This process is called "absorption". The extent of herbicide movement in a plant- "translocation"- after it has been absorbed is also a physiological process. Both absorption and translocation are important processes governing herbicide activity and vary markedly among plant species. Generally,

Biochemical and biophysical processes are also important plant factors determining herbicide selectivity. Herbicide adsorption can be responsible for differential herbicide susceptibility among plant species. During this process an herbicide is bound so tightly by cellular constit‐ uents (usually cell walls) that it cannot be translocated readily and thus is inactivated. Membrane stability is another biochemical/biophysical process that results in herbicide selectivity among plants. In this case, the cell membranes of tolerant plants can withstand the disruptive action of the herbicide. The ability of carrot to withstand the toxicity of certain oils

plant species that readily absorb and translocate herbicides are most easily killed.

application.

especially of contact action.

458 Herbicides - Current Research and Case Studies in Use

affect or modify weeds morphology and internal anatomy.

**9.4. Physiological and biochemical processes**

is an example of this form of herbicide selectivity.

Plant species within a genus usually respond to herbicides in a similar manner, while responses to herbicides by plants in different genera often vary. The reason is that plants with similar taxonomic traits often have similar morphogenetic and enzymatic components. Thus, crops and weeds that belong to the same genera are usually susceptible to the same herbicides and are similarly affected since they have similar biochemistry. This rule is not absolute, however, because varieties of many crops are known to respond differently to the same herbicide and weeds usually adopt different mechanisms of herbicide resistance while crop plants have lost many of their traits in breeding programs that present in wild relatives.

### **10. Herbicides and edaphic factors**

Soil factors affect herbicide performance and their effectiveness. These including soilorganic matter content, microorganism populations, soil water table and moisture con‐ tent and soil pH. Organic matter acts through adsorption and release of chemical molecules. Certain herbicides are tightly adsorbed on soil particles and thus become unavailable to weeds. These molecules may be totally inactivated upon their release. Therefore weed control may be complete or not based on the amount of the herbicide adsorbed and whether the held amount on soil colloids is compensated or not before applied. The higher the percentage of organic matter and clay particles, the greater the adsorption in amount and time of herbicide molecules and the lower the herbicide activity and *vice versa*. This requires that some operations should be well managed when soil applied herbicides are used including their incorporation or placement in/on the soil.

Activity of soil microorganisms is another factor affecting activity of soil- applied herbi‐ cides and persistence. Microorganisms may degrade herbicide molecules and feed on organic herbicides. In general, favorable soil factors to microorganism populations stimulate their activity and thus rapid herbicide degradation. Therefore, soil-microbe population is an important factor in increasing or decreasing herbicide persistence and weed control duration.

Soil water also affects herbicide activity and performance. When high amounts of soil water are available or at high soil water levels, herbicide molecules may by hydrated. On the other hand, moisture is necessary to transfer herbicide molecules into the root system and then translocate these upward to vegetative parts through the xylem.

Soil pH affects cation exchange capacity of soil particles. Salt or mineral forms of certain herbicides may interact with soil particles under these conditions by exchanging cations or anions and thus lead to breakdown of herbicide molecules and inactivation.

All above soil factors and others such as soil- root temperature and soil mechanical properties can affect herbicide activity and performance and their effectiveness in control‐ ling weed species and herbicide selectivity. Weeds may become adapted to certain soil conditions, escape control operations and lead to dominance of well adapted species or populations.

highest number of weed resistant species and biotypes came from the USA (141), Australia (61) and Canada (58). Most numbers of resistant species belong to the families Poaceae, Asteraceae and Amaranthaceae and most frequently mentioned are genera of *Amaranthus* (30 times and 11 species), *Echinochloa* (23 times and 6 species), *Lolium* (20 times and 4 species), *Alopecurus* (12 times and 3 species), *Avena* (11 times and 3 species), *Bromus* (11 times and 5 species), *Conyza* (10 times and 3 species), *Setaria* (9 times and 5 species), *Poa* (8 times and one species), *Ambrosia* (7 times and 2 species), *Digitaria* (6 times and 4 species), *Phalaris* (6 times and 3 species), *Hordeum* (5 times and 2 species) and *Sorghum* (6 times and 3 species). Most are of the grass family usually exhibiting distinct morphological features allowing wide dispersal and escape of herbicide treatment such as encased growing points, vertical leaf arrangement and thick waxy cuticle that reduce herbicide penetration and lead to herbicide droplets bouncing off leaves. Other genera reported are characterized by their prolific seed production and/ or seed polymorphism. All above mentioned genera however, showed multiple resistance to different herbicides groups. Most resisted are herbicides widely and repeatedly used including: glyphosate, paraquat, atrazine and 2,4-D and others used in fields cultivated by genetically modified crops. Some recently developed herbicides are also resisted including chlorsulfuron and sufonylurea group. This phenomenon demonstrates that the herbicide industry and development is far behind weed evolution. On the other hand, weed species and biotypes showing multiple resistance are most common and some are among the world's worst weeds [19] including: *Amaranthus* spp., *Echinochloa* spp., *Avena* spp. and *Chenopodium album* characterized by their polymorphic seed production and phenotypic plasticity. This reflects a great ability to maintain and exhibit high plasticity and possess various mechanisms of

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The precise molecular mechanism of resistance varies with different plants, but in general

**•** Avoiding the herbicide by not absorbing it or, if absorbed, the weed compartmentalizing it

**•** Reducing the uptake or herbicide uptake is not enough to injure the weed or reach lethal

**•** Changing the structure of the target site of the herbicide so the plant is no longer sensitive

**•** Target site mutation and changes in structure lead to insensitive plants and failure herbicide

**•** Deactivating the herbicide by chemical alteration or herbicide metabolism before reaching

However, resistance mechanisms through which different weed species resist herbicide

**•** Reduce herbicide translocation to the key site or binding it into certain plant constituent

**•** Sequestration by complete physical removal of the herbicide from the key site

treatments are many and varied but most are physio-chemically based (Table 2).

herbicide resistance.

level.

binding.

target site

away from its target site.

plants resist herbicides in one of the following ways:

### **11. Weed resistance and dormancy, avoidance and weed density**

Dormancy is the state at which seeds in the soil or buds are not germinating or growing due to external conditions exert influences on physiological and biochemical internal processes including enzymes activities, food transport to embryo and metabolism. This state is keeping seeds or buds safe until the cause of dormancy is over. This behavior is important to maintain genetic line and continuity of the species in changeable environment. Under conditions of herbicide application, some of these chemicals are absorbed by seeds or dormant buds while others are not. These result differences in germination, emergence and growth patterns of different weed species. However, some herbicides may stimulate seed germination while others inhibit this process or even kill seed embryo. Differences also exist in hardness and permeability of seed coat of different weed species at which species of Chenopodiaceae and Fabaceae are good examples. These characters cause differences in germination and growth of seedlings and may confer another cause of herbicide resistance. Avoidance of herbicide toxicity may result from seed interring into dormancy and not further responding to the applied herbicide with no absorption or translocation of the herbicide into the embryo. In addition, herbicide molecules may be deactivated or degraded inside the seed itself by some oxidative enzymes or may bound into certain constituent inside the seed.

On the other hand, stimulation of weed seeds to germinate using certain herbicides also exist and allows higher seedlings emergence and partitioning of herbicide molecules among individuals of weed species. Division of herbicide molecules among high number of emerged seedlings would further diluted herbicide inside weed plants.

All above mentioned factors should be considered when herbicide-resistance is discussed. These may cause great differences in weed growth patterns and distribution in the field.

### **12. Weed resistance updates and resistance mechanisms**

With continued dependence on herbicides for weed control and with the absence of other methods and herbicide rotation, the resistance problem is extenuated and the number of resistant weed species and biotypes is dramatically increased. At present, the reported herbicide resistant weeds are approaching 393 (species and their biotypes). These represent 211 species (124 dicots and 87 monocots) and detected from over 680,000 fields [21; 44] reported from 61 countries from all over the globe. However, the highest number of resistant species was reported from the advanced countries indicating efficient and rapid detection with available technology to diagnose, discover and deal with this issue. However, the highest number of weeds reported resist the main three groups of herbicides based on site of action including; the ALS (127 weeds), Photosystem II (69) and the ACCase (42) inhibitors. The highest number of weed resistant species and biotypes came from the USA (141), Australia (61) and Canada (58). Most numbers of resistant species belong to the families Poaceae, Asteraceae and Amaranthaceae and most frequently mentioned are genera of *Amaranthus* (30 times and 11 species), *Echinochloa* (23 times and 6 species), *Lolium* (20 times and 4 species), *Alopecurus* (12 times and 3 species), *Avena* (11 times and 3 species), *Bromus* (11 times and 5 species), *Conyza* (10 times and 3 species), *Setaria* (9 times and 5 species), *Poa* (8 times and one species), *Ambrosia* (7 times and 2 species), *Digitaria* (6 times and 4 species), *Phalaris* (6 times and 3 species), *Hordeum* (5 times and 2 species) and *Sorghum* (6 times and 3 species). Most are of the grass family usually exhibiting distinct morphological features allowing wide dispersal and escape of herbicide treatment such as encased growing points, vertical leaf arrangement and thick waxy cuticle that reduce herbicide penetration and lead to herbicide droplets bouncing off leaves. Other genera reported are characterized by their prolific seed production and/ or seed polymorphism. All above mentioned genera however, showed multiple resistance to different herbicides groups. Most resisted are herbicides widely and repeatedly used including: glyphosate, paraquat, atrazine and 2,4-D and others used in fields cultivated by genetically modified crops. Some recently developed herbicides are also resisted including chlorsulfuron and sufonylurea group. This phenomenon demonstrates that the herbicide industry and development is far behind weed evolution. On the other hand, weed species and biotypes showing multiple resistance are most common and some are among the world's worst weeds [19] including: *Amaranthus* spp., *Echinochloa* spp., *Avena* spp. and *Chenopodium album* characterized by their polymorphic seed production and phenotypic plasticity. This reflects a great ability to maintain and exhibit high plasticity and possess various mechanisms of herbicide resistance.

conditions, escape control operations and lead to dominance of well adapted species or

Dormancy is the state at which seeds in the soil or buds are not germinating or growing due to external conditions exert influences on physiological and biochemical internal processes including enzymes activities, food transport to embryo and metabolism. This state is keeping seeds or buds safe until the cause of dormancy is over. This behavior is important to maintain genetic line and continuity of the species in changeable environment. Under conditions of herbicide application, some of these chemicals are absorbed by seeds or dormant buds while others are not. These result differences in germination, emergence and growth patterns of different weed species. However, some herbicides may stimulate seed germination while others inhibit this process or even kill seed embryo. Differences also exist in hardness and permeability of seed coat of different weed species at which species of Chenopodiaceae and Fabaceae are good examples. These characters cause differences in germination and growth of seedlings and may confer another cause of herbicide resistance. Avoidance of herbicide toxicity may result from seed interring into dormancy and not further responding to the applied herbicide with no absorption or translocation of the herbicide into the embryo. In addition, herbicide molecules may be deactivated or degraded inside the seed itself by some

On the other hand, stimulation of weed seeds to germinate using certain herbicides also exist and allows higher seedlings emergence and partitioning of herbicide molecules among individuals of weed species. Division of herbicide molecules among high number of emerged

All above mentioned factors should be considered when herbicide-resistance is discussed. These may cause great differences in weed growth patterns and distribution in the field.

With continued dependence on herbicides for weed control and with the absence of other methods and herbicide rotation, the resistance problem is extenuated and the number of resistant weed species and biotypes is dramatically increased. At present, the reported herbicide resistant weeds are approaching 393 (species and their biotypes). These represent 211 species (124 dicots and 87 monocots) and detected from over 680,000 fields [21; 44] reported from 61 countries from all over the globe. However, the highest number of resistant species was reported from the advanced countries indicating efficient and rapid detection with available technology to diagnose, discover and deal with this issue. However, the highest number of weeds reported resist the main three groups of herbicides based on site of action including; the ALS (127 weeds), Photosystem II (69) and the ACCase (42) inhibitors. The

**11. Weed resistance and dormancy, avoidance and weed density**

oxidative enzymes or may bound into certain constituent inside the seed.

seedlings would further diluted herbicide inside weed plants.

**12. Weed resistance updates and resistance mechanisms**

populations.

460 Herbicides - Current Research and Case Studies in Use

The precise molecular mechanism of resistance varies with different plants, but in general plants resist herbicides in one of the following ways:


However, resistance mechanisms through which different weed species resist herbicide treatments are many and varied but most are physio-chemically based (Table 2).


Because weeds contain a tremendous amount of genetic variation that allows them to survive under a variety of environmental conditions, the development of a resistant species is brought about through selection pressure imposed by the continuous use of an herbicide or herbicides of similar mechanism of action. Long residual pre-emergence herbicides or repeated applica‐

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Factors in general that can lead to or accelerate the development of herbicide resistance include

Weed characteristics conducive to rapid development of resistance to a particular herbicide

**•** Relatively rapid turnover of the seed bank due to high percentage of seed germination each

**•** One weed which would normally be controlled but not controlled while others were

Herbicide characteristics which lead to rapid development of herbicide resistance in weed

Cultural practices can also increase the selection pressure for the development of herbicideresistant biotypes. In general, complete reliance on herbicides for weed control can greatly

**•** Little cultivation or zero tillage for weed control or no elimination of weeds that escape

**•** Continuous or repeated use of a single herbicide or several herbicides that have the same

tion of post-emergence herbicides will further increase selection pressure.

weed characteristics, chemical properties and cultural practices.

**•** Weeds having short life cycles (annuals).

year (i.e., little seed dormancy).

**•** High frequency of resistant gene (s).

**•** Broad spectrum of weed control. **•** Long residual activity in the soil.

**•** Level of selection pressure imposed by the herbicide

**•** Several reproductive generations per growing season.

**•** A single site of action of the same herbicide continuously is used.

enhance the occurrence of herbicide-resistant weeds. Other factors include:

**•** High herbicide use rate relative to the amount needed for weed control.

**•** Extreme susceptibility to a particular herbicide.

**•** Shift from crop rotations towards mono cropping.

**•** High seed production.

include:

removed.

biotypes include:

herbicide control.

mechanism of action.

**•** Complete weed control

**Table 2.** Herbicide resistant weeds summary table (Thursday, November 08, 2012)

### **13. Factors enhancing herbicide resistance**

All natural weed populations, regardless of the application of any herbicide, probably contain biotypes that resist herbicides. Repeated application of an herbicide exposes the weed population to a selection pressure which may lead to an increase in the number of surviving resistant individuals in the population. As a consequence, the resistant weed population may increase to a level that adequate weed control cannot be achieved by the application of that herbicide [18]. Factors enhancing herbicide resistance include: the use of a single herbicide or herbicides of same mechanism of action, same formulation, same method of application, time of application, weather conditions during spraying, weed-density and application rate, surfactants, herbicide family and mechanism of action, crop rotation, and employed control methods.

Because weeds contain a tremendous amount of genetic variation that allows them to survive under a variety of environmental conditions, the development of a resistant species is brought about through selection pressure imposed by the continuous use of an herbicide or herbicides of similar mechanism of action. Long residual pre-emergence herbicides or repeated applica‐ tion of post-emergence herbicides will further increase selection pressure.

Factors in general that can lead to or accelerate the development of herbicide resistance include weed characteristics, chemical properties and cultural practices.

Weed characteristics conducive to rapid development of resistance to a particular herbicide include:


**Herbicide Group Site of Action HRAC Group**

B

F3

K3

F1

F2

ALS inhibitors Inhibition of acetolactate synthase ALS (acetohydroxyacid synthase AHAS)

target)

acids)

4-HPPD inhibitors Bleaching: Inhibition of 4-hydroxyphenyl-pyruvatedioxygenase (4-HPPD)

**Table 2.** Herbicide resistant weeds summary table (Thursday, November 08, 2012)

**13. Factors enhancing herbicide resistance**

Carotenoid biosynthesis

462 Herbicides - Current Research and Case Studies in Use

Source: 21; Updated: November, 2012

inhibitors

methods.

Photosystem II inhibitors Inhibition of photosynthesis at photosystem II C1 ACCase inhibitors Inhibition of acetyl CoA carboxylase (ACCase) A Synthetic Auxins Synthetic auxins (action like indoleacetic acid) O Bipyridiliums Photosystem-I-electron diversion D Glycines Inhibition of EPSP synthase G Ureas and amides Inhibition of photosynthesis at photosystem II C2 Dinitroanilines and others Microtubule assembly inhibition K1 Thiocarbamates and others Inhibition of lipid synthesis - not ACCase inhibition N PPO inhibitors Inhibition of protoporphyrinogen oxidase (PPO) E Triazoles, ureas, isoxazolidiones Bleaching: Inhibition of carotenoid biosynthesis (unknown

Nitriles and others Inhibition of photosynthesis at photosystem II C3 Chloroacetamides and others Inhibition of cell division (Inhibition of very long chain fatty

phytoene desaturase step (PDS)

Glutamine synthase inhibitors Inhibition of glutamine synthetase H Arylaminopropionic acids Unknown Z Unknown Unknown Z

Mitosis inhibitors Inhibition of mitosis / microtubule polymerization inhibitor K2 Cellulose inhibitors Inhibition of cell wall (cellulose) synthesis L

All natural weed populations, regardless of the application of any herbicide, probably contain biotypes that resist herbicides. Repeated application of an herbicide exposes the weed population to a selection pressure which may lead to an increase in the number of surviving resistant individuals in the population. As a consequence, the resistant weed population may increase to a level that adequate weed control cannot be achieved by the application of that herbicide [18]. Factors enhancing herbicide resistance include: the use of a single herbicide or herbicides of same mechanism of action, same formulation, same method of application, time of application, weather conditions during spraying, weed-density and application rate, surfactants, herbicide family and mechanism of action, crop rotation, and employed control

Bleaching: Inhibition of carotenoid biosynthesis at the


Herbicide characteristics which lead to rapid development of herbicide resistance in weed biotypes include:


Cultural practices can also increase the selection pressure for the development of herbicideresistant biotypes. In general, complete reliance on herbicides for weed control can greatly enhance the occurrence of herbicide-resistant weeds. Other factors include:


### **14. Management of herbicide resistance**

Herbicide-resistant weed populations can be managed following an integrated weed control program. The following practices are important for an effective management strategy:

of how well particular herbicides have controlled them. Farmers should check for weedy patches in patterns consistent with application problems and hand-weeding these patches.

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**•** Always weed free crop seeds should be used that greatly minimize introduction seeds of

**•** Implementation of integrated weed management. This is important for effective control of

**•** Monitoring fields for weed escapes for resistant and susceptible biotypes. A resistance problem may not become visible until 30 percent or more of the weed population is no longer controlled. Check to see if the escapes are of one species or a mixture of species. lf a mixture, the problem is more likely related to the environment or the herbicide application. If only one species was not controlled, the problem is likely to be resistance, especially if the species was controlled by the herbicide in the past and if the same herbicide has been used repeat‐

**•** Implementation of prevention methods of weed control. All measures aimed at prevention of weed introduction to fields and their dispersal should be strictly followed including

**•** Alternating spring and winter crops, thus tillage and herbicides are used at different times in the different crops. Weed biotypes that survive in one crop could be killed in the other.

**•** Changing herbicide program, if weed resistance occurs, herbicides with other sites of action and other weed management practices must be used in an integrated management strategy. However, weed management strategies that discourage the evolution of herbicide resistance

**◦** Use herbicide only when necessary and where possible herbicide application should be

**◦** Apply herbicides in tank mixed, pre-packed, or sequential mixtures of multiple site of

**◦** Regularly monitor your crops so that resistant patches can be observed in time to be

**◦** Planting new herbicide-resistant crop varieties should not result in more than two consecutive applications of herbicides with the same site of action against the same weed unless other effective control practices are also included in the management system.

**◦** Respond quickly to changes in weed populations to restrict spread of weeds that may

**◦** Never use unregistered mixtures, follow label recommendation at all times

**◦** Apply the herbicide at the correct leaf stage of the weed and the crop.

herbicide-resistant biotypes.

edly in the field.

action.

all weeds including herbicide-resistance.

governmental quarantine regulations.

should include the following:

based on economic threshold.

controlled with, for instance, spot spraying.

have been selected for resistance.

**◦** Calibrate sprayer correctly before using herbicides


of how well particular herbicides have controlled them. Farmers should check for weedy patches in patterns consistent with application problems and hand-weeding these patches.

**•** Orchard and vineyard weeds.

464 Herbicides - Current Research and Case Studies in Use

**14. Management of herbicide resistance**

chemical families may have the same site of action.

and in the crop(s) planted within 3 years.

development of resistant weed populations.

mended to be combined with herbicide treatments.

compressed air to remove seeds).

at about the same time.

trums. This would help in managing evolution of weed resistance.

alone may not be enough to avoid resistance development in this case.

Herbicide-resistant weed populations can be managed following an integrated weed control program. The following practices are important for an effective management strategy:

**•** Herbicide rotation. Adopting this method, it should be known that herbicides of different

**•** Using mixtures of herbicides with different modes of action and overlapping weed spec‐

**•** Crop rotation. Crops differ in their competitiveness against weeds. Plant crops having a different season of growth, different registered herbicides and crops for which there are alternate methods of weed control. Rotation breaks down weed population and prevents the build up of resistance to herbicides. In addition, different crops may require different types of herbicides and thus herbicides may be rotated as well. However, some herbicide groups include different chemicals that can be used in different crops; therefore crop rotation

**•** Herbicides with the same site of action should not be applied or used in both fallow years

**•** Growers should keep rotating methods of weed control. Non-chemical control techniques including tillage, hand-weeding before flowering, mulching, soil solarization, prevention methods of weed dispersal (certified seed, clean equipments, use a power washer or

**•** Farmers should only use non- or short-residual herbicides and avoid using persistent chemicals and not applying them repeatedly within a growing season. This method would reduce the selection of herbicide-resistant weed biotypes. However, repeated applications within a single growing season of certain herbicides (paraquat, glyphosate) also lead to

**•** Where possible mechanical weed control such as rotary hoeing and cultivation is recom‐

**•** Weed escapes of resistant biotypes may be eliminated by cultivation in row crops. Fallow tillage can control herbicide-resistant and susceptible weed populations when they emerge

**•** Accurate record keeping. Farmers should be familiar with the history of herbicides use in their fields. Also keep tracking the weed species that have been present in a given field and

**•** Herbicide-resistant weeds should be controlled before flowering and seed setting.

**•** Roadside weeds.

	- **◦** Use herbicide only when necessary and where possible herbicide application should be based on economic threshold.
	- **◦** Apply herbicides in tank mixed, pre-packed, or sequential mixtures of multiple site of action.
	- **◦** Never use unregistered mixtures, follow label recommendation at all times
	- **◦** Regularly monitor your crops so that resistant patches can be observed in time to be controlled with, for instance, spot spraying.
	- **◦** Apply the herbicide at the correct leaf stage of the weed and the crop.
	- **◦** Calibrate sprayer correctly before using herbicides
	- **◦** Planting new herbicide-resistant crop varieties should not result in more than two consecutive applications of herbicides with the same site of action against the same weed unless other effective control practices are also included in the management system.
	- **◦** Respond quickly to changes in weed populations to restrict spread of weeds that may have been selected for resistance.

**◦** Encourage railroads, public utilities, highway departments and similar organizations that use total vegetation control programs and vegetation management systems that do not lead to selection of herbicide resistant weeds. Resistant weeds from total vegetation control areas frequently spread to cropland. Chemical companies, governmental agen‐ cies, and farm organizations can all help in this effort.

**15. Conclusion**

**Author details**

Jamal R. Qasem

**References**

Weeds either leave (disappear), adapt, tolerate or resist any unfavorable environmental conditions that influence their normal growth and life strategies. Herbicide resistance is a complex phenomenon resulting from altered herbicide target enzyme, enhanced herbicide metabolism or reduced herbicide absorption and/or translocation. It is a survival strategy through which many successful weed species and/or biotypes counteract or escape chemical hazards. Weeds expressing this phenomenon have developed some morpho- (behaviorist), physio-, and/ or biochemical mechanism/s allowing existence. However, two theories are mainly considered: the mutation and the natural selection [17]. Colonizers, as well as some specialist weeds of high seed production and polymorphic characteristics, have rapid re‐ sponses to prevailing environmental conditions and high ability to express herbicide-resistant genes and exhibit wide ecological variations [28]. This phenomenon is well documented in agricultural as well as other disturbed habitats while the list of weed resistant species gets longer with continued dependence on herbicides for weed control. From the information presented in this chapter, it is clearly demonstrated that herbicide resistance in weeds is far exceeding herbicide technology and industry. Most problematic weed species are genetically related to major food crops including wheat, rice and maize. This may pose another danger for the genetic industry and genetically engineered crops of wild relatives. Away from weed biology and resistance control, methods of weed control must be integrated and continuously rotated for effective weed control and prevention of weed resistance. This however, may not be achieved in absence of information and field data and well managed weed control strategies,

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considering all the factors that influence weed life and development.

Department of Plant Protection, Faculty of Agriculture, University of Jordan, Amman, Jordan

[1] Anonymous-I. (2010) Evolution. Retrieved December, 2010. Available at: *http://*

[2] Anonymous-II. (2010). Gene flow. Retrieved December, 2010. Available at: *http://*

Address all correspondence to: jrqasem@ju.edu.jo

*www.biologydaily.com/biology/Evolution*.

*en.wikipedia.org/wiki/Gene\_flow*.

	- **◦** Clean tillage and harvest equipments before moved from infested to clean fields from weed resistant species.
	- **◦** Total weed control in uncultivated places or sites
	- **◦** Close cultivation
	- **◦** Monitor hand weeding to insure more than 90% removal of weeds in the crop row.
	- **◦** Prevention of weed seed spread through:

– Watch for weed patches that do not have a regular shape that would indicate an herbicide application problem.

Herbicide resistance however, provides a basic understanding of the genetic basis of weedi‐ ness, while the development of weed genomics would provide three predictable and useful outcomes. The first is the identification of genes that could improve crop yields. The second is to improve our understanding of the evolution of herbicide resistance and the to aid in the identification of novel herbicide targets. Currently, there is little (if any) solid predictive capability of why some weeds develop resistance and others do not. Third, our understanding of weed biology would be exponentially expanded [6].

Research has recently been performed to assess the ability to cripple the effect of transgenes. The goal here is for the transgenic effect to not be as strong if it went to a wild relative. In one case, the genetic background of the crop weakened the weedy relative. In another case, the weakness was built into the genetic construct, called *transgenic mitigation*, in which an herbi‐ cide-resistant gene was paired with a dwarfing gene. In either case, transgenic weeds were less competitive than their non-transgenic parent weeds [39].

### **15. Conclusion**

**◦** Encourage railroads, public utilities, highway departments and similar organizations that use total vegetation control programs and vegetation management systems that do not lead to selection of herbicide resistant weeds. Resistant weeds from total vegetation control areas frequently spread to cropland. Chemical companies, governmental agen‐

**•** To keep herbicide-resistant weeds under control, the following strategies should be also

**◦** Clean tillage and harvest equipments before moved from infested to clean fields from

**◦** Monitor hand weeding to insure more than 90% removal of weeds in the crop row.

cies, and farm organizations can all help in this effort.

incorporated into a weed management plan:

**◦** Prevention of weed seed spread through:

– Enter the field with resistant plants last.

**◦** Total weed control in uncultivated places or sites

– Use a power washer or compressed air to remove seeds.

of weed biology would be exponentially expanded [6].

less competitive than their non-transgenic parent weeds [39].

– Recognizing patterns of weed escapes typical of resistant plants

– Watch for small weed patches that appear in the same place in the next crop.

– Watch for weed patches that do not have a regular shape that would indicate an herbicide

Herbicide resistance however, provides a basic understanding of the genetic basis of weedi‐ ness, while the development of weed genomics would provide three predictable and useful outcomes. The first is the identification of genes that could improve crop yields. The second is to improve our understanding of the evolution of herbicide resistance and the to aid in the identification of novel herbicide targets. Currently, there is little (if any) solid predictive capability of why some weeds develop resistance and others do not. Third, our understanding

Research has recently been performed to assess the ability to cripple the effect of transgenes. The goal here is for the transgenic effect to not be as strong if it went to a wild relative. In one case, the genetic background of the crop weakened the weedy relative. In another case, the weakness was built into the genetic construct, called *transgenic mitigation*, in which an herbi‐ cide-resistant gene was paired with a dwarfing gene. In either case, transgenic weeds were

weed resistant species.

466 Herbicides - Current Research and Case Studies in Use

**◦** Close cultivation

– Use of clean equipment.

application problem.

Weeds either leave (disappear), adapt, tolerate or resist any unfavorable environmental conditions that influence their normal growth and life strategies. Herbicide resistance is a complex phenomenon resulting from altered herbicide target enzyme, enhanced herbicide metabolism or reduced herbicide absorption and/or translocation. It is a survival strategy through which many successful weed species and/or biotypes counteract or escape chemical hazards. Weeds expressing this phenomenon have developed some morpho- (behaviorist), physio-, and/ or biochemical mechanism/s allowing existence. However, two theories are mainly considered: the mutation and the natural selection [17]. Colonizers, as well as some specialist weeds of high seed production and polymorphic characteristics, have rapid re‐ sponses to prevailing environmental conditions and high ability to express herbicide-resistant genes and exhibit wide ecological variations [28]. This phenomenon is well documented in agricultural as well as other disturbed habitats while the list of weed resistant species gets longer with continued dependence on herbicides for weed control. From the information presented in this chapter, it is clearly demonstrated that herbicide resistance in weeds is far exceeding herbicide technology and industry. Most problematic weed species are genetically related to major food crops including wheat, rice and maize. This may pose another danger for the genetic industry and genetically engineered crops of wild relatives. Away from weed biology and resistance control, methods of weed control must be integrated and continuously rotated for effective weed control and prevention of weed resistance. This however, may not be achieved in absence of information and field data and well managed weed control strategies, considering all the factors that influence weed life and development.

### **Author details**

#### Jamal R. Qasem

Address all correspondence to: jrqasem@ju.edu.jo

Department of Plant Protection, Faculty of Agriculture, University of Jordan, Amman, Jordan

### **References**


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**Chapter 18**

**Pesticide Tank Mixes: An Environmental Point of View**

During the last decades, human activity has affected the aquatic and terrestrial ecosystems' sustainability. None of these activities has damaged the environment as severely as agricul‐

Current agricultural practices have negatively affected aquatic and terrestrial ecosystems by

Pesticides are a heterogeneous category of chemical products destined to pest, disease and weed control including several types, such as insecticides, fungicides, herbicides, nematicides

Nowadays, such chemical product applications have been considered the most efficient plant protection procedures and have significantly contributed to the improvement of crop

Nevertheless, the claimed objective of supplying the population with enough food does not justify damaging the environment, just because small quantities of pesticides are known to efficiently control pests, diseases and weeds. However, most of them are rapidly spread out

The use of chemical molecules in agriculture increased after the Second World War with the advent of DDT (dichloro-diphenyl-trichloroethane). DDT was discovered in 1939 by Paul Müller (Swiss entomologist) and its worldwide use was rapidly expanded due to its large

> © 2013 Tornisielo 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 Tornisielo 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.

affecting all living beings (flora and fauna, including humans).

destroying habitats, deforesting to increase cropping areas and applying pesticides.

Valdemar Luiz Tornisielo, Rafael Grossi Botelho,

Additional information is available at the end of the chapter

Paulo Alexandre de Toledo Alves,

Eloana Janice Bonfleur and Sergio Henrique Monteiro

http://dx.doi.org/10.5772/55948

**1. Introduction**

tural practices.

and others.

productivity.

## **Pesticide Tank Mixes: An Environmental Point of View**

Valdemar Luiz Tornisielo, Rafael Grossi Botelho, Paulo Alexandre de Toledo Alves, Eloana Janice Bonfleur and Sergio Henrique Monteiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55948

### **1. Introduction**

During the last decades, human activity has affected the aquatic and terrestrial ecosystems' sustainability. None of these activities has damaged the environment as severely as agricul‐ tural practices.

Current agricultural practices have negatively affected aquatic and terrestrial ecosystems by destroying habitats, deforesting to increase cropping areas and applying pesticides.

Pesticides are a heterogeneous category of chemical products destined to pest, disease and weed control including several types, such as insecticides, fungicides, herbicides, nematicides and others.

Nowadays, such chemical product applications have been considered the most efficient plant protection procedures and have significantly contributed to the improvement of crop productivity.

Nevertheless, the claimed objective of supplying the population with enough food does not justify damaging the environment, just because small quantities of pesticides are known to efficiently control pests, diseases and weeds. However, most of them are rapidly spread out affecting all living beings (flora and fauna, including humans).

The use of chemical molecules in agriculture increased after the Second World War with the advent of DDT (dichloro-diphenyl-trichloroethane). DDT was discovered in 1939 by Paul Müller (Swiss entomologist) and its worldwide use was rapidly expanded due to its large

action range, low cost and efficiency in the control of tropical disease vectors, such as typhoid fever and malaria [1].

possibly providing a larger pest control range and efficacy, when compared to the single

Pesticide Tank Mixes: An Environmental Point of View

http://dx.doi.org/10.5772/55948

475

Nevertheless, the herbicide mixture might induce, for instance, interactions before or after reaching the target-plant, by altering the product action in synergistic, antagonistic or additive ways. One common practice is the simultaneous application of herbicides with and without residual effect in order to increase the weed species control range and/or the control period. Another practice is the addition of adjuvants to improve herbicide performance to control weeds. The simultaneous application of pesticides (concerning the species-target to be controlled) might induce undesirable (antagonistic, synergistic or additive) reactions, depend‐ ing on the herbicide type and plant species [4]. When the mixture induces an antagonistic reaction, it means that a lower weed control action than expected is observed. When the mixture induces a synergistic reaction, it means that a higher weed control than expected is observed. And, finally, when the mixture induces an additive reaction, it means that no change

Several studies have elucidated the questions about synergistic and antagonistic effects of active ingredient mixtures on weed control, for instance, the studies with glyphosate reported

The application of pesticides plus adjuvants has also been a usual practice. The adjuvant enhances the active ingredient action [6]. In other words, the adjuvant substance induces the herbicide molecule uptake by leaf tissues, by accelerating the product penetration through plant cuticles. The most common types are the biosurfactants, mineral or vegetal oils, synthetic

The tank mixture practice or different individual pesticide applications at short intervals might result in multiple pesticide residues on foods, as observed by Gebara et al. [8], when monitoring food samples in São Paulo metropolis, Brazil, during the period between 1994 and 2001. The authors found multiple pesticide residues in 5.8% of vegetable samples analyzed and 11.4%

Gebara et al. [9] alerted for the violation risk of the Theoretical Maximum Dietary Intake (TMDI), which is calculated by the relationship between the Limit of Maximum Residues (LMR, mg kg-1) established for a pesticide in a food and the daily consumption (DC, kg day-1), based on the individual diet. The presence of multiple pesticide residues in foods due to the use of tank mixtures, might lead to the extrapolation of toxicological parameters for the

Weed control with pesticide tank mixtures has been widely studied concerning mixture effectiveness, component antagonism and/or synergism. However, there is little information

or natural polymers, humectants, organic salts, buffer solutions, and others [7].

acceptable daily intake (ADI), mainly for children and nursing women.

**3. Pesticide tank mixtures environmental effects**

by Vidal et al. [4], Shaw and Arnold [3], Selleck and Baird [5].

product application. For these reasons, this technique is preferred by farmers [3].

in weed control is observed.

of fruit samples.

**3.1. Soil**

on environmental issues.

After the release of DDT, a large range of molecule groups destined to crop protection were developed and commercialized. In 1962, the book "Silent Spring" was the first act of environ‐ ment manifest against DDT, describing the bird population decrease (from the top of the food chain) attributed to its indiscriminate use.

After the 1960's, the use of chemical products in agriculture rapidly increased and it was associated with the appearance of environmental and human health problems.

The frequent and incorrect use of pesticides have caused soil, atmosphere, food and water resource (superficial/underwater) contaminations, negatively affecting aquatic and terrestrial organisms as well as frequently causing toxicity to the human population.

Therefore, studies are urgently needed to make environmental monitoring procedures viable in order to detect potential contamination risks and give support to public actions for envi‐ ronmental safety and agriculture sustainability.

Currently, product mixtures (associations between one or more molecules) are applied in agriculture instead of individual molecules; therefore, previous studies that focused on only one molecule should now consider molecule mixtures.

The existence of such a large variety of pests, diseases and weeds affecting yields have led farmers to use product mixtures, aiming at efficiently managing crop protection. Such mixtures, also called product associations, enter the environment in a different way compared to the individual product application. Thus, more studies are required about these mixtureenvironment interactions and possible interactions between molecules and consequent interferences in the environment.

Although mixtures have been intensively studied concerning their agronomic efficacy, little information is found about their implications on environmental safety.

In this chapter, the tank mixture subject is approached from an environmental point of view, explaining the chemical product mixture interactions and the possible contaminant effects. Studies on the product-environment interactions are presented to provide the main available information as support to future studies and decisions in environmental sustainability and safety.

### **2. Agronomic characteristics of tank mixtures**

Tank mixtures are associations among two or more chemical products (pesticides) or among chemical products and fertilizers in a unique tank for application in crops. This practice is common in Australia, Canada, U.S.A and United Kingdom, where there are recommendations on application procedures, incompatibilities, and safety [2].

Concerning agricultural practices, the tank mixture of two or more chemical products might be a good application strategy, saving fuel and labor-hours, causing less soil compaction, and possibly providing a larger pest control range and efficacy, when compared to the single product application. For these reasons, this technique is preferred by farmers [3].

Nevertheless, the herbicide mixture might induce, for instance, interactions before or after reaching the target-plant, by altering the product action in synergistic, antagonistic or additive ways. One common practice is the simultaneous application of herbicides with and without residual effect in order to increase the weed species control range and/or the control period. Another practice is the addition of adjuvants to improve herbicide performance to control weeds. The simultaneous application of pesticides (concerning the species-target to be controlled) might induce undesirable (antagonistic, synergistic or additive) reactions, depend‐ ing on the herbicide type and plant species [4]. When the mixture induces an antagonistic reaction, it means that a lower weed control action than expected is observed. When the mixture induces a synergistic reaction, it means that a higher weed control than expected is observed. And, finally, when the mixture induces an additive reaction, it means that no change in weed control is observed.

Several studies have elucidated the questions about synergistic and antagonistic effects of active ingredient mixtures on weed control, for instance, the studies with glyphosate reported by Vidal et al. [4], Shaw and Arnold [3], Selleck and Baird [5].

The application of pesticides plus adjuvants has also been a usual practice. The adjuvant enhances the active ingredient action [6]. In other words, the adjuvant substance induces the herbicide molecule uptake by leaf tissues, by accelerating the product penetration through plant cuticles. The most common types are the biosurfactants, mineral or vegetal oils, synthetic or natural polymers, humectants, organic salts, buffer solutions, and others [7].

The tank mixture practice or different individual pesticide applications at short intervals might result in multiple pesticide residues on foods, as observed by Gebara et al. [8], when monitoring food samples in São Paulo metropolis, Brazil, during the period between 1994 and 2001. The authors found multiple pesticide residues in 5.8% of vegetable samples analyzed and 11.4% of fruit samples.

Gebara et al. [9] alerted for the violation risk of the Theoretical Maximum Dietary Intake (TMDI), which is calculated by the relationship between the Limit of Maximum Residues (LMR, mg kg-1) established for a pesticide in a food and the daily consumption (DC, kg day-1), based on the individual diet. The presence of multiple pesticide residues in foods due to the use of tank mixtures, might lead to the extrapolation of toxicological parameters for the acceptable daily intake (ADI), mainly for children and nursing women.

### **3. Pesticide tank mixtures environmental effects**

#### **3.1. Soil**

action range, low cost and efficiency in the control of tropical disease vectors, such as typhoid

After the release of DDT, a large range of molecule groups destined to crop protection were developed and commercialized. In 1962, the book "Silent Spring" was the first act of environ‐ ment manifest against DDT, describing the bird population decrease (from the top of the food

After the 1960's, the use of chemical products in agriculture rapidly increased and it was

The frequent and incorrect use of pesticides have caused soil, atmosphere, food and water resource (superficial/underwater) contaminations, negatively affecting aquatic and terrestrial

Therefore, studies are urgently needed to make environmental monitoring procedures viable in order to detect potential contamination risks and give support to public actions for envi‐

Currently, product mixtures (associations between one or more molecules) are applied in agriculture instead of individual molecules; therefore, previous studies that focused on only

The existence of such a large variety of pests, diseases and weeds affecting yields have led farmers to use product mixtures, aiming at efficiently managing crop protection. Such mixtures, also called product associations, enter the environment in a different way compared to the individual product application. Thus, more studies are required about these mixtureenvironment interactions and possible interactions between molecules and consequent

Although mixtures have been intensively studied concerning their agronomic efficacy, little

In this chapter, the tank mixture subject is approached from an environmental point of view, explaining the chemical product mixture interactions and the possible contaminant effects. Studies on the product-environment interactions are presented to provide the main available information as support to future studies and decisions in environmental sustainability and

Tank mixtures are associations among two or more chemical products (pesticides) or among chemical products and fertilizers in a unique tank for application in crops. This practice is common in Australia, Canada, U.S.A and United Kingdom, where there are recommendations

Concerning agricultural practices, the tank mixture of two or more chemical products might be a good application strategy, saving fuel and labor-hours, causing less soil compaction, and

associated with the appearance of environmental and human health problems.

organisms as well as frequently causing toxicity to the human population.

information is found about their implications on environmental safety.

fever and malaria [1].

chain) attributed to its indiscriminate use.

474 Herbicides - Current Research and Case Studies in Use

ronmental safety and agriculture sustainability.

interferences in the environment.

safety.

one molecule should now consider molecule mixtures.

**2. Agronomic characteristics of tank mixtures**

on application procedures, incompatibilities, and safety [2].

Weed control with pesticide tank mixtures has been widely studied concerning mixture effectiveness, component antagonism and/or synergism. However, there is little information on environmental issues.

Knowledge on soil-herbicide interactions when herbicide mixtures are applied is extremely relevant. However, few studies on herbicide associations and their soil interactions can be found, because most studies are restricted to the individual molecule behavior.

Ke-Bin et al. [13] observed that atrazine and bentazon herbicides showed longer lag-phase and lower degradation rate when applied in tank mixture in a maize crop. Therefore, the associa‐ tion of atrazine-bentazon had longer soil persistence which means that higher environmental

Pesticide Tank Mixes: An Environmental Point of View

http://dx.doi.org/10.5772/55948

477

The effect of glyphosate on atrazine degradation was studied by Krutz et al. [14] in a silt clayey soil (pH 8.3 and 10.6 g kg-1 of organic-C) from the Texas region in USA. Atrazine degradation was inversely related to glyphosate rate and microbial activity during an eight-day period, evidencing that glyphosate enhanced microbial activity and inhibited atrazine degradation. The authors discussed that atrazine degradation, when in association, is mainly a microbial mechanism, and the degradation reduction might be explained by a lower enzymatic activity

Similar results were reported by Haney et al. [15] for the same soil type, demonstrating the atrazine and glyphosate effects on soil microbial activity evaluated through the soil carbon (C) and nitrogen (N) mineralization. Soil plots treated with the herbicide mixture showed higher microbial activity than plots treated with single atrazine. The evaluated soil C and N flows allowed understanding of the microbial preference for glyphosate because this herbicide's

Zablotowicz et al. [16] studied the effects of glufosinate (herbicide), ammonium sulfate (fertilizer) and both products in mixture on atrazine mineralization. The authors observed decreased atrazine mineralization when the product mixture was applied. The authors explained that an alteration in 14C-atrazine molecule partition into its metabolites and residues would occur caused by ammonium sulfate that would restrict the triazine ring cleavage. Such results evidenced that the application of glufosinate combined to a mineral N source might

Lancaster et al. [17] observed that glyphosate increased soil C mineralization and fluometuron microbial degradation. The authors suggested that the increasing C mineralization might be

Concerning the glyphosate and diflufenican association, Tejada [18] observed longer degra‐ dation periods for both herbicides in mixture than for the individual molecules. Furthermore, the glyphosate-diflufenican association increased both herbicide toxicities to the soil biological activity (measured by the microbial C biomass and enzyme activities - dehydrogenase, urease, β-glycosidase, phosphatase and arylsulfatase) and the individual herbicide persistence.

Pereira et al. [19] evaluated the application of isolated glyphosate and associated to endosulfan on the soil microbial activity in soybeans and observed reduced microbial activity and biomass,

In genetically modified glyphosate-tolerant maize cultivars, it is possible to mix glyphosate and atrazine. In the USA, there are a number of commercially available associations, among them, glufosinate or glyphosate mixed with atrazine [20]. Bonfleur et al. [21] observed that glyphosate mineralization was not affected by atrazine presence in a tropical soil. However, increased atrazine mineralization (measured by the 14CO2 release) was observed with increas‐

related to the increasing fluometuron degradation or to a priming glyphosate effect.

complete mineralization occurred in 14 days, followed by fast atrazine degradation.

potential contamination risks might be expected.

and/or by microbial population suppression by glyphosate.

increase soil atrazine persistence, increasing its residual effect.

and also, reduced metabolic quotient.

When a pesticide is released in the environment, it will probably enter the soil by direct application, or indirectly, by crop residue incorporation into the soil and molecule transport by spraying derivation. In the soil, several processes might occur, that is, molecule retention (adsorption, absorption), transformation (decomposition, degradation) and transport (spray‐ ing derivation, volatilization, lixiviation, superficial runoff). Such processes will determine the molecule destiny, persistence and agronomic efficiency. The main factors influencing those processes are the climatic conditions, the pesticide physical-chemical properties and the soil physical-chemical attributes. According to Oliveira [10], the complex molecule retention process by soil sorption/desorption directly or indirectly influences other factor activities.

Knowledge on pesticide physical-chemical properties is fundamental to predict soil interac‐ tions, potential contamination and transport risks when in the soil solution or associated to sediments. Studies on pesticide mixtures have been restricted to their phytotoxicity effects and few were dedicated to the interactions between two or more associated molecules.

Alves [11] demonstrated that ametryn mineralization half-life is longer when associated to glyphosate than when applied alone; but there was a synergistic effect in the soil, because ametryn half-life was 15 days for the ametryn + glyphosate mixture and 20 days for isolated ametryn in the soil. In the same study, the author observed increased glyphosate mineraliza‐ tion half-life from 55 to 119 days, when comparing single glyphosate and glyphosate + ametryn treatments, respectively; the glyphosate soil half-life could not be determined due to its strong soil sorption during extractions.

Yet in studies of soil microbial activity, Alves [11] observed that glyphosate (at a higher rate) enhanced microbial activity; meanwhile isolated ametryn (at a lower rate) negatively affected microbial activity, but a less negative effect of ametryn + glyphosate mixture (at a lower rate) was observed compared with single ametryn at the same rate. The ametryn + glyphosate mixture (at a higher rate) increased the microbial activity, evidencing a stronger mixture synergistic effect.

Alves [11] also studied the herbicide sorption/desorption in a red Ultisol. High glyphosate and low ametryn sorption were observed when herbicides were applied alone. Higher soil sorption was observed for both herbicides in mixture than for the single molecules. Low glyphosate desorption occurred at all rates in both application procedures (alone or in mixture), but ametryn desorption decreased when applied in mixture.

White et al. [12] studied the effects of chlorothalonil, tebuconazole, flutriafol and cyprocona‐ zole fungicides on the metolachlor herbicide dissipation kinetics. Significantly lower metola‐ chlor dissipation was observed with chlorothalonil, when compared with soil treatments without chlorothalonil or with other fungicides. The authors observed significant reduction in metolachlor metabolites probably attributed to the fungicide effect on glutathione Stransferase enzyme activity. Overall, chlorothalonil fungicide induced a two-fold increase in metolachlor persistence.

Ke-Bin et al. [13] observed that atrazine and bentazon herbicides showed longer lag-phase and lower degradation rate when applied in tank mixture in a maize crop. Therefore, the associa‐ tion of atrazine-bentazon had longer soil persistence which means that higher environmental potential contamination risks might be expected.

Knowledge on soil-herbicide interactions when herbicide mixtures are applied is extremely relevant. However, few studies on herbicide associations and their soil interactions can be

When a pesticide is released in the environment, it will probably enter the soil by direct application, or indirectly, by crop residue incorporation into the soil and molecule transport by spraying derivation. In the soil, several processes might occur, that is, molecule retention (adsorption, absorption), transformation (decomposition, degradation) and transport (spray‐ ing derivation, volatilization, lixiviation, superficial runoff). Such processes will determine the molecule destiny, persistence and agronomic efficiency. The main factors influencing those processes are the climatic conditions, the pesticide physical-chemical properties and the soil physical-chemical attributes. According to Oliveira [10], the complex molecule retention process by soil sorption/desorption directly or indirectly influences other factor activities.

Knowledge on pesticide physical-chemical properties is fundamental to predict soil interac‐ tions, potential contamination and transport risks when in the soil solution or associated to sediments. Studies on pesticide mixtures have been restricted to their phytotoxicity effects and

Alves [11] demonstrated that ametryn mineralization half-life is longer when associated to glyphosate than when applied alone; but there was a synergistic effect in the soil, because ametryn half-life was 15 days for the ametryn + glyphosate mixture and 20 days for isolated ametryn in the soil. In the same study, the author observed increased glyphosate mineraliza‐ tion half-life from 55 to 119 days, when comparing single glyphosate and glyphosate + ametryn treatments, respectively; the glyphosate soil half-life could not be determined due to its strong

Yet in studies of soil microbial activity, Alves [11] observed that glyphosate (at a higher rate) enhanced microbial activity; meanwhile isolated ametryn (at a lower rate) negatively affected microbial activity, but a less negative effect of ametryn + glyphosate mixture (at a lower rate) was observed compared with single ametryn at the same rate. The ametryn + glyphosate mixture (at a higher rate) increased the microbial activity, evidencing a stronger mixture

Alves [11] also studied the herbicide sorption/desorption in a red Ultisol. High glyphosate and low ametryn sorption were observed when herbicides were applied alone. Higher soil sorption was observed for both herbicides in mixture than for the single molecules. Low glyphosate desorption occurred at all rates in both application procedures (alone or in mixture), but

White et al. [12] studied the effects of chlorothalonil, tebuconazole, flutriafol and cyprocona‐ zole fungicides on the metolachlor herbicide dissipation kinetics. Significantly lower metola‐ chlor dissipation was observed with chlorothalonil, when compared with soil treatments without chlorothalonil or with other fungicides. The authors observed significant reduction in metolachlor metabolites probably attributed to the fungicide effect on glutathione Stransferase enzyme activity. Overall, chlorothalonil fungicide induced a two-fold increase in

few were dedicated to the interactions between two or more associated molecules.

soil sorption during extractions.

476 Herbicides - Current Research and Case Studies in Use

ametryn desorption decreased when applied in mixture.

synergistic effect.

metolachlor persistence.

found, because most studies are restricted to the individual molecule behavior.

The effect of glyphosate on atrazine degradation was studied by Krutz et al. [14] in a silt clayey soil (pH 8.3 and 10.6 g kg-1 of organic-C) from the Texas region in USA. Atrazine degradation was inversely related to glyphosate rate and microbial activity during an eight-day period, evidencing that glyphosate enhanced microbial activity and inhibited atrazine degradation. The authors discussed that atrazine degradation, when in association, is mainly a microbial mechanism, and the degradation reduction might be explained by a lower enzymatic activity and/or by microbial population suppression by glyphosate.

Similar results were reported by Haney et al. [15] for the same soil type, demonstrating the atrazine and glyphosate effects on soil microbial activity evaluated through the soil carbon (C) and nitrogen (N) mineralization. Soil plots treated with the herbicide mixture showed higher microbial activity than plots treated with single atrazine. The evaluated soil C and N flows allowed understanding of the microbial preference for glyphosate because this herbicide's complete mineralization occurred in 14 days, followed by fast atrazine degradation.

Zablotowicz et al. [16] studied the effects of glufosinate (herbicide), ammonium sulfate (fertilizer) and both products in mixture on atrazine mineralization. The authors observed decreased atrazine mineralization when the product mixture was applied. The authors explained that an alteration in 14C-atrazine molecule partition into its metabolites and residues would occur caused by ammonium sulfate that would restrict the triazine ring cleavage. Such results evidenced that the application of glufosinate combined to a mineral N source might increase soil atrazine persistence, increasing its residual effect.

Lancaster et al. [17] observed that glyphosate increased soil C mineralization and fluometuron microbial degradation. The authors suggested that the increasing C mineralization might be related to the increasing fluometuron degradation or to a priming glyphosate effect.

Concerning the glyphosate and diflufenican association, Tejada [18] observed longer degra‐ dation periods for both herbicides in mixture than for the individual molecules. Furthermore, the glyphosate-diflufenican association increased both herbicide toxicities to the soil biological activity (measured by the microbial C biomass and enzyme activities - dehydrogenase, urease, β-glycosidase, phosphatase and arylsulfatase) and the individual herbicide persistence.

Pereira et al. [19] evaluated the application of isolated glyphosate and associated to endosulfan on the soil microbial activity in soybeans and observed reduced microbial activity and biomass, and also, reduced metabolic quotient.

In genetically modified glyphosate-tolerant maize cultivars, it is possible to mix glyphosate and atrazine. In the USA, there are a number of commercially available associations, among them, glufosinate or glyphosate mixed with atrazine [20]. Bonfleur et al. [21] observed that glyphosate mineralization was not affected by atrazine presence in a tropical soil. However, increased atrazine mineralization (measured by the 14CO2 release) was observed with increas‐ ing glyphosate rates. The authors observed a 100-day variation in the atrazine half-life when associated with a two-fold glyphosate rate. Therefore, the glyphosate-atrazine tank mixture allowed atrazine persistence reduction in the soil. The authors said that a possible explanation is the glyphosate contribution to the microorganisms as source of N, and this N supply might decrease the initial atrazine immobilization when this is the only substrate, and then, increas‐ ing its mineralization.

Pesticides applied to field crops are released in the environment mainly through lixiviation (when molecules move into the soil and reach the underground waters), superficial runoff (when molecules move together with soil and water runoff), and spraying derivation (when

Pesticide Tank Mixes: An Environmental Point of View

http://dx.doi.org/10.5772/55948

479

The situation is complex once crop diversity allied to the high number and diversity of pesticide products usually applied to field crops, and the short distances between fields and aquatic areas have exposed the water resources not only to individual products but also to all

Several products, mainly herbicides and insecticides, are common superficial water contami‐ nants, due to their large application in agriculture and residential areas. Therefore, there is an increasing concern about superficial and underground water contamination, due to the lack

In Brazil, several studies have been carried out to determine the presence of pesticides in aquatic ecosystems. Armas et al. [31] evaluated the presence of herbicides in the superficial water and sediments of Corumbataí River (State of São Paulo, Brazil). The authors found several herbicides - ametryn, atrazine, simazine, hexazinone, glyphosate and clomazone – and triazines were specifically found in higher levels, above the limits allowed for potable water by Brazilian legislation. Dores et al. [32] found herbicide residues from the triazine group and their metabolites, as well as metribuzin, metolachlor and trifluralin residues. Among the Brazilian literature, the research works of Caldas et al. [33], Lanchote et al. [34], Filizola et al.

Other interesting results can be found in the literature: Benvenuto et al. [39] determined the presence of eleven pesticides in superficial waters of Italy and Spain and observed concentra‐ tion values varying between 0.002 and 0.087 μg L-1. Yu et al. [40] determined the presence of nine (among eleven pesticides evaluated) herbicides of the triazine group in all water samples analyzed. Similar determinations were made by Ma et al. [41], Palma et al. [42], Balinova and

Understanding of how pesticides affect aquatic environments has been a challenge to re‐ searchers, and the science of ecotoxicology has helped to answer many questions on this

The "ecotoxicology" term was first suggested by the French toxicologist René Truhaut, during the *Committee of the International Council of Scientific Unions* (ICSU) meeting, in June 1969, in Stockholm (Sweden) [45]. According to this author, Ecotoxicology is the science that studies the effects of natural or synthetic substances on living beings, populations and communities, animal or vegetal, terrestrial or aquatic, constituting the biosphere, including the substance

Usually, ecotoxicological experiments follow standardized protocols developed by interna‐ tional organizations, for example, the Environmental Protection Agency (EPA); the Organi‐ zation for Cooperation and Economical Development (OCDE); and the Brazilian Agency of

interaction with the environment where they live in an integrated context [46].

molecules are carried by wind during pesticide spraying).

of information on pesticide impacts mainly in aquatic systems.

[35], Laabs et al. [36], Dores et al. [37], Jacomini et al. [38] are pointed out.

their associations [30].

Mondesky [43] and Segura et al. [44].

Technical Norms (ABNT).

subject.

Fogg and Boxall [22] observed inhibitory effects of an isoproturon-chlorothalonil mixture on the isoproturon degradation in soils. Isoproturon half-life (DT50) values varied from 18.5 to 71.5 days when combined with chlorothalonil. This might be explained by the TPN-OH chlorothalonil metabolite inhibition and the reduction in the soil microorganism population involved in isoproturon degradation.

The soil degradation of pendimethalin (herbicide) was significantly reduced when mixed with mancozeb (fungicide) or mancozeb+thiamethoxam (insecticide) [23]. Pendimethalin herbicide half-life increased from 26.9 to 62.2 days when in single and combined (mancozeb + thiame‐ thoxam) applications, respectively, in a sandy soil. On the other hand, the same authors observed that pendimethalin degradation is not affected by the presence of isolated metribuzin or thiamethoxam.

Several studies have pointed out the adjuvant influence on pesticide destiny in the environ‐ ment, specifically their persistence and bioavailability. Cabrera [24], in laboratory studies, affirmed that metazachlor herbicide added to oil and surfactant showed reduced degradation rates and increased residues in the soil. Similar results to other pesticides were reported by Kucharski and Sadowski [25] and Rodríguez-Cruz et al. [26]. In a field experiment, Kucharski et al. [27] observed a 43% increase in lenacil herbicide residues in the superficial soil layer, with the addition of adjuvants (oil and surfactant).

High mobility pesticides used together with adjuvants present decreased movement along the soil profile. Reddy and Singh [28] evaluated bromacil and diuron herbicides lixiviation in soil columns. In treatments with adjuvant addition, the authors observed significant lower bromacil vertical movement and no effect on diuron movement. These two herbicides present distinct physical-chemical characteristics that explain their differential movement abilities in the soil. Thus, bromacil is an acidic molecule with high water solubility (815 mg L-1); meanwhile diuron is a non-ionic herbicide of low water solubility (42 mg L-1). From the environmental point of view, the adjuvant effect was positive in the case of bromacil, but the agronomic efficacy was restricted.

The results found in the literature have highlighted the interactions existing among several molecules, especially in the soil, but such interactions might be different under other environ‐ ment compartments. For this reason, studies on environmental pesticide behavior and destination must include all aspects, bringing together laboratory and field approaches.

#### **3.2. Water: An ecotoxicological approach for pesticide mixtures**

According to Botelho et al. [29], water resource contamination has currently been considered one of the greatest environmental problems on Earth.

Pesticides applied to field crops are released in the environment mainly through lixiviation (when molecules move into the soil and reach the underground waters), superficial runoff (when molecules move together with soil and water runoff), and spraying derivation (when molecules are carried by wind during pesticide spraying).

ing glyphosate rates. The authors observed a 100-day variation in the atrazine half-life when associated with a two-fold glyphosate rate. Therefore, the glyphosate-atrazine tank mixture allowed atrazine persistence reduction in the soil. The authors said that a possible explanation is the glyphosate contribution to the microorganisms as source of N, and this N supply might decrease the initial atrazine immobilization when this is the only substrate, and then, increas‐

Fogg and Boxall [22] observed inhibitory effects of an isoproturon-chlorothalonil mixture on the isoproturon degradation in soils. Isoproturon half-life (DT50) values varied from 18.5 to 71.5 days when combined with chlorothalonil. This might be explained by the TPN-OH chlorothalonil metabolite inhibition and the reduction in the soil microorganism population

The soil degradation of pendimethalin (herbicide) was significantly reduced when mixed with mancozeb (fungicide) or mancozeb+thiamethoxam (insecticide) [23]. Pendimethalin herbicide half-life increased from 26.9 to 62.2 days when in single and combined (mancozeb + thiame‐ thoxam) applications, respectively, in a sandy soil. On the other hand, the same authors observed that pendimethalin degradation is not affected by the presence of isolated metribuzin

Several studies have pointed out the adjuvant influence on pesticide destiny in the environ‐ ment, specifically their persistence and bioavailability. Cabrera [24], in laboratory studies, affirmed that metazachlor herbicide added to oil and surfactant showed reduced degradation rates and increased residues in the soil. Similar results to other pesticides were reported by Kucharski and Sadowski [25] and Rodríguez-Cruz et al. [26]. In a field experiment, Kucharski et al. [27] observed a 43% increase in lenacil herbicide residues in the superficial soil layer, with

High mobility pesticides used together with adjuvants present decreased movement along the soil profile. Reddy and Singh [28] evaluated bromacil and diuron herbicides lixiviation in soil columns. In treatments with adjuvant addition, the authors observed significant lower bromacil vertical movement and no effect on diuron movement. These two herbicides present distinct physical-chemical characteristics that explain their differential movement abilities in the soil. Thus, bromacil is an acidic molecule with high water solubility (815 mg L-1); meanwhile diuron is a non-ionic herbicide of low water solubility (42 mg L-1). From the environmental point of view, the adjuvant effect was positive in the case of bromacil, but the agronomic

The results found in the literature have highlighted the interactions existing among several molecules, especially in the soil, but such interactions might be different under other environ‐ ment compartments. For this reason, studies on environmental pesticide behavior and destination must include all aspects, bringing together laboratory and field approaches.

According to Botelho et al. [29], water resource contamination has currently been considered

**3.2. Water: An ecotoxicological approach for pesticide mixtures**

one of the greatest environmental problems on Earth.

ing its mineralization.

or thiamethoxam.

efficacy was restricted.

involved in isoproturon degradation.

478 Herbicides - Current Research and Case Studies in Use

the addition of adjuvants (oil and surfactant).

The situation is complex once crop diversity allied to the high number and diversity of pesticide products usually applied to field crops, and the short distances between fields and aquatic areas have exposed the water resources not only to individual products but also to all their associations [30].

Several products, mainly herbicides and insecticides, are common superficial water contami‐ nants, due to their large application in agriculture and residential areas. Therefore, there is an increasing concern about superficial and underground water contamination, due to the lack of information on pesticide impacts mainly in aquatic systems.

In Brazil, several studies have been carried out to determine the presence of pesticides in aquatic ecosystems. Armas et al. [31] evaluated the presence of herbicides in the superficial water and sediments of Corumbataí River (State of São Paulo, Brazil). The authors found several herbicides - ametryn, atrazine, simazine, hexazinone, glyphosate and clomazone – and triazines were specifically found in higher levels, above the limits allowed for potable water by Brazilian legislation. Dores et al. [32] found herbicide residues from the triazine group and their metabolites, as well as metribuzin, metolachlor and trifluralin residues. Among the Brazilian literature, the research works of Caldas et al. [33], Lanchote et al. [34], Filizola et al. [35], Laabs et al. [36], Dores et al. [37], Jacomini et al. [38] are pointed out.

Other interesting results can be found in the literature: Benvenuto et al. [39] determined the presence of eleven pesticides in superficial waters of Italy and Spain and observed concentra‐ tion values varying between 0.002 and 0.087 μg L-1. Yu et al. [40] determined the presence of nine (among eleven pesticides evaluated) herbicides of the triazine group in all water samples analyzed. Similar determinations were made by Ma et al. [41], Palma et al. [42], Balinova and Mondesky [43] and Segura et al. [44].

Understanding of how pesticides affect aquatic environments has been a challenge to re‐ searchers, and the science of ecotoxicology has helped to answer many questions on this subject.

The "ecotoxicology" term was first suggested by the French toxicologist René Truhaut, during the *Committee of the International Council of Scientific Unions* (ICSU) meeting, in June 1969, in Stockholm (Sweden) [45]. According to this author, Ecotoxicology is the science that studies the effects of natural or synthetic substances on living beings, populations and communities, animal or vegetal, terrestrial or aquatic, constituting the biosphere, including the substance interaction with the environment where they live in an integrated context [46].

Usually, ecotoxicological experiments follow standardized protocols developed by interna‐ tional organizations, for example, the Environmental Protection Agency (EPA); the Organi‐ zation for Cooperation and Economical Development (OCDE); and the Brazilian Agency of Technical Norms (ABNT).

The toxicity tests allow evaluating the environmental contamination by different pollutant sources, such as agricultural, industrial and domestic residues, sediments, medicines and chemical products overall, as well as the results of their synergistic and antagonistic effects [47-48]. The ecotoxicological tests can also detect the toxic agent or mixture capacity of causing deleterious effects on living organisms, allowing determination of the harmful concentration ranges, and how and where the effects are expressed [49].

several structure and biochemical alterations in rainbow trout hepatocytes submitted to a 20 component mixture, including pesticides. Delorenzo and Serrano [66] evaluated the effects of atrazine (herbicide), chlorpyrifos (insecticide) and chlorothalonil (fungicide) on the *Dunaliella tertiolecta* algae growth; the results of atrazine - chlorpyrifos mixture showed an additive toxicity pattern, meanwhile atrazine - chlorothalonil mixture showed a synergistic effect. Yet, the authors observed a two-fold higher toxicity effect of atrazine – Chlorothalonil mixture than the individual products. Choung et al. [67] observed that relatively high atrazine rates

Pesticide Tank Mixes: An Environmental Point of View

http://dx.doi.org/10.5772/55948

481

Pesticide tank mixtures are currently and frequently used not only in developed countries with specific regulatory legislation for the practice, but also in all agricultural countries where

From the agronomic point of view, an effective pest control with pesticide mixtures will depend on the molecule compatibility and also on specific control tests. When the farmer uses two chemically incompatible substances in tank mixture, high losses in crop yield and equipment problems might occur, for example, sprayer nozzle obstruction due to chemical

Although the pesticide tank mixture may appear to be an efficient pest control practice with synergistic results, the aspects concerning environmental safety must be considered. Little specific information on associated pesticide residues is available in the literature concerning

When a single pesticide is applied, the expected environmental results should be similar to previous results reported for the pesticide registration and before its commercial release. The environment (mainly aquatic and soil medium) is a large contaminant reservoir, where the chemical compounds used in agriculture can be found together. In spite of that, it is important to reinforce that a single pesticide interacts quite differently with the medium, compared to

In light of the large global demand for food and the increasing crop productivity in the same cropping area, it is imperative to consider the environmental safety questions concerning tank

This is a relatively new science area that demands urgent studies on environmental safety, ecotoxicology and toxicology, in order to make highly prevalent the declaration of the United Nation Organization about the planet environment: "*The man has the fundamental right to liberty, equality and enjoyment of adequate life conditions, under an environment of such quality that allows him living a dignifying life and well-being, and he is carrier of the solemn duty of protecting and*

increased the terbufos (insecticide) toxicity to *Ceriodaphnia dubia* microcrustacean.

information on harmful effects do not directly reach farmers.

withholding periods and overall environmental behavior.

the mixture interaction, as already discussed in this chapter.

*improving the environment for the present and future generations"* [68].

chemical mixture applications in agriculture.

reaction between molecules and subsequent compound precipitation.

**4. Final remarks**

Several parameters have been used to determine the xenobiotic effects in different organisms. Among these variables, the lethality [50-51], immobility [52], gill alterations [53-56], and reproduction [57-59] are pointed out.

The ecotoxicological experiments consist of exposing living organisms to several concentra‐ tions of a specific product and evaluating the results that might be expressed according to the test type. For instance, the acute test consists of short-term exposure of organisms to several product concentrations, and then, the species life cycle is evaluated; the toxicity indicative parameters more frequently used are: lethality (expressed by the average lethal concentration - LC50), and immobility (expressed by the observable toxic concentration effect - EC50). It is important to highlight that both parameters take into consideration the effects for 50% of the organisms tested under the specific experiment conditions [60-61]. In the case of a chronic test, the organism is submitted to long-term product exposure and the observable effects are usually focused on organism reproduction, behavior, morphology, and size, among others.

Water quality tests have been important tools aiming to minimize the pollution effects on aquatic ecosystems and to implement remediation and monitoring programs, and for that, the ecotoxicological tests have been used.

In the case of pesticide mixtures, the ecotoxicological tests to determine toxicity effects are difficult to interpret, because toxicity symptoms might depend on interactions occurring among different chemical molecules in solution and their accumulative quantities in organisms [61].

When analyzing mixture toxicity effects, some approaches and definitions must be established. In the aquatic ecotoxicology, two different models have been used to describe the relationships between single compound effects and their mixtures: concentration addition model (CA) and independent action model (IA) [62]. In the CA model, each mixture component toxicity effect is induced through a same mechanism, meanwhile in the IA model, the combined components show different actions, inducing a unique toxicological response, but via distinct reactions within the organisms [63]. Nevertheless, both models are used as references to predict the expected mixture toxicity effect, based on the known toxicity of the individual compounds [62].

For a long time, there has been concern about mixture impacts on aquatic ecosystems, not only from pesticides but also from other compound groups, and several discussions and reviews have been reported. In 1984, Hermens and collaborators investigated organic mixture effects on mortality and reproduction of *Daphnia magna* microcrustacean, after exposure to 14 products with different modes of action. The authors observed more severe toxicity effects on mixture-treated organisms than with individual products, although the chronic test results with the mixture showed less severe symptoms [64]. Strmac and Braunbeck [65] observed several structure and biochemical alterations in rainbow trout hepatocytes submitted to a 20 component mixture, including pesticides. Delorenzo and Serrano [66] evaluated the effects of atrazine (herbicide), chlorpyrifos (insecticide) and chlorothalonil (fungicide) on the *Dunaliella tertiolecta* algae growth; the results of atrazine - chlorpyrifos mixture showed an additive toxicity pattern, meanwhile atrazine - chlorothalonil mixture showed a synergistic effect. Yet, the authors observed a two-fold higher toxicity effect of atrazine – Chlorothalonil mixture than the individual products. Choung et al. [67] observed that relatively high atrazine rates increased the terbufos (insecticide) toxicity to *Ceriodaphnia dubia* microcrustacean.

### **4. Final remarks**

The toxicity tests allow evaluating the environmental contamination by different pollutant sources, such as agricultural, industrial and domestic residues, sediments, medicines and chemical products overall, as well as the results of their synergistic and antagonistic effects [47-48]. The ecotoxicological tests can also detect the toxic agent or mixture capacity of causing deleterious effects on living organisms, allowing determination of the harmful concentration

Several parameters have been used to determine the xenobiotic effects in different organisms. Among these variables, the lethality [50-51], immobility [52], gill alterations [53-56], and

The ecotoxicological experiments consist of exposing living organisms to several concentra‐ tions of a specific product and evaluating the results that might be expressed according to the test type. For instance, the acute test consists of short-term exposure of organisms to several product concentrations, and then, the species life cycle is evaluated; the toxicity indicative parameters more frequently used are: lethality (expressed by the average lethal concentration - LC50), and immobility (expressed by the observable toxic concentration effect - EC50). It is important to highlight that both parameters take into consideration the effects for 50% of the organisms tested under the specific experiment conditions [60-61]. In the case of a chronic test, the organism is submitted to long-term product exposure and the observable effects are usually

focused on organism reproduction, behavior, morphology, and size, among others.

Water quality tests have been important tools aiming to minimize the pollution effects on aquatic ecosystems and to implement remediation and monitoring programs, and for that, the

In the case of pesticide mixtures, the ecotoxicological tests to determine toxicity effects are difficult to interpret, because toxicity symptoms might depend on interactions occurring among different chemical molecules in solution and their accumulative quantities in

When analyzing mixture toxicity effects, some approaches and definitions must be established. In the aquatic ecotoxicology, two different models have been used to describe the relationships between single compound effects and their mixtures: concentration addition model (CA) and independent action model (IA) [62]. In the CA model, each mixture component toxicity effect is induced through a same mechanism, meanwhile in the IA model, the combined components show different actions, inducing a unique toxicological response, but via distinct reactions within the organisms [63]. Nevertheless, both models are used as references to predict the expected mixture toxicity effect, based on the known toxicity of the individual compounds [62]. For a long time, there has been concern about mixture impacts on aquatic ecosystems, not only from pesticides but also from other compound groups, and several discussions and reviews have been reported. In 1984, Hermens and collaborators investigated organic mixture effects on mortality and reproduction of *Daphnia magna* microcrustacean, after exposure to 14 products with different modes of action. The authors observed more severe toxicity effects on mixture-treated organisms than with individual products, although the chronic test results with the mixture showed less severe symptoms [64]. Strmac and Braunbeck [65] observed

ranges, and how and where the effects are expressed [49].

reproduction [57-59] are pointed out.

480 Herbicides - Current Research and Case Studies in Use

ecotoxicological tests have been used.

organisms [61].

Pesticide tank mixtures are currently and frequently used not only in developed countries with specific regulatory legislation for the practice, but also in all agricultural countries where information on harmful effects do not directly reach farmers.

From the agronomic point of view, an effective pest control with pesticide mixtures will depend on the molecule compatibility and also on specific control tests. When the farmer uses two chemically incompatible substances in tank mixture, high losses in crop yield and equipment problems might occur, for example, sprayer nozzle obstruction due to chemical reaction between molecules and subsequent compound precipitation.

Although the pesticide tank mixture may appear to be an efficient pest control practice with synergistic results, the aspects concerning environmental safety must be considered. Little specific information on associated pesticide residues is available in the literature concerning withholding periods and overall environmental behavior.

When a single pesticide is applied, the expected environmental results should be similar to previous results reported for the pesticide registration and before its commercial release. The environment (mainly aquatic and soil medium) is a large contaminant reservoir, where the chemical compounds used in agriculture can be found together. In spite of that, it is important to reinforce that a single pesticide interacts quite differently with the medium, compared to the mixture interaction, as already discussed in this chapter.

In light of the large global demand for food and the increasing crop productivity in the same cropping area, it is imperative to consider the environmental safety questions concerning tank chemical mixture applications in agriculture.

This is a relatively new science area that demands urgent studies on environmental safety, ecotoxicology and toxicology, in order to make highly prevalent the declaration of the United Nation Organization about the planet environment: "*The man has the fundamental right to liberty, equality and enjoyment of adequate life conditions, under an environment of such quality that allows him living a dignifying life and well-being, and he is carrier of the solemn duty of protecting and improving the environment for the present and future generations"* [68].

### **Acknowledgements**

The authors are grateful to the Research Foundation of the State of São Paulo (FAPESP) and to the National Council for Scientific and Technological Development (CNPQ).

[9] Gebara, A. B. Ciscato CHP, Monteiro SH, Souza GS. Pesticide Residues in some Com‐ modities: Dietary Risk for Children. Bulletin of Environmental Contamination and

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483

[10] Oliveira, M. F. Comportamento de herbicidas no ambiente. In: Oliveira Jr., Constan‐ tin RSJ. (Eds.) Plantas daninhas e seu manejo. Guaíba: Agropecuária; (2001). ,

[11] Alves PATComportamento dos herbicidas ametrina e glifosato aplicados em associa‐ ção em solo de cultivo de cana-de-açúcar. PhD Thesis. University of São Paulo;

[12] White, P. M, Potter, T. L, & Culbreath, A. K. Fungicide dissipation and impact on me‐ tolachlor aerobic soil degradation and soil microbial dynamics. Science of the Total

[13] Ke-bin, L. I, Cheng, J, Wang, X, Zhou, Y, & Liu, W. Degradation of herbicides atra‐ zine and bentazone applied alone and in combination in soils. Pedosphere (2008). ,

[14] Krutz, L. J, Senseman, S. A, & Haney, R. L. Effect of Roundup Ultra on atrazine deg‐

[15] Haney, R. L, Senseman, S. A, Krutz, L. J, & Hons, F. M. Soil carbon and nitrogen min‐ eralization as affected by atrazine and glyphosate. Biology and Fertility of Soils

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[17] Lancaster, S. H, Haney, R. L, Senseman, S. A, Kenerley, C. M, & Hons, F. M. Microbi‐ al degradation of Fluometuron is influenced by Roundup Weather MAX. Journal of

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[19] Pereira, J. L, Picanço, M. C, Silva, A. A, & Santos, E. A. Tomé HVV, Olarte JB. Effects of glyphosate and endosulfan on soil microorganisms in soybean crop. Planta Dani‐

[20] Owen MDKCurrent use of transgenic herbicide-resistant soybean and corn in the

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### **Author details**

Valdemar Luiz Tornisielo, Rafael Grossi Botelho, Paulo Alexandre de Toledo Alves, Eloana Janice Bonfleur and Sergio Henrique Monteiro

Laboratory of Ecotoxicology, Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, SP, Brazil

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**Acknowledgements**

482 Herbicides - Current Research and Case Studies in Use

**Author details**

**References**

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Paulo, Piracicaba, SP, Brazil

The authors are grateful to the Research Foundation of the State of São Paulo (FAPESP) and

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**Chapter 19**

**Characterization, Modes of**

Maria A. Marin-Morales

http://dx.doi.org/10.5772/55169

**1. Introduction**

Thaís C. C. Fernandes, Marcos A. Pizano and

Additional information is available at the end of the chapter

cause damage to human health [1], [6] and [7].

phosphates, and dinitroanilines, among others [8].

**Action and Effects of Trifluralin: A Review**

The use of chemicals to control human diseases, plagues and weeds in agriculture started in the late 19th century, but only after the Second World War did this practice follow rather scientific criteria [1]. According to targets against which they are designated, the chemicals used in agriculture are called insecticides, fungicides, herbicides, nematicides, among others [2].

All pesticides have the common priority of stopping a metabolic process essential to undesir‐ able organisms, for which they are toxic. These chemicals act directly upon the organisms,

Among agricultural pesticides, herbicides comprise the most employed group in agriculture. The main function of these chemicals is to control weeds, weed competition reduces produc‐ tivity, without significantly impacting crop yield. Weeds tend to compete with crops by extracting essential elements from the soil, water, intercepting light and CO2, interfering in the culture development and affecting agricultural production practices including harvest [4]. Herbicides are also used for eliminating plants from both road, railways, and riversides [3].

The mechanism of action of some herbicides on organisms is not completely understood [5]. Lack of detailed information about the action of herbicides on the biological environment may

Herbicides may be classified according to different criteria related to their properties, charac‐ teristics, use, efficiency, permanence in the environment and mechanism of action. As for their chemical features, herbicides may be classified as carbamates, amides, diphenyl ethers, amino

> © 2013 Fernandes 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 Fernandes 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.

eliminating or controlling them, such as interfering in their reproductive process [3].

### **Chapter 19**

## **Characterization, Modes of Action and Effects of Trifluralin: A Review**

Thaís C. C. Fernandes, Marcos A. Pizano and Maria A. Marin-Morales

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55169

### **1. Introduction**

The use of chemicals to control human diseases, plagues and weeds in agriculture started in the late 19th century, but only after the Second World War did this practice follow rather scientific criteria [1]. According to targets against which they are designated, the chemicals used in agriculture are called insecticides, fungicides, herbicides, nematicides, among others [2].

All pesticides have the common priority of stopping a metabolic process essential to undesir‐ able organisms, for which they are toxic. These chemicals act directly upon the organisms, eliminating or controlling them, such as interfering in their reproductive process [3].

Among agricultural pesticides, herbicides comprise the most employed group in agriculture. The main function of these chemicals is to control weeds, weed competition reduces produc‐ tivity, without significantly impacting crop yield. Weeds tend to compete with crops by extracting essential elements from the soil, water, intercepting light and CO2, interfering in the culture development and affecting agricultural production practices including harvest [4]. Herbicides are also used for eliminating plants from both road, railways, and riversides [3].

The mechanism of action of some herbicides on organisms is not completely understood [5]. Lack of detailed information about the action of herbicides on the biological environment may cause damage to human health [1], [6] and [7].

Herbicides may be classified according to different criteria related to their properties, charac‐ teristics, use, efficiency, permanence in the environment and mechanism of action. As for their chemical features, herbicides may be classified as carbamates, amides, diphenyl ethers, amino phosphates, and dinitroanilines, among others [8].

© 2013 Fernandes 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 Fernandes 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.

Classification of herbicides based on their mechanism of action has changed over time, both according to the discovery of new herbicides and the elucidation of site of action of the herbicide on plants. The internationally accepted classification is the one proposed by the Herbicide Resistance Action Committee (HRAC). In it, the herbicides are classified in alpha‐ betical order in accordance with their sites of action and chemical classes (Table 1). Herbicides having unknown site of action are grouped under Z until identification. The (numeric) Weed Science Society of America (WSSA) classification system is also listed in Table 1 [5].

**HRAC SITES OF ACTION CHEMICAL GROUP WSSA**

G Inhibition of EPSP synthase Glycines 9 H Inhibition of glutamine synthase Phosphinic acid 10 I Inhibition of DHP (dihydropteroate synthase) Carbamates 18

K2 Inhibition of mitosis Carbamates 23

M Decouplers (cell membrane disruptors) Dinitrophenols 24

Triazoles Isoxazolidinones Diphenyl ethers

Dinitroanilines Phosphoramidates Pyridines Benzamides Benzoic acid

Chloroacetamides Acetamides Tetrazolinones Others

Nitriles Benzamides Triazolocarboxamides Quinolinocarboxylic acid

Tiocarbamates Phosphoroditioates Benzofurans Chlorocarbonic acid

Others

Ftalamates Semicarbazones

Pirazoliuns Organoarsenicals Others

Phenoxicarboxylic acid Benzoic acid Pyridinecarboxylic acid Quinolinocarboxylic acid

Arylamino Propionic acid

F3 Inhibition of carotenoid biosynthesis (unknown target)

K1 Inhibition of microtubule assembly

L Inhibition of cell wall (cellulose) synthesis

Inhibition of lipid synthesis (different from ACCase

R ... ... S ... ... . ... ...

**WSSA.** Weed Science Society of America; **HRAC.** Herbicide Resistance Action Committee.

**Table 1.** Herbicide Classification in accordance with their mechanism of action.

K3 Inhibition of cell cycle

inhibitors)

Q Auxin transport inhibitors

P Auxin mimics

Z Unknown

N

Others 27

Characterization, Modes of Action and Effects of Trifluralin: A Review

11 13 11 491

http://dx.doi.org/10.5772/55169

19 19

25 26 17



**WSSA.** Weed Science Society of America; **HRAC.** Herbicide Resistance Action Committee.

**Table 1.** Herbicide Classification in accordance with their mechanism of action.

Classification of herbicides based on their mechanism of action has changed over time, both according to the discovery of new herbicides and the elucidation of site of action of the herbicide on plants. The internationally accepted classification is the one proposed by the Herbicide Resistance Action Committee (HRAC). In it, the herbicides are classified in alpha‐ betical order in accordance with their sites of action and chemical classes (Table 1). Herbicides having unknown site of action are grouped under Z until identification. The (numeric) Weed

Science Society of America (WSSA) classification system is also listed in Table 1 [5].

A Inhibition of Acetyl-CoA Carboxylase (ACCase)

490 Herbicides - Current Research and Case Studies in Use

C1 Inhibition of Phtosynthesis in photosystem II

C2 Inhibition of Phtosynthesis in photosystem II

C3 Inhibition of Phtosynthesis in photosystem II

E Inhibition of Protoporphyrinogen Oxidase (PPO)

Inhibition of carotenoid biosynthesis in naphytoenedesaturase (PDS)

pyruvate-dioxygenase (4HPPD)

Inhibition of carotenoid biosynthesis in 4-hydroxyphenyl-

acid synthase AHAS)

Inhibition of Acetolactate Synthase (ALS) (or acetohydrxy

B

F1

F2

**HRAC SITES OF ACTION CHEMICAL GROUP WSSA**

Aryloxyphenoxypropionates (FOPs) Ciclohexanodiones (DIMs) Phenylpyrazolones (DENs)

Sulfonylaminocarbonyl-triazolinones

Sulfonylureas Imidazolinones Triazolopyrimidines Pirimidinil(tio)benzoates

Triazines Triazinones Triazolinones Uracils Pyridazinone Phenyl Carbamates

Ureas Amides

Nitriles

Others

Others

Triacetones Isoxazoles Pyrazoles

Pyridazinones Pyridine Carboxamides

D Inhibition of Phtosynthesis in photosystem I Bipiridiliuns 22

Benzotiadiazinones Phenyl-pyridazines

Diphenyl ethers Phenylpyrazoles N-phenylftalimidas Thiadiazoles Oxadiazoles Triazolinones Oxazolidinediones Pyirimidinediones

1 1 1

7 7

6 6 6

12 12 12

27 27 27

### **2. Trifluralin identification and characterisitcs**

Trifluralin belongs to the dinitroaniline group which has the aniline structure as a basis, containing NO2 molecules at 2 and 6 or 3 and 5 positions of the benzene ring. This group has more than ten different herbicides, among which are trifluralin, dinitramine, oryzalin and pendimethalin [8].

USEPA (1999) [16] classifies trifluralin as group C: possibly carcinogenic to humans, based on

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493

Trifluralin is strongly adsorbed by organic matter colloids and not much by clay ones. In organic matter rich soils, adsorption prevents absorption of the product by plant roots. Therefore, the use of this herbicide under such conditions is not advisable [10]. Leaching, as well as soil lateral movement is quite reduced compared to some pesticides [17]. Its main characteristic is soil persistence resulting from low mobility, which can cause damage to crops

Such herbicides as trifluralin, applied in pre-emergence, act better when soil humidity is between high and elevated. Therefore, the herbicide may at least be partially solubilized and

This herbicide degradation in soil occurs through chemical, microbial pathways and photol‐ ysis. Chemical degradation promotes dealkylation of the amino group, reduction from the nitro to the amino group, partial oxidation from the trifluoromethyl to the carboxyl group and,

Microbial degradation may occur under aerobic and anaerobic conditions (Figure 3). However, it is observed that degradation occurs mainly under anaerobic conditions, as the ones observed in poorly drained soils, when there is subsequent rainfall. Under anaerobic conditions, within the same time period, 98% of trifluralin degrades, whereas under aerobic conditions only 25% of the product decomposes. Among the fungi capable of decomposing trifluralin are *Sclero‐ tiumrolfsii*, *Aspergillusniger*, *Fusarium*sp and *Tricoderma*sp [10]. According to Carter and Camper

distributed in the first layers of the soil surface, which will protect it from losses [8].

subsequently, degradation into smaller fragments (Figure 2).

**Figure 2.** Possible sequence of events that occur during trifluralin chemical degradation.

[18], trifluralin may also be degraded by *Pseudomonas* sp.

evidences with animals, not with humans.

**3.1. Behavior in soil**

following its application [12].

**3. Trifluralin behavior in the environment**

Trifluralin has been used in agriculture since 1963 [9]. This herbicide is registered separately or in mixtures, and used in the following crops: *Glycine max*, citrus, *Coffea arábica* under formation, *Gossypium hirsutum*, *Arachis hypogaea*, *Phaseolus vulgaris*, *Allium sativum*, *Ricinus communis*, *Manihot esculenta*, *Helianthus annuus*, *Solanum melongena*, *Daucus carota*, *Abelmoschus esculentus*, *Brassica oleracea*, *Brassica oleracea capitata, Brassica oleracea botrytis*, *Capsicum ann‐ uum*, *Lycopersicon esculentum*, and ornamental plants [10].

Trifluralin is available either in emulsifiable concentrate or in crystalline solid both formula‐ tions of the yellow-orange color. It is not quite soluble in water (0.3 to 0.6 mg/L solubility at 25°C) [9], it is mildly volatile (1.1. 10-4 mmHg pressure vapor at 25°C), its density is 1.36 g/cm3 at 22°C, it is considered alkaline and long-lasting in the environment (120-240 days) [8]. Trifluralin has a high affinity to soil [11], is relatively immobile and has a half-life of 3 to 18 weeks, depending on the soil and the geographical location [12].

Trifluralin chemical composition is α,α,α–trifluoro–2–6-dinitro–N–N– dipropyl–p–toluidine [13]. The chemical structure formula is shown in Figure 1.

**Figure 1.** Trifluralin chemical structure formule.

Trifluralin commercial products contain nitrosodipropylamine, a carcinogenic contaminant (NDPA) [14]. This compound reacts with 06 -guanine DNA and may cause mutation [15]. On account of concerns about this characteristic, the *Environmental Protection Agency* (EPA) demanded that industries make sure products containing trifluralin active principle had nitrosodipropylamine 0.5 ppm concentrations at the most [14].

USEPA (1999) [16] classifies trifluralin as group C: possibly carcinogenic to humans, based on evidences with animals, not with humans.

### **3. Trifluralin behavior in the environment**

#### **3.1. Behavior in soil**

**2. Trifluralin identification and characterisitcs**

492 Herbicides - Current Research and Case Studies in Use

*uum*, *Lycopersicon esculentum*, and ornamental plants [10].

weeks, depending on the soil and the geographical location [12].

[13]. The chemical structure formula is shown in Figure 1.

**Figure 1.** Trifluralin chemical structure formule.

(NDPA) [14]. This compound reacts with 06

nitrosodipropylamine 0.5 ppm concentrations at the most [14].

pendimethalin [8].

Trifluralin belongs to the dinitroaniline group which has the aniline structure as a basis, containing NO2 molecules at 2 and 6 or 3 and 5 positions of the benzene ring. This group has more than ten different herbicides, among which are trifluralin, dinitramine, oryzalin and

Trifluralin has been used in agriculture since 1963 [9]. This herbicide is registered separately or in mixtures, and used in the following crops: *Glycine max*, citrus, *Coffea arábica* under formation, *Gossypium hirsutum*, *Arachis hypogaea*, *Phaseolus vulgaris*, *Allium sativum*, *Ricinus communis*, *Manihot esculenta*, *Helianthus annuus*, *Solanum melongena*, *Daucus carota*, *Abelmoschus esculentus*, *Brassica oleracea*, *Brassica oleracea capitata, Brassica oleracea botrytis*, *Capsicum ann‐*

Trifluralin is available either in emulsifiable concentrate or in crystalline solid both formula‐ tions of the yellow-orange color. It is not quite soluble in water (0.3 to 0.6 mg/L solubility at 25°C) [9], it is mildly volatile (1.1. 10-4 mmHg pressure vapor at 25°C), its density is 1.36 g/cm3 at 22°C, it is considered alkaline and long-lasting in the environment (120-240 days) [8]. Trifluralin has a high affinity to soil [11], is relatively immobile and has a half-life of 3 to 18

Trifluralin chemical composition is α,α,α–trifluoro–2–6-dinitro–N–N– dipropyl–p–toluidine

Trifluralin commercial products contain nitrosodipropylamine, a carcinogenic contaminant

account of concerns about this characteristic, the *Environmental Protection Agency* (EPA) demanded that industries make sure products containing trifluralin active principle had


Trifluralin is strongly adsorbed by organic matter colloids and not much by clay ones. In organic matter rich soils, adsorption prevents absorption of the product by plant roots. Therefore, the use of this herbicide under such conditions is not advisable [10]. Leaching, as well as soil lateral movement is quite reduced compared to some pesticides [17]. Its main characteristic is soil persistence resulting from low mobility, which can cause damage to crops following its application [12].

Such herbicides as trifluralin, applied in pre-emergence, act better when soil humidity is between high and elevated. Therefore, the herbicide may at least be partially solubilized and distributed in the first layers of the soil surface, which will protect it from losses [8].

This herbicide degradation in soil occurs through chemical, microbial pathways and photol‐ ysis. Chemical degradation promotes dealkylation of the amino group, reduction from the nitro to the amino group, partial oxidation from the trifluoromethyl to the carboxyl group and, subsequently, degradation into smaller fragments (Figure 2).

**Figure 2.** Possible sequence of events that occur during trifluralin chemical degradation.

Microbial degradation may occur under aerobic and anaerobic conditions (Figure 3). However, it is observed that degradation occurs mainly under anaerobic conditions, as the ones observed in poorly drained soils, when there is subsequent rainfall. Under anaerobic conditions, within the same time period, 98% of trifluralin degrades, whereas under aerobic conditions only 25% of the product decomposes. Among the fungi capable of decomposing trifluralin are *Sclero‐ tiumrolfsii*, *Aspergillusniger*, *Fusarium*sp and *Tricoderma*sp [10]. According to Carter and Camper [18], trifluralin may also be degraded by *Pseudomonas* sp.

Trifluralin is also sensitive to degradation by ultraviolet rays, and its volatility is one of the main factors of product loss in the soil as well [19] and [20]. Trifluralin photodecomposition generally involves three processes: propylamine oxidative dealkylation, cyclization and nitro group reduction (Figure 4) [21].

The first product of trifluralin photolysis, according to Dimou et al. [21] and illustrated in Figure 3, seems to be a mono-dealkylate deriving from the main compound, originating compound 1. Dealkylation is attributed to the free radical oxidation. Another intermediate of photodegradation appears to be formed by cyclization reactions. The compounds 4 and 5 are apparently formed by reaction among trifluralinpropylamine α carbon and the NO2 group of compound 1, ant they are identified as 2– ethyl -7nitro-1-propyl-5 (trifluoromethyl)-1*H*benzimidazole and 2-ethyl-4 nitro-6- (trifluoromethyl)-1*H*-enzimidazole, respectively. The benzimidazoledealkylate (compound 4) is the most stable photoproduct, which can last in the environment longer, making its detection possible. This product may be formed by the reaction of compound 5 dealkylation.

**Figure 4.** Trifluralin photodegradation. \*ND = Substance not detected in the source. Modified scheme by Dimou et al. [21].

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Compounds 4 and 5 can be reduced in water by not so clear mechanisms [23], straight from the aryl hydroxylamine formation [24] to form compound 7 and 6, respectively. According to the same author these products have also been formed during trifluralin chemical degradation. Compound 2 and 3 are formed from NO2 to NH2 group reduction of compound 1 and 2,6 dinitro-4- (trifluoromethyl) benzenamine (compound ND), respectively. These compounds

**Figure 3.** Trifluralin microbial degradation by aerobic (A) and anaerobic (B) pathways. Source: Audus [22].

Trifluralin is also sensitive to degradation by ultraviolet rays, and its volatility is one of the main factors of product loss in the soil as well [19] and [20]. Trifluralin photodecomposition generally involves three processes: propylamine oxidative dealkylation, cyclization and nitro

The first product of trifluralin photolysis, according to Dimou et al. [21] and illustrated in Figure 3, seems to be a mono-dealkylate deriving from the main compound, originating compound 1. Dealkylation is attributed to the free radical oxidation. Another intermediate of photodegradation appears to be formed by cyclization reactions. The compounds 4 and 5 are apparently formed by reaction among trifluralinpropylamine α carbon and the NO2 group of compound 1, ant they are identified as 2– ethyl -7nitro-1-propyl-5 (trifluoromethyl)-1*H*benzimidazole and 2-ethyl-4 nitro-6- (trifluoromethyl)-1*H*-enzimidazole, respectively. The benzimidazoledealkylate (compound 4) is the most stable photoproduct, which can last in the environment longer, making its detection possible. This product may be formed by the reaction

**Figure 3.** Trifluralin microbial degradation by aerobic (A) and anaerobic (B) pathways. Source: Audus [22].

group reduction (Figure 4) [21].

494 Herbicides - Current Research and Case Studies in Use

of compound 5 dealkylation.

**Figure 4.** Trifluralin photodegradation. \*ND = Substance not detected in the source. Modified scheme by Dimou et al. [21].

Compounds 4 and 5 can be reduced in water by not so clear mechanisms [23], straight from the aryl hydroxylamine formation [24] to form compound 7 and 6, respectively. According to the same author these products have also been formed during trifluralin chemical degradation. Compound 2 and 3 are formed from NO2 to NH2 group reduction of compound 1 and 2,6 dinitro-4- (trifluoromethyl) benzenamine (compound ND), respectively. These compounds have also been identified during trifluralin chemical degradation [24], showing that this pathway also happens in other processes, besides photodegradation [21].

Trifluralin's main mechanism of action is the inhibition of cell mitosis. This herbicide typically acts on the meristems and tissues of underground organs, such as roots, epicotyls, hypocotyls,

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The inhibition of radicle development by trifluralin action, both on main root growth and the emission of secondary roots, is quite evident in some dicotyledons. Thickening of the hypo‐ cotyls also commonly occurs [8], as well as swollen root tips [36]. According to Almeida [25], trifluralin induces several biochemical changes in higher plants, including alterations of carbohydrate, lipid, nitrogen concentrations and, especially, nucleic acid alterations. There‐ fore, the product affects cell division in meristematic tissues, thus inhibiting seed germination

Bayer et al. [37] report that trifluralin promotes a decrease in the zone of meristematic tissues and the interruption of mitosis in the roots of wheat, cotton and onions. The onion cells treated with trifluralin showed to be small, dense and multinucleated, abnormal, weak and aberrant [38].Studies conducted by Fernandes [39] using *Allium cepa* showed that the toxicity of trifluralin residual concentrations might induce changes in that plant. The author observed that the herbicide promoted plant growth inhibition, higher turgidity, weakness and thickness

Plants grown in soils treated with trifluralin exhibited residues on the roots only. No residue was found on the leaves, fruit and seeds [25]. These results indicate that trifluralin is not

Plant growth and development depend on mitosis in their meristematic regions. Cell division is a process that requires different cell organelles, structures and the products of many genes to be working correctly. Dinitroanilines, the family to which trifluralin, phosphoride amides and N-phenyl carbonates belong, are microtubule-depolymerizing chemical compounds [5], [40], [41], [42] and[43]. According to Senseman [36], the herbicide-trifluralin complex inhibits microtubule polymerization, leading to physical misconfiguration and loss of function. As a consequence, the mitotic spindle does not form, causing misalignment and chromosome separation during mitosis. In addition to that, the so-called spindle apparatus is not formed.

Microtubules are subcellular structure filaments, basically made up of heterodimeric tubulin protein (Figure 5A) [44]. They have important cellular functions, which are directly related to mitosis and indirectly related to organism development. These structures are involved in several cellular processes such as chromosome migration, cellular structure maintenance, cellulose microfibril orientation and organization, cell wall formation, intracellular movement, as well as cellular differentiation [42] and [45]. Most sets of cell microtubules are labile and their functions depend on this lability.The mitotic spindle is one of the most extraordinary examples, whose formation is brought about after disorganization of cytoplasmic microtubule at the beginning of mitosis. For this reason, the mitotic spindle is targeted by various specific

plumules, rhizomes, bulbs and seeds [8].

and the formation of new radicle and hypocotyl cells.

of the roots, in relation to the control treatment.

transported by sap into other plat tissues.

**4. Trifluralin mechanisms of action**

Trifluralin average persistence in soil for the recommended doses under field conditions is of 1.8 ppm residue after 180 days following application [25]. However, according to the same author, this persistence may vary in accordance with the kind of soil and climatic conditions.

#### **3.2. Herbicide behavior in water**

Water contamination with trifluralin may occur by sediment leaching while equipment is being cleaned, or due to accidental spills. Nevertheless, only 0.5% of the quantity applied to the soil in field conditions is leached and may consequently contaminate water sources. This percentage means a rather low water contamination, representing smaller concentrations than 1.0 μg L-1. As a consequence, trifluralin is not commonly detected in surface water [9] and [26].

While Zimmerman et al. [26], Dayama and Coupe [27], Thurman et al. [28] and were carrying out analyses in the Mississippi River, they detected extremely low levels of trifluralin (lower than 0.1 g/L). Once this herbicide is widely used, the authors ascertain that low concentrations of it detected in surface water may be attributed to its low mobility in soil and low solubility in water (lower than 1 mg/L). USEPA [29] and the European Community legislation [30] established limits of 2μg/ L and 0.1μg/ Ltrifluralin in drinking water, respectively. According to Dimou et al. [21], trifluralin degradation in water is influenced by the presence of nitrate ions, which accelerate photolysis reaction. Products derived from this reaction have either low or no toxicity, when compared to the whole product.

#### **3.3. Herbicide behavior in the air**

Grover et al. [31] ascertain that trifluralin is quickly dissipated in the atmosphere. Depending on the season of the year, about 25% of the product applied is volatilized, but only 2-3 μg/m3 at the most of trifluralin is found in the air, soon after its application, to less than 100ng/m3 a few hours later [32]. According to the United States Environment Protection Agency (1993) [33], an average 0.27 ng/m3 concentration of herbicide, varying from 0 to 3.4 ng/m3 , was found in the Canadian atmosphere between 1988 and 1989.

Mongar and Miller [34] state that low concentrations of this herbicide found in the atmosphere are due to both trifluralin quick reaction with the hydroxyl radical (OH) and the photolysis reaction, which promotes the product degradation.Nonetheless, Waite et al. [32] verified that of the five most used herbicides on the Canadian prairies, trifluralin was the most frequently found in the air (79% of samples).

#### **3.4. Herbicide behavior in plants**

Trifluralin is a pre-emergence herbicide which must be incorporated into the soil and applied soon after sowing, when the plant seeds are beginning the germination process [36]. The herbicide absorption occurs mainly by the hypocotyl, then by the seedling radicles, at the beginning of germination [10].

Trifluralin's main mechanism of action is the inhibition of cell mitosis. This herbicide typically acts on the meristems and tissues of underground organs, such as roots, epicotyls, hypocotyls, plumules, rhizomes, bulbs and seeds [8].

The inhibition of radicle development by trifluralin action, both on main root growth and the emission of secondary roots, is quite evident in some dicotyledons. Thickening of the hypo‐ cotyls also commonly occurs [8], as well as swollen root tips [36]. According to Almeida [25], trifluralin induces several biochemical changes in higher plants, including alterations of carbohydrate, lipid, nitrogen concentrations and, especially, nucleic acid alterations. There‐ fore, the product affects cell division in meristematic tissues, thus inhibiting seed germination and the formation of new radicle and hypocotyl cells.

Bayer et al. [37] report that trifluralin promotes a decrease in the zone of meristematic tissues and the interruption of mitosis in the roots of wheat, cotton and onions. The onion cells treated with trifluralin showed to be small, dense and multinucleated, abnormal, weak and aberrant [38].Studies conducted by Fernandes [39] using *Allium cepa* showed that the toxicity of trifluralin residual concentrations might induce changes in that plant. The author observed that the herbicide promoted plant growth inhibition, higher turgidity, weakness and thickness of the roots, in relation to the control treatment.

Plants grown in soils treated with trifluralin exhibited residues on the roots only. No residue was found on the leaves, fruit and seeds [25]. These results indicate that trifluralin is not transported by sap into other plat tissues.

### **4. Trifluralin mechanisms of action**

have also been identified during trifluralin chemical degradation [24], showing that this

Trifluralin average persistence in soil for the recommended doses under field conditions is of 1.8 ppm residue after 180 days following application [25]. However, according to the same author, this persistence may vary in accordance with the kind of soil and climatic conditions.

Water contamination with trifluralin may occur by sediment leaching while equipment is being cleaned, or due to accidental spills. Nevertheless, only 0.5% of the quantity applied to the soil in field conditions is leached and may consequently contaminate water sources. This percentage means a rather low water contamination, representing smaller concentrations than 1.0 μg L-1. As a consequence, trifluralin is not commonly detected in surface water [9] and [26].

While Zimmerman et al. [26], Dayama and Coupe [27], Thurman et al. [28] and were carrying out analyses in the Mississippi River, they detected extremely low levels of trifluralin (lower than 0.1 g/L). Once this herbicide is widely used, the authors ascertain that low concentrations of it detected in surface water may be attributed to its low mobility in soil and low solubility in water (lower than 1 mg/L). USEPA [29] and the European Community legislation [30] established limits of 2μg/ L and 0.1μg/ Ltrifluralin in drinking water, respectively. According to Dimou et al. [21], trifluralin degradation in water is influenced by the presence of nitrate ions, which accelerate photolysis reaction. Products derived from this reaction have either low

Grover et al. [31] ascertain that trifluralin is quickly dissipated in the atmosphere. Depending on the season of the year, about 25% of the product applied is volatilized, but only 2-3 μg/m3 at the most of trifluralin is found in the air, soon after its application, to less than 100ng/m3

few hours later [32]. According to the United States Environment Protection Agency (1993)

Mongar and Miller [34] state that low concentrations of this herbicide found in the atmosphere are due to both trifluralin quick reaction with the hydroxyl radical (OH) and the photolysis reaction, which promotes the product degradation.Nonetheless, Waite et al. [32] verified that of the five most used herbicides on the Canadian prairies, trifluralin was the most frequently

Trifluralin is a pre-emergence herbicide which must be incorporated into the soil and applied soon after sowing, when the plant seeds are beginning the germination process [36]. The herbicide absorption occurs mainly by the hypocotyl, then by the seedling radicles, at the

concentration of herbicide, varying from 0 to 3.4 ng/m3

a

, was found

pathway also happens in other processes, besides photodegradation [21].

**3.2. Herbicide behavior in water**

496 Herbicides - Current Research and Case Studies in Use

or no toxicity, when compared to the whole product.

in the Canadian atmosphere between 1988 and 1989.

**3.3. Herbicide behavior in the air**

[33], an average 0.27 ng/m3

found in the air (79% of samples).

**3.4. Herbicide behavior in plants**

beginning of germination [10].

Plant growth and development depend on mitosis in their meristematic regions. Cell division is a process that requires different cell organelles, structures and the products of many genes to be working correctly. Dinitroanilines, the family to which trifluralin, phosphoride amides and N-phenyl carbonates belong, are microtubule-depolymerizing chemical compounds [5], [40], [41], [42] and[43]. According to Senseman [36], the herbicide-trifluralin complex inhibits microtubule polymerization, leading to physical misconfiguration and loss of function. As a consequence, the mitotic spindle does not form, causing misalignment and chromosome separation during mitosis. In addition to that, the so-called spindle apparatus is not formed.

Microtubules are subcellular structure filaments, basically made up of heterodimeric tubulin protein (Figure 5A) [44]. They have important cellular functions, which are directly related to mitosis and indirectly related to organism development. These structures are involved in several cellular processes such as chromosome migration, cellular structure maintenance, cellulose microfibril orientation and organization, cell wall formation, intracellular movement, as well as cellular differentiation [42] and [45]. Most sets of cell microtubules are labile and their functions depend on this lability.The mitotic spindle is one of the most extraordinary examples, whose formation is brought about after disorganization of cytoplasmic microtubule at the beginning of mitosis. For this reason, the mitotic spindle is targeted by various specific anti-mitotic drugs, which interfere in the exchange of tubulin subunits between the microtu‐ bules and the pool of free tubulins [46].

Anthony et al. [49] ascertained that, as a rule, the tubulin sequence is the most preserved among the different organisms; and this preservation is related to the basic functions of microtubules. Mahresh and Larry [50], however, believe that, depending on the organism, dinitroaniline herbicides have different affinities to tubulins, since they do not interact with vertebrate tubulins, although they interact with plant and *Chlamydomonas* tubulins. This situation is reinforced with data from Anthony and Hussey [47], Baird et al. [51], Breviário and Nick [52] and Yemets and Blume [53], who ascertain that dinitroaniline herbicides are compounds with

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Studies on plant resistance to dinitroanilines showed that some plant species own a natural mutation which bring about a change in base pairs, and consequently in their genetic code. One of these alterations of base causes a change in the amino acids of the tubulin protein. Threonine, a normal amino acid at position 239, is changed into isoleucine, stopping group NO2 of the dinitroaniline herbicides from binding the tubulin molecule, thus preventing its

**Figure 6.** Alignment of amino acid sequence of α-tubulins, evidencing the position of substitution in the mutating tubulin from *Eleusine indica* (Thr 239 into Ile- represented in black and indicated with an arrow). Modified from Blume

From these pieces of information, it would be intuitive to hypothesize the idea that the smallest affinity of trifluralin to vertebrates should be owing to the fact they do not have the amino acid at position 239, seemingly the herbicide target site. Nevertheless, it can be seen in Figure 7 that the threonine amino acid at position 239 of the α-tubulin protein is present in plants, parasites

However, Hashim et al. [58] found mutations in the α-tubulin gene expression which changed the amino acid synthesis at a different position than that found by Anthony and Hussey [47]. According to Hashim et al. [58], *Alopecurus aequalis* plants that underwent mutations, which altered the amino acid synthesis at positions 202, 136 and 125 of the α-tubulin, also brought

Sree et al. [59], Hansen et al. [60] and Vidakovié-Cifrek et al. [61], ascertain that trifluralin can inhibit microtubule polymerization by binding tubulin. However, it can also cause changes in

higher specificity for binding plant tubulins than to those of vertebrates.

mechanism of action (Figure 6) [47].

and vertebrates, including man.

about resistance to trifluralin.

et al. [54].

*In-vitro* analyses of *Chlamydomonas reinhardii* showed that trifluralin specifically binds tubulins, demonstrating that it is the first subcellular target of dinitroaniline action [47]. Trifluralinsub‐ micromolar concentrations totally blocked cytokinesis and inhibit nuclear division in *Toxo‐ plasma gondii* by interfering in intracellular spindle and in other cytoskeletal components [48].

According to Anthony and Hussey [47], the herbicide-tubulin complex is related to the suppression of microtubule growth. With minus-end specific microtubule depolymerization, the tubules progressively start to get shorter, eventually leading to total loss of microtubule (Figure 5B). The author still states that cortical microtubules are among the most resistant to trifluralin action and microtubule spindles and fragments are among the most sensitive to the herbicide action.

**Figure 5. A.** tubulin dimers forming the microtubule; **B.** herbicide-tubulin complex preventing microtubule polymerization.

Anthony et al. [49] ascertained that, as a rule, the tubulin sequence is the most preserved among the different organisms; and this preservation is related to the basic functions of microtubules. Mahresh and Larry [50], however, believe that, depending on the organism, dinitroaniline herbicides have different affinities to tubulins, since they do not interact with vertebrate tubulins, although they interact with plant and *Chlamydomonas* tubulins. This situation is reinforced with data from Anthony and Hussey [47], Baird et al. [51], Breviário and Nick [52] and Yemets and Blume [53], who ascertain that dinitroaniline herbicides are compounds with higher specificity for binding plant tubulins than to those of vertebrates.

anti-mitotic drugs, which interfere in the exchange of tubulin subunits between the microtu‐

*In-vitro* analyses of *Chlamydomonas reinhardii* showed that trifluralin specifically binds tubulins, demonstrating that it is the first subcellular target of dinitroaniline action [47]. Trifluralinsub‐ micromolar concentrations totally blocked cytokinesis and inhibit nuclear division in *Toxo‐ plasma gondii* by interfering in intracellular spindle and in other cytoskeletal components [48].

According to Anthony and Hussey [47], the herbicide-tubulin complex is related to the suppression of microtubule growth. With minus-end specific microtubule depolymerization, the tubules progressively start to get shorter, eventually leading to total loss of microtubule (Figure 5B). The author still states that cortical microtubules are among the most resistant to trifluralin action and microtubule spindles and fragments are among the most sensitive to the

**Figure 5. A.** tubulin dimers forming the microtubule; **B.** herbicide-tubulin complex preventing microtubule polymerization.

bules and the pool of free tubulins [46].

498 Herbicides - Current Research and Case Studies in Use

herbicide action.

Studies on plant resistance to dinitroanilines showed that some plant species own a natural mutation which bring about a change in base pairs, and consequently in their genetic code. One of these alterations of base causes a change in the amino acids of the tubulin protein. Threonine, a normal amino acid at position 239, is changed into isoleucine, stopping group NO2 of the dinitroaniline herbicides from binding the tubulin molecule, thus preventing its mechanism of action (Figure 6) [47].

**Figure 6.** Alignment of amino acid sequence of α-tubulins, evidencing the position of substitution in the mutating tubulin from *Eleusine indica* (Thr 239 into Ile- represented in black and indicated with an arrow). Modified from Blume et al. [54].

From these pieces of information, it would be intuitive to hypothesize the idea that the smallest affinity of trifluralin to vertebrates should be owing to the fact they do not have the amino acid at position 239, seemingly the herbicide target site. Nevertheless, it can be seen in Figure 7 that the threonine amino acid at position 239 of the α-tubulin protein is present in plants, parasites and vertebrates, including man.

However, Hashim et al. [58] found mutations in the α-tubulin gene expression which changed the amino acid synthesis at a different position than that found by Anthony and Hussey [47]. According to Hashim et al. [58], *Alopecurus aequalis* plants that underwent mutations, which altered the amino acid synthesis at positions 202, 136 and 125 of the α-tubulin, also brought about resistance to trifluralin.

Sree et al. [59], Hansen et al. [60] and Vidakovié-Cifrek et al. [61], ascertain that trifluralin can inhibit microtubule polymerization by binding tubulin. However, it can also cause changes in the ion calcium concentration in cytoplasm and influence polymerization and depolymeriza‐ tion regulation of microtubules. According to Hertel et al. [62], changes in the quantity of free Ca2+ in cytoplasm, due to trifluralin action, can alter calcium-dependant biochemical and physiological processes, in addition to causing problems to microtubules, either in animals or in plants. Vidakovié-Cifrek et al. [61] report that trifluralin may increase the concentration of Ca2+ ions in cytoplasm, influencing onion root mitosis.

Another important factor to be considered is the derivate generation through pesticide biodegradation [66] and [67]. One of the byproducts of trifluralin biodegradation is an aniline:

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Anilines are compounds that cause a variety of toxic effects depending on the structural changes they undergo. Several studies demonstrate that anilines and halogens can induce metahemoglobin formation and also be toxic to the kidneys and the liver, either treated *in vitro* or *in vivo* [69] and [70]. Aminophenols, the primary products of aniline metabolism, are

Although many researchers and international governmental agencies have investigated and published trifluralin toxic effects on different fields, whether they are related to either acute or chronic toxicity, cytotoxicity, genotoxicity, mutagenicity and carcinogenicity, the results

According to the W.H.O (World Health Organization) [70], trifluralin causes hemoglobin oxidation (by forming metahemoglobin), red blood cell destruction, besides being toxic to the kidneys and the liver, and stimulating depression in the central nervous system. It may cause vomiting, diarrhea, weakness, profuse sweating, loss of sight, memory and concentration, and dermatitis as well. This herbicide is considered to be neurotoxic and gastrointestinal irritant. It can lead to death because of ventricular fibrillation [71], although several authors [10], [72],

Trifluralin lethal concentrations and doses for vertebrates and invertebrates are shown in Table 2.

[73], [74], [75] and [76], ascertain that trifluralin is a low toxicity substance.

2,6dinitroaniline (Figure 8) [68].

**Figure 8.** Chemical structure of 2,6 dinitroaniline.

**5. Trifluralin toxic effect**

compounds related to neurotoxicity induction [69].

shown are confusing and often contradictory.

Due to trifluralin chemical structure, this herbicide tends to receive two electrons, which significantly increases its toxicity, since the group NH2 hydrogen of trifluralin tends to bind the polar group of cellular membranes and cause disorganization to its structure, eventually bringing function disorders [63]. This disorganization in the membrane structure seems to interfere mainly in the permeability of plasma and mitochondrial membranes. Trifluralin changes the permeability of membranes because it promotes a collapse in their electric potential, making Ca+2 efflux of the mitochondrial inner membranes and Ca+2 go from the outer to the inner surface of the cell membrane via uniporters, thus increasing the concentrations of such ions in the inner cytoplasmic membrane.

Since low levels of calcium are needed for polymerization, Hepler [64] ascertains that mitotic spindles may undergo disorders due to the high levels of this ion. Low concentrations of free calcium in the cytoplasm (0.1-0.2 μM) are essential to prevent phosphorus precipitation, compete with Mg2+ for binding sites and act as a secondary messenger [65].

According to Alberts et al. [46], Ca+2 is important for regulating mitochondrial enzyme activity, and it is imported from the cytosol through an H+ electrostatic gradient. It is also believed that this process is important to remove Ca+2 from the cytosol when cytosolic Ca+2 levels get dangerously high.

**Figure 7.** Comparisons among sequences of α-tubulin amino acids of species *Zea mays* (vegetable)*, Hordeumvulgare* (vegetable)*, Arabidopsis thaliana* (vegetable) *Prunus amygdalus* (vegetable)*, Pisum sativum* (vegetable),Leishmania d*onovani* (parasite)*, Trypanosoma cruzi* (vegetable),*Mus musculus* (vertebrate)*, Sus scrofa* (vertebrate) and Homo sapi‐ *ens*. The sequences were obtained from the data base at NCBI (National Center of Biotechnology Information) in ac‐ cordance with the codes P14641, Y08490, P29511, P33629, U12589, U09612, M97956, P05213, P02550 and P04687, respectively [55]. The sequences were aligned by means of the ClustalW program [56], using default parameters. The alignment was then analyzed using the MPALign program [57].

Another important factor to be considered is the derivate generation through pesticide biodegradation [66] and [67]. One of the byproducts of trifluralin biodegradation is an aniline: 2,6dinitroaniline (Figure 8) [68].

**Figure 8.** Chemical structure of 2,6 dinitroaniline.

the ion calcium concentration in cytoplasm and influence polymerization and depolymeriza‐ tion regulation of microtubules. According to Hertel et al. [62], changes in the quantity of free Ca2+ in cytoplasm, due to trifluralin action, can alter calcium-dependant biochemical and physiological processes, in addition to causing problems to microtubules, either in animals or in plants. Vidakovié-Cifrek et al. [61] report that trifluralin may increase the concentration of

Due to trifluralin chemical structure, this herbicide tends to receive two electrons, which significantly increases its toxicity, since the group NH2 hydrogen of trifluralin tends to bind the polar group of cellular membranes and cause disorganization to its structure, eventually bringing function disorders [63]. This disorganization in the membrane structure seems to interfere mainly in the permeability of plasma and mitochondrial membranes. Trifluralin changes the permeability of membranes because it promotes a collapse in their electric potential, making Ca+2 efflux of the mitochondrial inner membranes and Ca+2 go from the outer to the inner surface of the cell membrane via uniporters, thus increasing the concentrations of

Since low levels of calcium are needed for polymerization, Hepler [64] ascertains that mitotic spindles may undergo disorders due to the high levels of this ion. Low concentrations of free calcium in the cytoplasm (0.1-0.2 μM) are essential to prevent phosphorus precipitation,

According to Alberts et al. [46], Ca+2 is important for regulating mitochondrial enzyme activity, and it is imported from the cytosol through an H+ electrostatic gradient. It is also believed that this process is important to remove Ca+2 from the cytosol when cytosolic Ca+2 levels get

**Figure 7.** Comparisons among sequences of α-tubulin amino acids of species *Zea mays* (vegetable)*, Hordeumvulgare* (vegetable)*, Arabidopsis thaliana* (vegetable) *Prunus amygdalus* (vegetable)*, Pisum sativum* (vegetable),Leishmania d*onovani* (parasite)*, Trypanosoma cruzi* (vegetable),*Mus musculus* (vertebrate)*, Sus scrofa* (vertebrate) and Homo sapi‐ *ens*. The sequences were obtained from the data base at NCBI (National Center of Biotechnology Information) in ac‐ cordance with the codes P14641, Y08490, P29511, P33629, U12589, U09612, M97956, P05213, P02550 and P04687, respectively [55]. The sequences were aligned by means of the ClustalW program [56], using default parameters. The

compete with Mg2+ for binding sites and act as a secondary messenger [65].

Ca2+ ions in cytoplasm, influencing onion root mitosis.

500 Herbicides - Current Research and Case Studies in Use

such ions in the inner cytoplasmic membrane.

alignment was then analyzed using the MPALign program [57].

dangerously high.

Anilines are compounds that cause a variety of toxic effects depending on the structural changes they undergo. Several studies demonstrate that anilines and halogens can induce metahemoglobin formation and also be toxic to the kidneys and the liver, either treated *in vitro* or *in vivo* [69] and [70]. Aminophenols, the primary products of aniline metabolism, are compounds related to neurotoxicity induction [69].

### **5. Trifluralin toxic effect**

Although many researchers and international governmental agencies have investigated and published trifluralin toxic effects on different fields, whether they are related to either acute or chronic toxicity, cytotoxicity, genotoxicity, mutagenicity and carcinogenicity, the results shown are confusing and often contradictory.

According to the W.H.O (World Health Organization) [70], trifluralin causes hemoglobin oxidation (by forming metahemoglobin), red blood cell destruction, besides being toxic to the kidneys and the liver, and stimulating depression in the central nervous system. It may cause vomiting, diarrhea, weakness, profuse sweating, loss of sight, memory and concentration, and dermatitis as well. This herbicide is considered to be neurotoxic and gastrointestinal irritant. It can lead to death because of ventricular fibrillation [71], although several authors [10], [72], [73], [74], [75] and [76], ascertain that trifluralin is a low toxicity substance.

Trifluralin lethal concentrations and doses for vertebrates and invertebrates are shown in Table 2.


According to the Occupational Health Service [79], prolonged skin contact with trifluralin may cause allergic dermatitis. The WSSA [80] states that administering trifluralin to dogs while washing them for two years does not cause toxic effects. However, in trifluralin chronic assays conducted with 60 animals (F344 mice), which received 0.813, 3250 and 6500 ppm dietary does

Characterization, Modes of Action and Effects of Trifluralin: A Review

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503

Worthing [71] states that trifluralin is highly toxic and neurotoxic. The author ascertains that the herbicide is capable of accumulating in the adipose tissue and inhibiting the immunologic function of the thymus. Trifluralin is regarded as possibly teratogenic and fetal toxicity.It has the property of altering the endocrine and reproductive system, and it reduces the quantity of

In studies conducted by Ovidi et al. [82], they tested trifluralin concentration of 1.53 mg/ml and observed that the herbicide exerts a specific effect on the reproductive system in plants, by direct action on the formation of the pollinic tubes, since it causes complete microtubule depolymerization. The authors even suggest that pollinic microtubule cytoskeleton may be used as bioindicators for studies on toxicity induced by aneugenic agents such as trifluralin.

As a general rule, the effects of pesticides may be diversified, such as the direct reaction with nuclear DNA; incorporation of DNA during cellular replication; interference in mitosis or

Genotoxic effects may lead to DNA breaks, causing loss of genetic material and mutations which lead to cell death or result in carcinogenesis. Genotoxicity is assessed by different tests, carry out with several organisms and provide safe, precise information regarding their potential to damage the DNA.There are a number of reports evaluating trifluralin for geno‐ toxicity, immunotoxicity, and reproductive toxicity, although the results are not entirely

Chromosome aberration tests have shown evidences of trifluralin mutagenicity for different plant species [85], [86], [87], [88], [89] and [90]. Könen and Çavas [91], Peña [92] and Canevari [93] ascertained that the herbicide is capable of inducing significant microtubule rates in *Oreochromis niloticus*. Kaya et al. [94] also ascertained that the herbicide may be considered genotoxic to *Drosophila melanogaster*, since it exhibited positive outcomes for the Somatic Mutation and Recombination Test (SMART). Tests conducted in the bone marrow of mice exposed to trifluralin showed that it is potentially genotoxic [95] and it is also capable of influencing serum concentration of reproductive and metabolic hormones, especially thyroxin [96]. Nonetheless, tests performed on bacteria [14], on *Drosophyla melanogaster* conducted by Bryant and Murnik [97] and Foureman [98], on cells taken from the bone marrow of mice conducted by Nehéz et al. [99], Pilinkaya [100], Gebel et al., [95], and on cell culture conducted by IARC [101] and Ribas et al. [35 and 102] demonstrated contradictory results. According to Chan and Fong [103], Bhattacharya et al. [104] and Esteves et al. [105], due to its characteristics, mechanisms of action and, especially its reduced effects on human cells, trifluralin can be regarded as a promising substance for fighting Leishmaniasis. There is also research that confirms the use of trifluralin as a powerful antiparasitic to treat *Trypanosoma* [106] and [107],

for two years, damage to their liver and kidneys were observed [81].

semen, besides increasing the number of abnormal sperm.

meiosis, resulting from incorrect cell division [83].

*Toxoplasma* [48] and *Plasmodium* [108].

consistent, trifluralin does not appear to be strongly genotoxic [84].

**Table 2.** TrifluralinCL50 and DL50 for different organisms

Meister [78] conducted tests with animals and verified that trifluralin does not have any toxic effect on them when they are exposed to the product either through ingestion, inhalation or when in contact with the skin. Nauseas and severe gastrointestinal discomfort may occur after trifluralin ingestion. When placed in the rabbit eyes, it produced a mild irritation, which was reverted within seven days. In humans, it may induce skin allergies and, when inhaled, it may irritate the throat and the lungs.

Table 3 shows some information regarding trifluralin chronic, sub-acute and sub- chronic toxicity to different organisms.


\*Liver weight of the animals submitted to the 50 and 100mg Kg-1 diet somehow showed to be higher, when compared to the control animals. Data extracted from Gangolli [77].

**Table 3.** Data on trifluralin sub-acute, chronic and sub-chronic toxicity.

According to the Occupational Health Service [79], prolonged skin contact with trifluralin may cause allergic dermatitis. The WSSA [80] states that administering trifluralin to dogs while washing them for two years does not cause toxic effects. However, in trifluralin chronic assays conducted with 60 animals (F344 mice), which received 0.813, 3250 and 6500 ppm dietary does for two years, damage to their liver and kidneys were observed [81].

**Treatment Species Group Popular Name Toxicity** Cl50 (48h) *Lepomis macrochirus* Fish Bluegill 19 µg L-1 CL50 (48h) *Mola mola* Fish Ocean sunfish 19µg L-1 CL50 (48h) *Cyprinus carpio* Fish Common carp 1.0mg L-1 CL50 (96h) *Oncorhynchus mykiss* Fish Rainbow trout 0,21mgL-1

(Young)fish

Meister [78] conducted tests with animals and verified that trifluralin does not have any toxic effect on them when they are exposed to the product either through ingestion, inhalation or when in contact with the skin. Nauseas and severe gastrointestinal discomfort may occur after trifluralin ingestion. When placed in the rabbit eyes, it produced a mild irritation, which was reverted within seven days. In humans, it may induce skin allergies and, when inhaled, it may

Table 3 shows some information regarding trifluralin chronic, sub-acute and sub- chronic


*Ratus norvegicus* Mammal Mouse 25, 50 e 100

\*Liver weight of the animals submitted to the 50 and 100mg Kg-1 diet somehow showed to be higher, when compared

CL50 (48h) *Daphnia magma* Micro-crustacean - 0,56 mgL-1 CL50 (96h) *Procambarus clarkia* Crustacean Lobster 12mgL-1 DL50 (oral) *Apis mellifera* Insect Honey bee 0,011mg bee-1 DL50 (oral) *Mus musculus* Mammal Laboratory mice >500 mg kg -1 DL50 (oral) *Ratus norvegicus* Mammal Laboratory mice > 10.000 mg kg-1 DL50 (oral) - Mammal Dog > 200 mg kg-1 DL50 (oral) - Mammal Rabbit > 200 mg kg-1 DL50 (oral) - Bird Hen > 200 mg kg-1

Rainbow trout, Bluegill, Ocean sunfish

**Name Toxicity Symptoms**

mg kg-1 dia-1

erythema

no effects produced on either survival or appearance \*

10-90µg L-1

CL50 (96h)

*Oncorhynchus mykiss*, *Lepomis macrochirus, Mola*

**Table 2.** TrifluralinCL50 and DL50 for different organisms

**Treatment Species Group Popular**

to the control animals. Data extracted from Gangolli [77].

**Table 3.** Data on trifluralin sub-acute, chronic and sub-chronic toxicity.

LOEC *Amphiprion percula* Fish clownfish 5µg L-1 - NOEL *Amphiprion percula* Fish clownfish 2µ L-1 - CE50 (10 days) *Chlorococcum* sp Protozoa - 2,5 mg L-1 -

*mola*

502 Herbicides - Current Research and Case Studies in Use

Data extracted from Gangolli [77].

irritate the throat and the lungs.

toxicity to different organisms.

Sub-acute(dermis

chronic(ingestion - 3 months)

Sub-

Worthing [71] states that trifluralin is highly toxic and neurotoxic. The author ascertains that the herbicide is capable of accumulating in the adipose tissue and inhibiting the immunologic function of the thymus. Trifluralin is regarded as possibly teratogenic and fetal toxicity.It has the property of altering the endocrine and reproductive system, and it reduces the quantity of semen, besides increasing the number of abnormal sperm.

In studies conducted by Ovidi et al. [82], they tested trifluralin concentration of 1.53 mg/ml and observed that the herbicide exerts a specific effect on the reproductive system in plants, by direct action on the formation of the pollinic tubes, since it causes complete microtubule depolymerization. The authors even suggest that pollinic microtubule cytoskeleton may be used as bioindicators for studies on toxicity induced by aneugenic agents such as trifluralin.

As a general rule, the effects of pesticides may be diversified, such as the direct reaction with nuclear DNA; incorporation of DNA during cellular replication; interference in mitosis or meiosis, resulting from incorrect cell division [83].

Genotoxic effects may lead to DNA breaks, causing loss of genetic material and mutations which lead to cell death or result in carcinogenesis. Genotoxicity is assessed by different tests, carry out with several organisms and provide safe, precise information regarding their potential to damage the DNA.There are a number of reports evaluating trifluralin for geno‐ toxicity, immunotoxicity, and reproductive toxicity, although the results are not entirely consistent, trifluralin does not appear to be strongly genotoxic [84].

Chromosome aberration tests have shown evidences of trifluralin mutagenicity for different plant species [85], [86], [87], [88], [89] and [90]. Könen and Çavas [91], Peña [92] and Canevari [93] ascertained that the herbicide is capable of inducing significant microtubule rates in *Oreochromis niloticus*. Kaya et al. [94] also ascertained that the herbicide may be considered genotoxic to *Drosophila melanogaster*, since it exhibited positive outcomes for the Somatic Mutation and Recombination Test (SMART). Tests conducted in the bone marrow of mice exposed to trifluralin showed that it is potentially genotoxic [95] and it is also capable of influencing serum concentration of reproductive and metabolic hormones, especially thyroxin [96]. Nonetheless, tests performed on bacteria [14], on *Drosophyla melanogaster* conducted by Bryant and Murnik [97] and Foureman [98], on cells taken from the bone marrow of mice conducted by Nehéz et al. [99], Pilinkaya [100], Gebel et al., [95], and on cell culture conducted by IARC [101] and Ribas et al. [35 and 102] demonstrated contradictory results. According to Chan and Fong [103], Bhattacharya et al. [104] and Esteves et al. [105], due to its characteristics, mechanisms of action and, especially its reduced effects on human cells, trifluralin can be regarded as a promising substance for fighting Leishmaniasis. There is also research that confirms the use of trifluralin as a powerful antiparasitic to treat *Trypanosoma* [106] and [107], *Toxoplasma* [48] and *Plasmodium* [108].

Studies carried out by Peña [92] and Canevari [93] indicate that low trifluralin concentrations may induce mutagenic effects. These authors observed significant presence of micronuclei in erythrocytes of fish submitted to acute treatments with this herbicide. When the micronuclei diameters were measured by Canevari [93], data indicated that they could be derived from losses of whole chromosomes, thus proving the aneugenic effect of the herbicide due to the pesticide interference in the mitotic spindle.

Fernandes et al. [89] ascertained that, among the root meristems of *Allium cepa* under division, trifluralin promotes a significant increase in the irregular metaphase rate. These data corrob‐ orate the statement of Lignowski and Scott [85], Lee et al. [109], Dow et al. [110], Werbovetz et al. [111] and Ovidi et al. [82], who characterized trifluralin as a powerful microtubule inhibitor, which is therefore capable of accumulating a large amount of meristematic cells in metaphase.

Characterization, Modes of Action and Effects of Trifluralin: A Review

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505

Genotoxicity tests using the comet assay in human lymphocyte cultures showed that trifluralin produced a significant increase in the length of the comet's tail. This increase is due to DNA breaks, since there is an induction of nucleotide excision repair, resulting from damage caused by the herbicide action [103]. As for the frequency of comet-bearing cells, the author observed that, after 48 hours of exposure to the herbicide, few tailed nucleoides were found. These results

According to Ribas et al. [35], trifluralin has a genotoxic effect on human cell cultures because it causes a decrease in cell proliferation. The same author ascertains that this herbicide has not revealed carcinogenic effects, since it caused little induction exchange between sister chroma‐ tids.The micronucleus test conducted by Ribas et al. [35], used for detecting aneugenic activity, has also produced a negative response, which contradicts studies carried out by several other authors [88], [89], [91], [92], [97], [112], among others) who ascertain that trifluralin brings about chromosome aberrations and nuclear alterations resulting from problems in the mitotic spindle

According to Kang et al. [113], trifluralin is not associated with bladder, kidney, liver, leukemia, colorectal or hematopoietic-lymphatic cancers. The authors only suggest a possible connection between trifluralin exposure and the risk of colon cancer in human beings, but the inconsis‐ tency per exposure level and a small number of colon cancers indicate that this could be an

Data from the National Cancer Institute (NCI) [114] report that mice subjected to trifluralin chronic exposure, at low concentrations, had an increase in hepatocellular carcinoma and higher incidence of alveolar bronchial adenomas. An increase in bladder cancer was also verified in mice exposed to low trifluralin concentrations. It was observed that, when male mice were submitted to high doses of trifluralin, they presented higher incidence of follicular cell and thyroid gland tumors [115]. Trifluralin has been reported to cause a significant increase

**Figure 10.** Meristematic cells of *Allium* cepa treated with trifluralin. **A.** cell with micronucleus; **B.** cell with micronu‐

proved to be statistically significant, though.

cleus and an adjacent mini cell; **C.**polynucleated cell.

incidental finding.

*Allium cepa*meristematic cells treated with trifluralin also presented problems during mitosis, such as polyploidies, C-metaphases, multipolar anaphases, anaphase-telophase chromatin bridges, chromosome delay and loss of genetic material [89]. (Figure 9).

**Figure 9.** Meristematic cells of *Allium cepa* treated with trifluralin. **A.** C-metaphase; **B.**polyploid cell; **C.** multipolar cell; **D**. loss of genetic material; **E.**chromosome bridge; **F.**telophase with chromosome delay.

According to Fernandes et al. [88], in the bioassays with root meristems of *Allium cepa* treated with trifluralin, a large amount of interphase cells with more than one nucleus and cells with micronuclei and a mini cell were observed (Figure 10).

Lignowski and Scott [85] observed C-metaphases, micronuclei, amoeboid nuclei and poly‐ ploidies in root meristems of wheat and onion submitted to trifluralin action. Due to the occurrence of irregular metaphases, they concluded that the mitotic spindle might have been broken owing to the herbicide action on it.

Bioassays performed with trifluralin, using *Pisum sativum* as test material revealed a positive action of the herbicide with the increase in chromosome alterations, C-mitosis and anti-mitosis effects [87].

Fernandes et al. [89] ascertained that, among the root meristems of *Allium cepa* under division, trifluralin promotes a significant increase in the irregular metaphase rate. These data corrob‐ orate the statement of Lignowski and Scott [85], Lee et al. [109], Dow et al. [110], Werbovetz et al. [111] and Ovidi et al. [82], who characterized trifluralin as a powerful microtubule inhibitor, which is therefore capable of accumulating a large amount of meristematic cells in metaphase.

Studies carried out by Peña [92] and Canevari [93] indicate that low trifluralin concentrations may induce mutagenic effects. These authors observed significant presence of micronuclei in erythrocytes of fish submitted to acute treatments with this herbicide. When the micronuclei diameters were measured by Canevari [93], data indicated that they could be derived from losses of whole chromosomes, thus proving the aneugenic effect of the herbicide due to the

*Allium cepa*meristematic cells treated with trifluralin also presented problems during mitosis, such as polyploidies, C-metaphases, multipolar anaphases, anaphase-telophase chromatin

**Figure 9.** Meristematic cells of *Allium cepa* treated with trifluralin. **A.** C-metaphase; **B.**polyploid cell; **C.** multipolar cell;

According to Fernandes et al. [88], in the bioassays with root meristems of *Allium cepa* treated with trifluralin, a large amount of interphase cells with more than one nucleus and cells with

Lignowski and Scott [85] observed C-metaphases, micronuclei, amoeboid nuclei and poly‐ ploidies in root meristems of wheat and onion submitted to trifluralin action. Due to the occurrence of irregular metaphases, they concluded that the mitotic spindle might have been

Bioassays performed with trifluralin, using *Pisum sativum* as test material revealed a positive action of the herbicide with the increase in chromosome alterations, C-mitosis and anti-mitosis

**D**. loss of genetic material; **E.**chromosome bridge; **F.**telophase with chromosome delay.

micronuclei and a mini cell were observed (Figure 10).

broken owing to the herbicide action on it.

effects [87].

bridges, chromosome delay and loss of genetic material [89]. (Figure 9).

pesticide interference in the mitotic spindle.

504 Herbicides - Current Research and Case Studies in Use

Genotoxicity tests using the comet assay in human lymphocyte cultures showed that trifluralin produced a significant increase in the length of the comet's tail. This increase is due to DNA breaks, since there is an induction of nucleotide excision repair, resulting from damage caused by the herbicide action [103]. As for the frequency of comet-bearing cells, the author observed that, after 48 hours of exposure to the herbicide, few tailed nucleoides were found. These results proved to be statistically significant, though.

According to Ribas et al. [35], trifluralin has a genotoxic effect on human cell cultures because it causes a decrease in cell proliferation. The same author ascertains that this herbicide has not revealed carcinogenic effects, since it caused little induction exchange between sister chroma‐ tids.The micronucleus test conducted by Ribas et al. [35], used for detecting aneugenic activity, has also produced a negative response, which contradicts studies carried out by several other authors [88], [89], [91], [92], [97], [112], among others) who ascertain that trifluralin brings about chromosome aberrations and nuclear alterations resulting from problems in the mitotic spindle

According to Kang et al. [113], trifluralin is not associated with bladder, kidney, liver, leukemia, colorectal or hematopoietic-lymphatic cancers. The authors only suggest a possible connection between trifluralin exposure and the risk of colon cancer in human beings, but the inconsis‐ tency per exposure level and a small number of colon cancers indicate that this could be an incidental finding.

Data from the National Cancer Institute (NCI) [114] report that mice subjected to trifluralin chronic exposure, at low concentrations, had an increase in hepatocellular carcinoma and higher incidence of alveolar bronchial adenomas. An increase in bladder cancer was also verified in mice exposed to low trifluralin concentrations. It was observed that, when male mice were submitted to high doses of trifluralin, they presented higher incidence of follicular cell and thyroid gland tumors [115]. Trifluralin has been reported to cause a significant increase

**Figure 10.** Meristematic cells of *Allium* cepa treated with trifluralin. **A.** cell with micronucleus; **B.** cell with micronu‐ cleus and an adjacent mini cell; **C.**polynucleated cell.

in thyroid follicular cell tumors in male Fischer 344 rats only at the highest dietary dose of 6500ppm in a 2-year chronic study [115].

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[13] Bellinaso M De. L., Henrique L.A., Gaylarde C.C., Greer C.W. Genes similar tonaph‐ thalenedioxygenase genes in trifluralin-degrading bacteria. Pest Manag. Sci., Sus‐

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### **6. Final considerations**

The increase in agricultural productivity has occurred thanks to several factors, among which are improvements in genetics, agricultural machinery and the use of substances that allow control of weeds in agriculture.

The use of pesticides has generated discussions and controversy among the scientific com‐ munity and its users, registering advantageous and disadvantageous recommendations in different ways. Among contrary recommendations to the use of pesticides, we can point out lack of detailed studies on the action of such chemicals on the exposed organisms, making it impossible to associate their action with the emergence of eventual problems. In the soil, trifluralin is moderately persistent, which might jeopardize organisms that are eventually exposed to it. Trifluralin is a substance that has a microtubule-depolymerizing activity, which prevents cell division, a fact that might compromise organism development.

Existing reports characterize trifluralin as a highly acute toxic substance to fish, but there are not enough descriptions of its chronic toxicity and cytotoxic effect. Studies mainly related to its genotoxic, mutagenic and carcinogenic potential are mostly inconclusive or even contra‐ dictory. There is little information about the toxicity of products derived from trifluralin degradation and its effects on the organisms.

### **Author details**

Thaís C. C. Fernandes, Marcos A. Pizano and Maria A. Marin-Morales\*

\*Address all correspondence to: mamm@rc.unesp.br

Universidade Estadual Paulista, IB-Campus de Rio Claro, Rio Claro/SP, Brasil

### **References**


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The increase in agricultural productivity has occurred thanks to several factors, among which are improvements in genetics, agricultural machinery and the use of substances that allow

The use of pesticides has generated discussions and controversy among the scientific com‐ munity and its users, registering advantageous and disadvantageous recommendations in different ways. Among contrary recommendations to the use of pesticides, we can point out lack of detailed studies on the action of such chemicals on the exposed organisms, making it impossible to associate their action with the emergence of eventual problems. In the soil, trifluralin is moderately persistent, which might jeopardize organisms that are eventually exposed to it. Trifluralin is a substance that has a microtubule-depolymerizing activity, which

Existing reports characterize trifluralin as a highly acute toxic substance to fish, but there are not enough descriptions of its chronic toxicity and cytotoxic effect. Studies mainly related to its genotoxic, mutagenic and carcinogenic potential are mostly inconclusive or even contra‐ dictory. There is little information about the toxicity of products derived from trifluralin

prevents cell division, a fact that might compromise organism development.

Thaís C. C. Fernandes, Marcos A. Pizano and Maria A. Marin-Morales\*

Universidade Estadual Paulista, IB-Campus de Rio Claro, Rio Claro/SP, Brasil

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\*Address all correspondence to: mamm@rc.unesp.br

**Author details**

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Res., Amsterdam v. 344, p. 41-54, 1995.

514 Herbicides - Current Research and Case Studies in Use

try Letters. v.12, p. 2395–2398, 2002.

47, p.133-281, 1998.

ologica, Gênova v. 73, p. 707-721, 1981.

53-65, 1999.

dicinal Chemistry. v. 18, p. 274–281. 2010.

gotes. Parasitology Research. v. 85, p. 475–480, 1999.

[115] Emmerson, J.L., Pierce, E.C., Mcgrath, J.P. The chronic toxicity of compound 36352 (trifluralin) given as a compound of the diet to the fischer 344 rats for two years. Studies r-87 and R-97 (unpublished study received September 18, 1980 under 1471-35; submitted by Elanco Products Co., Division of Eli Lilly and Co., Indianapo‐ lis, IN), 1980.

**Chapter 20**

**Allelochemicals as Bioherbicides — Present and**

Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploi‐ tation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active com‐ pounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes [1]. To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified [2]. Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (*Lolium rigidum* Gaud.), wild oat (*Avena fatua* L.) and redroot pigweed (*Amaranthus*

Evolution of weeds resistant to herbicides demands new solutions to cope with the problem since economic losses generated by weeds can be higher than those caused by other pests. Due to the fact that abandoning chemical weed control is, with current agricultural practices, rather impossible, it is necessary to create new classes of herbicides with new mechanisms of action and target sites not previously exploited. Presently used synthetic herbicides are not approved for use in organic agriculture. Moreover, using crop protection chemicals also need public acceptance. [3]. The number of synthetic chemicals with new target sites are decreasing

> © 2013 Soltys 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 Soltys 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.

**Perspectives**

Dorota Soltys, Urszula Krasuska,

http://dx.doi.org/10.5772/56185

**1. Introduction**

*retroflexus* L.).

Renata Bogatek and Agnieszka Gniazdowska

Additional information is available at the end of the chapter

## **Allelochemicals as Bioherbicides — Present and Perspectives**

Dorota Soltys, Urszula Krasuska, Renata Bogatek and Agnieszka Gniazdowska

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56185

### **1. Introduction**

Since the first implementation of synthetic herbicides in crop protection systems, weeds have continuously developed resistance. As a main reason of such evolution, long-lasting exploi‐ tation of herbicides with one target site in plants is considered. This has been the case with the first widely-used triazine herbicides, photosynthesis inhibitors, which have effectively eliminated a wide range of weeds. Unfortunately, inappropriate adjustment of herbicides to weed species occupying fields, application of herbicides at the incorrect developmental stage and in unsuitable weather conditions have contributed to the accumulation of active com‐ pounds in the soil, accumulation of weed species and acceleration evolution of resistant biotypes [1]. To date, there have been 211 species and 393 biotypes of herbicide resistant weeds identified [2]. Most of them are resistant to B, C1 and A groups of herbicides, inhibitors of: acetolactate synthase (ALS), photosystem II and acetyl CoA carboxylase, respectively. Ten species pose the biggest threat for crops due to causing yield losses, including the most important herbicide-resistant species which are characterized by multiple resistances: rigid ryegrass (*Lolium rigidum* Gaud.), wild oat (*Avena fatua* L.) and redroot pigweed (*Amaranthus retroflexus* L.).

Evolution of weeds resistant to herbicides demands new solutions to cope with the problem since economic losses generated by weeds can be higher than those caused by other pests. Due to the fact that abandoning chemical weed control is, with current agricultural practices, rather impossible, it is necessary to create new classes of herbicides with new mechanisms of action and target sites not previously exploited. Presently used synthetic herbicides are not approved for use in organic agriculture. Moreover, using crop protection chemicals also need public acceptance. [3]. The number of synthetic chemicals with new target sites are decreasing

© 2013 Soltys 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 Soltys 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.

dramatically. Eco-friendly trends in weed management force scientists to reach for innovative sources and tools. Natural compounds pose a great field for the discovery of new environ‐ mentally safe herbicides, so called "bioherbicides", which are based on compounds produced by living organisms. According to the CAS (Chemical Abstracts Service) registry, among the 24 million organic compounds, a large group of secondary plant metabolites is represented. Some of these compounds take part in allelopathic interactions.

**9.** tannins;

**10.** steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).

juglone, benzoxazolin-2-one (BOA), 2-amino-3-H-phenoxazin-3-one (APO).

these compounds on target plants are also classified as "phytotoxic".

can significantly decrease bioactivity of the whole compound.

**3. Advantages and disadvantages of allelopathins as bioherbicides**

Mode of action of some allelochemicals is similar to synthetic herbicides. These features have allowed them to be considered for possible use in weed management as bioherbicides. However, the field of knowledge is poorly studied but it is a very attractive area to explore. Allelochemicals are highly attractive as new classes of herbicides due to a variety of advan‐ tages. However, in the perspective of bioherbicides based on allelopathins, effects caused by

Most of allelopathins are totally or partially water-soluble which makes them easier to apply without additional surfactants [3, 10]. Their chemical structure is more environmentally friendly than synthetic ones. They possess higher oxygen- and nitrogen-rich molecules with relatively few so called 'heavy atoms', a halogen substitute, and are characterized by the absence of 'unnatural' rings. These properties decrease a chemical's environmental half-life, prevent accumulation of the compound in soil and eventual influence on non-target organ‐ isms. On the other hand, these properties are an allelochemical's Achille's heel due to less than satisfactory duration of activity. Structure complexity generates more stereocenters making them more reactive and unstable. Therefore, rapid degradation of one of the chemical groups

The diversity of allelopathins makes them promising tools possessing specific properties in discovering novel, specific target sites in acceptor plants. Even if they inhibit photosynthesis

Allelochemicals are released into the environment by plant organs such as roots, rhizomes, leaves, stems, bark, flowers, fruits and seeds (Figure 1a). The huge number of allelopathic interactions is typically negative in character, with positive relations being rare. Allelopathic compounds affect germination and growth of neighboring plants by disruption of various physiological processes including photosynthesis, respiration, water and hormonal balance. The underlying cause of their action is mainly inhibition of enzyme activity. Ability of an allelochemical to inhibit or delay plant growth and/or seed germination is usually defined as its "allelopathic (or phytotoxic) potential". An excellent example of allelopathic interaction is seen in soil exhaustion due to the accumulation of allelopathins that can be prevented by using fertilizers and rotating crops. Plants producing allelopathins are considered as "donor" organisms while the plants which allelopathins are directed to are referred to as "target" plants or "acceptors". The after-effects and strength of allelopathic interactions are diverse due to modifications of the allelopathins taking place in soil (Fig 1b). Most of the allelochemicals penetrate the soil as already plant-active compounds, e.g. phenolic acids, cyanamide, momi‐ lactones, heliannuols etc. Some have to be modified into the active form by microorganisms or by specific environmental conditions (pH, moisture, temperature, light, oxygen etc.), e.g.

Allelochemicals as Bioherbicides — Present and Perspectives

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519

### **2. Allelopathic interactions and allelopathic compounds**

Allelopathy is considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems [4]. It is defined as the interaction between plants and micro‐ organisms by a variety of compounds usually referred to as allelopathins, allelochemicals, or allelopathic compounds. This review is focused mainly on compounds taking part in complex allelopathic interactions between higher plants. However, determination of quality, quantity, direct or indirect effects of allelopathins on plant or microorganism communities in the natural environment is very difficult owing to the multi-dimensional character of those interactions. The development of analytical techniques allowing better specification of direct effects of allelopathins, have moved the exploration (or the research on) of this phenomenon from fields into laboratories. The term "allelopathy" refers rather to interactions occurring in the natural environment [5]. For studies with plant extracts, allelopathins isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones, it was established the term ''phytotoxicity'' to distinguish allelopathy (as a phenomenon occurring in natural environment) from studies conducted in laboratory.

Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites [6,7]. These compounds belong to numerous chemical groups including: trike‐ tones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, tannins lignin, fatty acids and nonprotein aminoacids. A wide range of these biochem‐ icals are synthesized during the shikimate pathway [8] or, in the case of essential oils, from the soprenoid pathway. Allelochemicals can be classified into 10 categories [9] according to their different structures and properties:


#### **9.** tannins;

dramatically. Eco-friendly trends in weed management force scientists to reach for innovative sources and tools. Natural compounds pose a great field for the discovery of new environ‐ mentally safe herbicides, so called "bioherbicides", which are based on compounds produced by living organisms. According to the CAS (Chemical Abstracts Service) registry, among the 24 million organic compounds, a large group of secondary plant metabolites is represented.

Allelopathy is considered a multi-dimensional phenomenon occurring constantly in natural and anthropogenic ecosystems [4]. It is defined as the interaction between plants and micro‐ organisms by a variety of compounds usually referred to as allelopathins, allelochemicals, or allelopathic compounds. This review is focused mainly on compounds taking part in complex allelopathic interactions between higher plants. However, determination of quality, quantity, direct or indirect effects of allelopathins on plant or microorganism communities in the natural environment is very difficult owing to the multi-dimensional character of those interactions. The development of analytical techniques allowing better specification of direct effects of allelopathins, have moved the exploration (or the research on) of this phenomenon from fields into laboratories. The term "allelopathy" refers rather to interactions occurring in the natural environment [5]. For studies with plant extracts, allelopathins isolated from plant tissue, collected from exudates or even synthetic compounds identical to natural ones, it was established the term ''phytotoxicity'' to distinguish allelopathy (as a phenomenon occurring

Allelopathins are products of the secondary metabolism and are non-nutritional primary metabolites [6,7]. These compounds belong to numerous chemical groups including: trike‐ tones, terpenes, benzoquinones, coumarins, flavonoids, terpenoids, strigolactones, phenolic acids, tannins lignin, fatty acids and nonprotein aminoacids. A wide range of these biochem‐ icals are synthesized during the shikimate pathway [8] or, in the case of essential oils, from the soprenoid pathway. Allelochemicals can be classified into 10 categories [9] according to their

**1.** water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, and ketones;

Some of these compounds take part in allelopathic interactions.

518 Herbicides - Current Research and Case Studies in Use

in natural environment) from studies conducted in laboratory.

**4.** quinines (benzoquinone, anthraquinone and complex quinines);

different structures and properties:

**6.** cinnamic acid and its derivatives;

**3.** long-chain fatty acids and polyacetylenes;

**2.** simple lactones;

**5.** phenolics;

**7.** coumarins; **8.** flavonoids;

**2. Allelopathic interactions and allelopathic compounds**

**10.** steroids and terpenoids (sesquiterpene lactones, diterpenes, and triterpenoids).

Allelochemicals are released into the environment by plant organs such as roots, rhizomes, leaves, stems, bark, flowers, fruits and seeds (Figure 1a). The huge number of allelopathic interactions is typically negative in character, with positive relations being rare. Allelopathic compounds affect germination and growth of neighboring plants by disruption of various physiological processes including photosynthesis, respiration, water and hormonal balance. The underlying cause of their action is mainly inhibition of enzyme activity. Ability of an allelochemical to inhibit or delay plant growth and/or seed germination is usually defined as its "allelopathic (or phytotoxic) potential". An excellent example of allelopathic interaction is seen in soil exhaustion due to the accumulation of allelopathins that can be prevented by using fertilizers and rotating crops. Plants producing allelopathins are considered as "donor" organisms while the plants which allelopathins are directed to are referred to as "target" plants or "acceptors". The after-effects and strength of allelopathic interactions are diverse due to modifications of the allelopathins taking place in soil (Fig 1b). Most of the allelochemicals penetrate the soil as already plant-active compounds, e.g. phenolic acids, cyanamide, momi‐ lactones, heliannuols etc. Some have to be modified into the active form by microorganisms or by specific environmental conditions (pH, moisture, temperature, light, oxygen etc.), e.g. juglone, benzoxazolin-2-one (BOA), 2-amino-3-H-phenoxazin-3-one (APO).

### **3. Advantages and disadvantages of allelopathins as bioherbicides**

Mode of action of some allelochemicals is similar to synthetic herbicides. These features have allowed them to be considered for possible use in weed management as bioherbicides. However, the field of knowledge is poorly studied but it is a very attractive area to explore.

Allelochemicals are highly attractive as new classes of herbicides due to a variety of advan‐ tages. However, in the perspective of bioherbicides based on allelopathins, effects caused by these compounds on target plants are also classified as "phytotoxic".

Most of allelopathins are totally or partially water-soluble which makes them easier to apply without additional surfactants [3, 10]. Their chemical structure is more environmentally friendly than synthetic ones. They possess higher oxygen- and nitrogen-rich molecules with relatively few so called 'heavy atoms', a halogen substitute, and are characterized by the absence of 'unnatural' rings. These properties decrease a chemical's environmental half-life, prevent accumulation of the compound in soil and eventual influence on non-target organ‐ isms. On the other hand, these properties are an allelochemical's Achille's heel due to less than satisfactory duration of activity. Structure complexity generates more stereocenters making them more reactive and unstable. Therefore, rapid degradation of one of the chemical groups can significantly decrease bioactivity of the whole compound.

The diversity of allelopathins makes them promising tools possessing specific properties in discovering novel, specific target sites in acceptor plants. Even if they inhibit photosynthesis

on microbial ecology and non-target plants, possible toxic properties on human health and

Allelochemicals as Bioherbicides — Present and Perspectives

http://dx.doi.org/10.5772/56185

521

A high number of limitations does not exclude allelochemicals as possible herbicides. In particular, they can be alternatives in weed management strategy. Widely developed bioin‐ formatics and cheminformatics support development of new herbicides [3, 15, 16]. Identified chemical structure of a particular allelochemical is a starting point to design a product with the compound-like properties using computer programs. Thanks to cheminformatics we are able to predict the potential structure of analogues and make several modifications, which make it more or less active, with higher environmental stability, as it was done for leptosper‐ mone. We may also predict the target site of compound action in plants due to comparison studies. Similar structure of a compound to a commercialized herbicide or other natural compound whose mode of action is well-known may allow us to predict the target site.

Plant protection is effective but rather costly and problematic due to environmental pollution. Exploration of the allelopathic potential of some species allows the introduction of alternative techniques for weed management, e.g. extracts from allelopathic plants can be applied as foliar sprays. Apart from decreasing the costs of herbicide application, this method also improves

The best known examples of natural bioherbicides are phytotoxic water extracts from herbage of sorghum (*Sorghum bicolor* (L.) Moench.) (sorgaab) and sunflower (*Helianthus annuus* L.)

Effects of sorgaab on weeds is time- and dose-depend but is typically used at 5% or 10% (w/v) concentration as double spray 20/30 and 40/60 days after sowing (DAS) or after seedling transplantation (AT) [17-19]. The best results to account for net profits have been elicited with a double spray of 10% extract in cotton (*Gossypim hirsutum* L.), soybean (*Glycine max* L.), wheat (*Triticum aestivum* L.) or rice (*Oryza sativa* L.). The highest efficacy of such extract applications has been verified in rice on reduction of barnyard grass (*Echinochloa cruss-galli* L.) biomass by 40%, without significant changes in weed density and accompanied yield increase by 18%.

Sunfaag has been widely used in wheat. The extract has been usually applied three times at 7 day intervals starting between 3-4 weeks post-emergence. This system of application has reduced biomass of the two most commonly occurring weeds, lambsquarters (*Chenopodium album* L.) and toothed dock (*Rumex dentatus* L.), by 70% and 97% respectively, although it has not eliminated all weed species in field. It has improved wheat biomass by 7-8% in comparison to weed free control without significant changes in number of tillers and total seed biomass. The herbicidal efficiency calculated as the effectiveness of sunfaag in comparison to synthetic herbicides showed a quite high value, 60% efficiency index. Weed management systems require high concentrations of sunfaag ranging up to 80% and can generate economic losses due to the necessity of cultivating higher amounts of sorghum or sunflower that also required

(sunfaag) which can be effectively used in plant protection without yield losses.

profitability of production on a commercial scale [16].

**4. Allelopathic plant extracts as bioherbicides**

crop production.

**Figure 1.** Multi-dimensional nature of allelopathic interacions. (1a) Plant A releases allelochemicals X and F which di‐ rectly affect growth of plant B. (1b) left side; Plant A releases allelochemical X which is modified or activated by micro‐ organisms to allelochemical Y that affects growth of plant B. (1b) right side; Plant A releases allelochemical X which stimulates microorganisms to produce allelochemical Z that affects growth of plant B.

or respiration, they may also bind to proteins at different sites than synthetic herbicides [11, 12]. This provides the opportunity to eliminate weeds that are already resistant to commer‐ cialized herbicides with the same mode of action. Allelochemicals are also characterized by multi-site action in plants without high specificity which is achieved in the case of synthetic herbicides. Therefore, this feature excludes the application of an allelopathic compound as a selective herbicide or totally prohibits its usage in weed management. On the other hand, effects of allelopathins in acceptor plants are highly dose-dependent [13]. This allows the opportunity to search out compounds exhibiting selectivity. Generally, monocotyledonous plants are more resistant to allelochemicals than dicotyledonous ones. Therefore, usage of a compound as a potential herbicide is possible but rather restricted to cultivation of exact crops with a defined weed composition.

The route of discovery is much more complicated with allelopathins. In contrast to synthetic herbicides where synthesis, bioassay, evaluation and quantitative structure-active relationship follow Quantitative Structure-Activity Relationship (QSAR), allelochemicals have to be first isolated from plant extracts [14]. The amount of recovered compounds is usually low in comparison to chemical synthesis. After extraction, purification and selection of the most attractive compound and determination of its mode of action in plants is done. At the end of the process, similar to synthetic herbicides, allelpathins are subjected to QSAR. The long discovery process is usually offset by a shorter, less expensive track of registration [15]. It is worth noting that before an allelochemical can become an herbicide, the following conditions have to be performed: phytotoxic activity at the range between 10-5 and 10-7 M, identified chemical structure, known mode of action in plants, time of residence in soil, possible influence on microbial ecology and non-target plants, possible toxic properties on human health and profitability of production on a commercial scale [16].

A high number of limitations does not exclude allelochemicals as possible herbicides. In particular, they can be alternatives in weed management strategy. Widely developed bioin‐ formatics and cheminformatics support development of new herbicides [3, 15, 16]. Identified chemical structure of a particular allelochemical is a starting point to design a product with the compound-like properties using computer programs. Thanks to cheminformatics we are able to predict the potential structure of analogues and make several modifications, which make it more or less active, with higher environmental stability, as it was done for leptosper‐ mone. We may also predict the target site of compound action in plants due to comparison studies. Similar structure of a compound to a commercialized herbicide or other natural compound whose mode of action is well-known may allow us to predict the target site.

### **4. Allelopathic plant extracts as bioherbicides**

or respiration, they may also bind to proteins at different sites than synthetic herbicides [11, 12]. This provides the opportunity to eliminate weeds that are already resistant to commer‐ cialized herbicides with the same mode of action. Allelochemicals are also characterized by multi-site action in plants without high specificity which is achieved in the case of synthetic herbicides. Therefore, this feature excludes the application of an allelopathic compound as a selective herbicide or totally prohibits its usage in weed management. On the other hand, effects of allelopathins in acceptor plants are highly dose-dependent [13]. This allows the opportunity to search out compounds exhibiting selectivity. Generally, monocotyledonous plants are more resistant to allelochemicals than dicotyledonous ones. Therefore, usage of a compound as a potential herbicide is possible but rather restricted to cultivation of exact crops

stimulates microorganisms to produce allelochemical Z that affects growth of plant B.

**Figure 1.** Multi-dimensional nature of allelopathic interacions. (1a) Plant A releases allelochemicals X and F which di‐ rectly affect growth of plant B. (1b) left side; Plant A releases allelochemical X which is modified or activated by micro‐ organisms to allelochemical Y that affects growth of plant B. (1b) right side; Plant A releases allelochemical X which

The route of discovery is much more complicated with allelopathins. In contrast to synthetic herbicides where synthesis, bioassay, evaluation and quantitative structure-active relationship follow Quantitative Structure-Activity Relationship (QSAR), allelochemicals have to be first isolated from plant extracts [14]. The amount of recovered compounds is usually low in comparison to chemical synthesis. After extraction, purification and selection of the most attractive compound and determination of its mode of action in plants is done. At the end of the process, similar to synthetic herbicides, allelpathins are subjected to QSAR. The long discovery process is usually offset by a shorter, less expensive track of registration [15]. It is worth noting that before an allelochemical can become an herbicide, the following conditions have to be performed: phytotoxic activity at the range between 10-5 and 10-7 M, identified chemical structure, known mode of action in plants, time of residence in soil, possible influence

with a defined weed composition.

520 Herbicides - Current Research and Case Studies in Use

Plant protection is effective but rather costly and problematic due to environmental pollution. Exploration of the allelopathic potential of some species allows the introduction of alternative techniques for weed management, e.g. extracts from allelopathic plants can be applied as foliar sprays. Apart from decreasing the costs of herbicide application, this method also improves crop production.

The best known examples of natural bioherbicides are phytotoxic water extracts from herbage of sorghum (*Sorghum bicolor* (L.) Moench.) (sorgaab) and sunflower (*Helianthus annuus* L.) (sunfaag) which can be effectively used in plant protection without yield losses.

Effects of sorgaab on weeds is time- and dose-depend but is typically used at 5% or 10% (w/v) concentration as double spray 20/30 and 40/60 days after sowing (DAS) or after seedling transplantation (AT) [17-19]. The best results to account for net profits have been elicited with a double spray of 10% extract in cotton (*Gossypim hirsutum* L.), soybean (*Glycine max* L.), wheat (*Triticum aestivum* L.) or rice (*Oryza sativa* L.). The highest efficacy of such extract applications has been verified in rice on reduction of barnyard grass (*Echinochloa cruss-galli* L.) biomass by 40%, without significant changes in weed density and accompanied yield increase by 18%.

Sunfaag has been widely used in wheat. The extract has been usually applied three times at 7 day intervals starting between 3-4 weeks post-emergence. This system of application has reduced biomass of the two most commonly occurring weeds, lambsquarters (*Chenopodium album* L.) and toothed dock (*Rumex dentatus* L.), by 70% and 97% respectively, although it has not eliminated all weed species in field. It has improved wheat biomass by 7-8% in comparison to weed free control without significant changes in number of tillers and total seed biomass. The herbicidal efficiency calculated as the effectiveness of sunfaag in comparison to synthetic herbicides showed a quite high value, 60% efficiency index. Weed management systems require high concentrations of sunfaag ranging up to 80% and can generate economic losses due to the necessity of cultivating higher amounts of sorghum or sunflower that also required an appropriate cultivation system [20, 21]. Therefore, sunfaag can be applied as a preemergence herbicide with much lower doses. The most promising application system has considered usage of 10% (w/v) extract at pre-emergence + 25 DAS + 35 DAS. Following the application, there has been noted a remarkably reduced population of wild oat, lesser swi‐ necress (*Coronopus didymus* L.) and littleseed canarygrass *(Phalaris minor* Retz.) without affecting germination of wheat and increased wheat yield in 7% [22]. However, the inhibitory effect on weed growth and crop yield is selective and highly dependent on duration or term of sorgaab and sunfaag application.

tition. Sunfaag has been applied when wheat seedlings were 3-4 weeks old while lambsquar‐

Allelochemicals as Bioherbicides — Present and Perspectives

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High allelopathic potential conditioned by glucosinolates and isothiocyanates is present in Brassica sp. [27, 28]. Isothiocyanates have been strong suppressants of germination of spiny sowthistle (*Sonchus asper* L. Hill), scentless mayweed (*Matricaria inodora* L.), smooth pigweed (*Amaranthus hybridus* L.), barnyardgrass, blackgrass (*Alopecurus myosuroides* Huds.) and wheat [28]. Black mustard (*Brassica nigra* L.) extract of different plant parts like leaf, stem, flower and root have inhibited germination and radicle length of wild oat. Inhibitory effects on germina‐ tion increased with increasing concentration of extract solution of the fresh plant parts [29]. Some experiments were conducted also using garden radish (*Raphanus sativus* L.) extract on germination of 25 weed and 32 crop species [30]. Garden radish extracts totally inhibited germination of 11 weeds such as Johnsongrass (*Sorghum halelense* L. Pers.), *Alhagi* spp., blackgrass (*Alopecurus myosuroides* Huds.), shepherd's-purse (*Capsella bursa-pastoris* L. Medik.), field bindweed (*Convolvulus arvensis* L.), dodder (*Cuscuta* sp.), carrot (*Daucus carota* L.), shortpod mustard (*Hirschfeldia incana* L.), *Ochtodium aegyptiacum* (L.), and shortfruit hedgemustard (*Sisymbrium polyceratium* L.), and 4 crop species namely lettuce (*Lactuca sativa* L.), tobacco (*Nicotiana tabacum* L.), bean (*Phaseolus vulgaris* L.), and clover (*Trifolium* sp.). Garden radish extracts at different rates (100, 66, 50 and 33% of pure extract) did not affect germination of wheat, cotton, and maize (*Zea mays* L.), but affected soybean germination at the 100% extract rate in vitro. Rhizome regeneration of Johnsongrass was inhibited by 54-99% depending on extract concentration. Regeneration of bermudagrass (*Cynodon dactylon* L. Pers.) rhizomes was inhibited to a lower extent at all concentrations; for instance, 54% inhibition occurred at the highest extract concentration. Lower extract rates stimulated redroot pigweed germination, while 66 and 100% extracts inhibited germination by 21 and 42%, respectively. Inhibition reached only 56 and 49% at the highest extract concentration for common purslane (*Portulaca oleracea* L.) and cocklebur (*Xanthium strumarium* L.), respectively. Garden radish residues which were cut into pieces and incorporated into the growing medium decreased weed

Legumes crops may also be applied as a source of allelochemicals useful in weed suppression. Mulch of dead pea plants could be used to control growth of weeds. Pea cover crop has regulated germination and growth of lady's thumb (*Polygonum persicaria* L.), smooth pigweed, smallflower galinsoga, and common lambsquarters. Similarly, the aqueous leachates (1%) of all four legumes, velvetbean (*Mucuna deeringiana* (Bort.) Merr.), jackbean (*Canavalia ensifor‐ mis* (L.) DC.), jumbiebean (*Leucaena leucocephala* (Lam.) de Wit), and wild tamarind (*Lysiloma latisiliquum* (L.) Benth.), have been shown to suppress weeds [32]. These plants exhibited strong phytotoxic effects on the radicle growth of barnyardgrass, alegría (*Amaranthus* ssp.) and amaranth (*Amaranthus hypochondriacus* L.) [33]. Russian knapweed (*Acroptilon repens*) control is difficult in many crops. Allelopathic effects of extracts and plant parts of alfalfa (*Medicago sativa* L.) on Russian knapweed were reported both in Petri dishes and pot experiments [34]. Alfalfa has been recommended in fields with high mugwort (*Artemisia vulgaris* L.) infestation, as it decreased mugwort to 89% under field conditions, while extracts of alfalfa vegetative

parts inhibited mugwort germination up to 83% in Petri dish assays.

ters and toothed dock 1-week old at the stage of three to four leaf [20, 21].

intensity and increased maize yield [31].

Aqueous extracts of sorghum and sunflower are effective on weed growth but unfortunately might not be profitable enough in crop production; however, crop allelopathy can be manip‐ ulated for achieving sustainable weed management. Combination of phytotoxic crop water extracts with lower rates of herbicides may provide reduced weed control levels with reduced herbicide usage. The interesting review of allelopathic crop plants in weed management strategy is presented in reference [23]. Two field studies were conducted utilizing water extracts of sorghum, sunflower and rapeseed (*Brassica napus* L.) with reduced glyphosate dosage for controlling purple nutsedge (*Cyperus rotundus* L) in cotton [24]. Sorghum and rapeseed water extracts were tank mixed (at 15 or 18 L ha-1) in different combinations with reduced rates of glyphosate by 767 and 575 g active substance (a.s.) ha-1 and sprayed as directed post emergence at 21 DAS. Purple nutsedge density and dry weight were suppressed by 78% to 95% and 83% to 95%, respectively, when different crop water extracts were used in combi‐ nation with a reduced rate of glyphosate. Seed cotton yield was improved from 15-21% in sorgaab and rape water extract combinations with reduced rates of glyphosate (67-75%). Similar research has been conducted on water extracts of sorghum with sunflower in combi‐ nation with herbicides in wheat, soybean, rice, and canola (*Brassica* sp.) [25, 26]. Both extracts, in combination with herbicides, have the same or even better effect on inhibition of growth of the following weeds: littleseed canarygrass and lesser swinecress, compared to single synthetic herbicide applications [25, 26]. Spraying of wheat seedlings 30 DAS with sorgaab+sunfaag (18 L each ha-1) with mesosulfuron+idosulfuron (4.32 g a.s. ha-1) has the same effect on total weed density (reduction up to 90% in relation to control) as application of mesosulfuron+idosulfuron used alone, but with higher doses (120 g a.s. ha-1). Herbicidal solution has also improved yield parameters, both in relation to control and in relation to single herbicide application: fertile tillers (10%), spikelets per spike (11%) and grains per spike (10%) [26]. In cotton, application of both extracts at 18 L ha-1 each with glyphosate (767 g a.s. ha-1) 21 DAS has been the most effective in density reduction of the highly competitive weed purple nutsedge up to 93% [24]. However, the greatest benefit in wheat is the usage of a sorgaab/sunfaag combination which lowered by 70% doses of metribuzin and phenaxaprop (at 57 g a.s. ha-1), applied at 18 L each ha-1. In turn, in cotton, application of the same rates of extracts per ha with glyphosate (767 g a.s. ha-1) seems to be the most economically reasonable costs of following weed management method [24, 25].

Selectivity of plant extracts on weeds without any negative implications on crop productivity is probably due to differences in the physiological stage of plants and following plant compe‐ tition. Sunfaag has been applied when wheat seedlings were 3-4 weeks old while lambsquar‐ ters and toothed dock 1-week old at the stage of three to four leaf [20, 21].

an appropriate cultivation system [20, 21]. Therefore, sunfaag can be applied as a preemergence herbicide with much lower doses. The most promising application system has considered usage of 10% (w/v) extract at pre-emergence + 25 DAS + 35 DAS. Following the application, there has been noted a remarkably reduced population of wild oat, lesser swi‐ necress (*Coronopus didymus* L.) and littleseed canarygrass *(Phalaris minor* Retz.) without affecting germination of wheat and increased wheat yield in 7% [22]. However, the inhibitory effect on weed growth and crop yield is selective and highly dependent on duration or term

Aqueous extracts of sorghum and sunflower are effective on weed growth but unfortunately might not be profitable enough in crop production; however, crop allelopathy can be manip‐ ulated for achieving sustainable weed management. Combination of phytotoxic crop water extracts with lower rates of herbicides may provide reduced weed control levels with reduced herbicide usage. The interesting review of allelopathic crop plants in weed management strategy is presented in reference [23]. Two field studies were conducted utilizing water extracts of sorghum, sunflower and rapeseed (*Brassica napus* L.) with reduced glyphosate dosage for controlling purple nutsedge (*Cyperus rotundus* L) in cotton [24]. Sorghum and rapeseed water extracts were tank mixed (at 15 or 18 L ha-1) in different combinations with reduced rates of glyphosate by 767 and 575 g active substance (a.s.) ha-1 and sprayed as directed post emergence at 21 DAS. Purple nutsedge density and dry weight were suppressed by 78% to 95% and 83% to 95%, respectively, when different crop water extracts were used in combi‐ nation with a reduced rate of glyphosate. Seed cotton yield was improved from 15-21% in sorgaab and rape water extract combinations with reduced rates of glyphosate (67-75%). Similar research has been conducted on water extracts of sorghum with sunflower in combi‐ nation with herbicides in wheat, soybean, rice, and canola (*Brassica* sp.) [25, 26]. Both extracts, in combination with herbicides, have the same or even better effect on inhibition of growth of the following weeds: littleseed canarygrass and lesser swinecress, compared to single synthetic herbicide applications [25, 26]. Spraying of wheat seedlings 30 DAS with sorgaab+sunfaag (18 L each ha-1) with mesosulfuron+idosulfuron (4.32 g a.s. ha-1) has the same effect on total weed density (reduction up to 90% in relation to control) as application of mesosulfuron+idosulfuron used alone, but with higher doses (120 g a.s. ha-1). Herbicidal solution has also improved yield parameters, both in relation to control and in relation to single herbicide application: fertile tillers (10%), spikelets per spike (11%) and grains per spike (10%) [26]. In cotton, application of both extracts at 18 L ha-1 each with glyphosate (767 g a.s. ha-1) 21 DAS has been the most effective in density reduction of the highly competitive weed purple nutsedge up to 93% [24]. However, the greatest benefit in wheat is the usage of a sorgaab/sunfaag combination which lowered by 70% doses of metribuzin and phenaxaprop (at 57 g a.s. ha-1), applied at 18 L each ha-1. In turn, in cotton, application of the same rates of extracts per ha with glyphosate (767 g a.s. ha-1) seems to be the most economically reasonable costs of following weed management

Selectivity of plant extracts on weeds without any negative implications on crop productivity is probably due to differences in the physiological stage of plants and following plant compe‐

of sorgaab and sunfaag application.

522 Herbicides - Current Research and Case Studies in Use

method [24, 25].

High allelopathic potential conditioned by glucosinolates and isothiocyanates is present in Brassica sp. [27, 28]. Isothiocyanates have been strong suppressants of germination of spiny sowthistle (*Sonchus asper* L. Hill), scentless mayweed (*Matricaria inodora* L.), smooth pigweed (*Amaranthus hybridus* L.), barnyardgrass, blackgrass (*Alopecurus myosuroides* Huds.) and wheat [28]. Black mustard (*Brassica nigra* L.) extract of different plant parts like leaf, stem, flower and root have inhibited germination and radicle length of wild oat. Inhibitory effects on germina‐ tion increased with increasing concentration of extract solution of the fresh plant parts [29]. Some experiments were conducted also using garden radish (*Raphanus sativus* L.) extract on germination of 25 weed and 32 crop species [30]. Garden radish extracts totally inhibited germination of 11 weeds such as Johnsongrass (*Sorghum halelense* L. Pers.), *Alhagi* spp., blackgrass (*Alopecurus myosuroides* Huds.), shepherd's-purse (*Capsella bursa-pastoris* L. Medik.), field bindweed (*Convolvulus arvensis* L.), dodder (*Cuscuta* sp.), carrot (*Daucus carota* L.), shortpod mustard (*Hirschfeldia incana* L.), *Ochtodium aegyptiacum* (L.), and shortfruit hedgemustard (*Sisymbrium polyceratium* L.), and 4 crop species namely lettuce (*Lactuca sativa* L.), tobacco (*Nicotiana tabacum* L.), bean (*Phaseolus vulgaris* L.), and clover (*Trifolium* sp.). Garden radish extracts at different rates (100, 66, 50 and 33% of pure extract) did not affect germination of wheat, cotton, and maize (*Zea mays* L.), but affected soybean germination at the 100% extract rate in vitro. Rhizome regeneration of Johnsongrass was inhibited by 54-99% depending on extract concentration. Regeneration of bermudagrass (*Cynodon dactylon* L. Pers.) rhizomes was inhibited to a lower extent at all concentrations; for instance, 54% inhibition occurred at the highest extract concentration. Lower extract rates stimulated redroot pigweed germination, while 66 and 100% extracts inhibited germination by 21 and 42%, respectively. Inhibition reached only 56 and 49% at the highest extract concentration for common purslane (*Portulaca oleracea* L.) and cocklebur (*Xanthium strumarium* L.), respectively. Garden radish residues which were cut into pieces and incorporated into the growing medium decreased weed intensity and increased maize yield [31].

Legumes crops may also be applied as a source of allelochemicals useful in weed suppression. Mulch of dead pea plants could be used to control growth of weeds. Pea cover crop has regulated germination and growth of lady's thumb (*Polygonum persicaria* L.), smooth pigweed, smallflower galinsoga, and common lambsquarters. Similarly, the aqueous leachates (1%) of all four legumes, velvetbean (*Mucuna deeringiana* (Bort.) Merr.), jackbean (*Canavalia ensifor‐ mis* (L.) DC.), jumbiebean (*Leucaena leucocephala* (Lam.) de Wit), and wild tamarind (*Lysiloma latisiliquum* (L.) Benth.), have been shown to suppress weeds [32]. These plants exhibited strong phytotoxic effects on the radicle growth of barnyardgrass, alegría (*Amaranthus* ssp.) and amaranth (*Amaranthus hypochondriacus* L.) [33]. Russian knapweed (*Acroptilon repens*) control is difficult in many crops. Allelopathic effects of extracts and plant parts of alfalfa (*Medicago sativa* L.) on Russian knapweed were reported both in Petri dishes and pot experiments [34]. Alfalfa has been recommended in fields with high mugwort (*Artemisia vulgaris* L.) infestation, as it decreased mugwort to 89% under field conditions, while extracts of alfalfa vegetative parts inhibited mugwort germination up to 83% in Petri dish assays.

Application of plant extracts as pre-emergence or as early post emergence herbicides resulted in reduction of doses of synthetic herbicide due to their synergistic or additive action. How‐ ever, not all phytotoxic extracts are effective enough to inhibit weed growth or germination when applied as spray even when plants show high allelopathic potential as mulch, inter‐ cropping system or in rotation. This may be the result of masking the activity of one compound by another in water solution or other factors such as impossibility of extract penetration through the cuticle [12]. A new opportunity to enhance effectiveness of usage of bioherbicides based on natural extracts is associated with extraction of individual allelochemicals and/or its comparison with synthetic herbicides. The extraction of sesquiterpene lactone, dehydrozalu‐ zanin C (DHZ) produced among Compositae family serves as an example [34]. Comparison studies of isolated DHZ (1 mM) and the commercial herbicide Logran® showed high inhibitory activity of DHZ on dicotyledonous plants while the synthetic herbicide showed no activity [34]. Also pure 2-benzoxazolinone (BOA) isolated from several graminaceous crops such as rye (*Secale cereale* L.), maize and wheat was active similarly as herbicide but its stability in the environment was much shorter than the synthetic herbicide [35].

**Compounds Botanical source** S**ensitive weeds**

garden radish (*Raphanus sativus*)

spiny sowthistle (*Sonchus asper* L. Hill), scentless mayweed (*Matricaria inodora* L.), smooth pigweed (*Amaranthus hybridus* L.), barnyardgrass (*Echinochloa cruss-galli* L. Beauv.), slender meadow foxtail or blackgrass (*Alopecurus myosuroides* Huds.), *Alhagi* spp., *Cachia maritime*, Shepherd's-purse(*Capsella bursapastoris* L.), morning glory (*Convolvulus arvensis* L.), dodders (*Cuscuta* spp.), wild carrot or bird's nest (*Daucus carota* L.), shortpod mustard, buchanweed or hoary mustard(*Hirschfeldia incana* L.), *Ochtodium aegyptiacum* (L.), shortfruit hedgemustard (*Sisymbrium*

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525

littleseed canarygrass (*Phalaris minor* Retz.), lesser swinecress (*Coronopus didymus* L.), purple nutsedge (*Cyperus rotundus* L.), black nightshade (*Solanum nigrum* L.), redroot pigweed (*Amaranthus retroflexus* L.), common ragweed (*Ambrosia atrtemisiflora* L.), sicklepod

barnyardgrass, (*Echinochloa colonum* L.), livid amaranth(*Amaranthus lividus* L.), hairy crabgrass (*Digitaria sanguinalis* L.), annual meadow grass, annual

redroot pigweed, pitted morning-glory (*Ipomoea lacunose* L.), common purslane (*Portulaca oleracea* L.), annual wormwood, duckweed (*Lemna minor* L.), algae

barnyard grass, hairy crabgrass, yellow foxtail (*Setaria glauca* L.), california red oat (*Avena sativa* L.), Indian mustard (*Brassica juncea* L.), curly dock (*Rumex crispus*

Sprangletop (*Leptochloa filiformis* Lam.), dandelion (*Taraxacum* sp.), lambsquarter or wild spinach (*Chenopodium album* L.), annual bluegrass or poa, morning glory or bindweed, wild mustard, curly dock

*polyceratium* L.)

(*Cassia obtusifolia* L.)

bluegrass or poa (*Poa annua* L.)

(*Pseudokirchneriella subcapitata*)

L.)

(*Lolium rigidum*)

Essential oils eucalyptus *(Eucalyptus* sp.) barnyard grass, *Cassia occidentalis*, annual ryegrass

**Table 1.** Allelopathic compounds isolated from plants that exhibit inhibitory potential on seed germination and

Sarmentine pepper *(Piper* sp.) barnyard grass, redroot pigweed, crabgrass,

mustard (*Brassica* sp.)

Sorgoleone sorghum (*Sorghum bicolor* L. Moench)

Momilactone rice (*Oryza sativa L.*), moss (*Hypnum plumaeform*)

Artemisinin annual wormwood (*Artemisia annua* L.)

Leptospermone bottle brush (*Callistemon citrinus*),

growth of weeds

J.R., G. Forst)

manuka (*Leptospermum scoparium*

Glucosinolates, Isothiocyanates

### **5. Plant allelopathins as sources of bioherbicides**

Plant phytotoxic extracts, after evaluation, can be successfully used in integrated weed management. However, as was aforementioned, not all systems of its application under field conditions are suitable and profitable enough. To circumvent masking effects of one allelopa‐ thin by another in plant extract, research is now focused on isolation and application of a single, specific compound for the purpose of weed elimination. The list of allelochemicals isolated from various plants that may act as inhibitors of weed seed germination and/or weed growth are summarized in Table 1. A purified allelopathic compound may act on target plants with much higher or much lower strength. Even in situations when an allelopathin is active at unprofitably high doses but has a favorable environmental profile, it still may be a source to explore due to several reasons such as biodegradability. Modifications of chemical structure can make a compound more active on target plants while preserving desire properties.

Herein, examples of purified allelopathins with possible roles as herbicides are described. Some herbicides based on modified allelopathins already launched on the market are also included.

#### **5.1. Sorgoleone**

The inhibitory effect of sorghum on various plant species has been known for many years. Accumulation of sorghum phytotoxins in soil affects crop growth and imposes the need for a crop rotation system. Besides crops, weeds are also vulnerable to its allelopathic influence [16, 36]. Sorghum toxicity is mainly determined by both hydrophilic phenols in herbage, as well as hydrophobic sorgoleone and its analogs exuded by the root hairs [37, 38]. Therefore, sorghum herbage reach can be successfully used against weeds as a foliar spray as it is discussed in detail in the previous chapter.


Application of plant extracts as pre-emergence or as early post emergence herbicides resulted in reduction of doses of synthetic herbicide due to their synergistic or additive action. How‐ ever, not all phytotoxic extracts are effective enough to inhibit weed growth or germination when applied as spray even when plants show high allelopathic potential as mulch, inter‐ cropping system or in rotation. This may be the result of masking the activity of one compound by another in water solution or other factors such as impossibility of extract penetration through the cuticle [12]. A new opportunity to enhance effectiveness of usage of bioherbicides based on natural extracts is associated with extraction of individual allelochemicals and/or its comparison with synthetic herbicides. The extraction of sesquiterpene lactone, dehydrozalu‐ zanin C (DHZ) produced among Compositae family serves as an example [34]. Comparison studies of isolated DHZ (1 mM) and the commercial herbicide Logran® showed high inhibitory activity of DHZ on dicotyledonous plants while the synthetic herbicide showed no activity [34]. Also pure 2-benzoxazolinone (BOA) isolated from several graminaceous crops such as rye (*Secale cereale* L.), maize and wheat was active similarly as herbicide but its stability in the

Plant phytotoxic extracts, after evaluation, can be successfully used in integrated weed management. However, as was aforementioned, not all systems of its application under field conditions are suitable and profitable enough. To circumvent masking effects of one allelopa‐ thin by another in plant extract, research is now focused on isolation and application of a single, specific compound for the purpose of weed elimination. The list of allelochemicals isolated from various plants that may act as inhibitors of weed seed germination and/or weed growth are summarized in Table 1. A purified allelopathic compound may act on target plants with much higher or much lower strength. Even in situations when an allelopathin is active at unprofitably high doses but has a favorable environmental profile, it still may be a source to explore due to several reasons such as biodegradability. Modifications of chemical structure can make a compound more active on target plants while preserving desire properties.

Herein, examples of purified allelopathins with possible roles as herbicides are described. Some herbicides based on modified allelopathins already launched on the market are also

The inhibitory effect of sorghum on various plant species has been known for many years. Accumulation of sorghum phytotoxins in soil affects crop growth and imposes the need for a crop rotation system. Besides crops, weeds are also vulnerable to its allelopathic influence [16, 36]. Sorghum toxicity is mainly determined by both hydrophilic phenols in herbage, as well as hydrophobic sorgoleone and its analogs exuded by the root hairs [37, 38]. Therefore, sorghum herbage reach can be successfully used against weeds as a foliar spray as it is

environment was much shorter than the synthetic herbicide [35].

524 Herbicides - Current Research and Case Studies in Use

**5. Plant allelopathins as sources of bioherbicides**

included.

**5.1. Sorgoleone**

discussed in detail in the previous chapter.

**Table 1.** Allelopathic compounds isolated from plants that exhibit inhibitory potential on seed germination and growth of weeds

However, allelochemical sorgoleone has enormous potential as an herbicide due to its high activity against various weed species. Studies conducted under laboratory conditions have shown that low doses of sorgoleone (100 μM) inhibit growth of the following weeds by 80%, black nightshade (*Solanum nigrum* L.), redroot pigweed, common ragweed (*Am‐ brosia atrtemisiflora* L.), and by 40% of sicklepod (*Cassia obtusifolia* L.), hairy crabgrass (*Dig‐ itaria sanguinalis* L.), velvetleaf (*Abutilon theophrasti* Medik.), barnyardgrass and tef (*Eragrostis tef* Zucc., Trotter) [11, 16].

physiological effects as applications of atrazine in redroot pigweed-susceptible biotypes [11]. These properties make sorgoleone a potential early post-emergence herbicide when applied as a spray with much less environmental implications than atrazine. Therefore, inhibition of photosynthesis is the main target site of sorgoleone action in young seed‐ lings but its mode of action in older plants may be different [12]. Sorgoleone can be a useful inhibitor of *p*-hydroxyphenylpyruvate dioxygenase (HPPD), which takes part in αtocopherol and plastoquinone synthesis. Inhibition of that enzyme leads to a decreased pool of available plastoquinone and indirectly affects activity of phytoene desaturase, a key enzyme in carotenoid synthesis. Such sequence of events causes declining carotenoid levels and affects photosynthesis [45]. Currently used triketone herbicides (e.g. sulco‐ trione, isoxaflutole) have the same mechanism of action on HPPD as sorgoleone, irreversi‐ ble competitive inhibition, with I50 = 0.4 μM. Triketone herbicides are considered by the U.S. Environmental Protection Agency (EPA) to be a low environmental risk. They are usually utilized as selective herbicides to eliminate broadleaf weeds in corn [10]. It fol‐ lows, due to similar action and chemical structure and environmental friendly profile, sorgoleone might also be useful as a selective herbicide; however, such comparison stud‐ ies have yet to be conducted. Then, its mode of action also cannot explain whether it is

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527

Extracts and residues of rice, the well-known cereal plant, also have allelopathic potential. Among isolated secondary metabolites, phenolic acids, hydroxamic acids, fatty acids, terpenes and indoles were identified [46]. The key role in rice allelopathy plays momilactone A and B isolated from root exudates. High allelopathic rice varieties release up to 2-3 μg of momilactone B per day [3]. These compounds inhibited the growth of typical weeds in rice, e.g. barnyard grass and awnless barnyard grass (*Echinochloa colona* (L.) Link.) at concentrations higher than 1 μM and 10 μM, respectively. Furthermore, phytotoxic abilities of momilacton A and B were also demonstrated on livid pigweed (*Amaranthus lividus* L.), hairy crabgrass and annual bluegrass (*Poa annua* L.) at concentrations higher than 60 μM and 12 μM, respectively [47]. The experiment has shown that momilactone B is secreted by rice roots into the rhizosphere over the entire life cycle [48]. Momilactone A and B belong to the diterpenoid phytoalexins which are known as antimicrobial secondary metabolites generated in response to signal molecules called elicitors (especially biotic elicitors) [49]. Both compounds thought to be unique to rice, recently have been found in the moss (*Hypnum plumaeforme* Wils.), a taxonomically distinct plant [49]. Despite the ability of momilactone A and B to inhibit plant growth, its mode of

Artemisinin is a sesquiterpenoid lactone of annual wormwood (*Artemisia annua* L.). It is synthesized and sequestered in glandular trichomes located on the leaves and flowers [51]. It can also be excreted by the roots or root hairs, but only at the beginning of the growing season; therefore, dead leaves are the major source of artemisinin in soils [52]. Artemisinin is also lost

more or less active on broadleaf or grass weeds species [44].

**5.2. Momilactones**

action in plants is still unknown.

**5.3. Artemisinin**

Sorgoleone released into the soil may act as a pre-emergence herbicide. Its persistence in the soil during or after sorghum cultivation inhibits germination and growth of small-seeded weeds, e.g. hairy crabgrass and green bristlegrass (*Setaria viridis* (L.) Beauv.), due to its better absorption and translocation within the small seeds than in large seeds [39]. However, strength and final effect on seeds or seedling physiology is multifactor-dependent. Sorgoleone sorbs strongly to the organic matter. This allows an extended persistence in the soil but unfortu‐ nately, significantly reduces its bioavailability. Moreover, the dynamics of decomposition significantly influences sorgoleone bioactivity, e.g. the methoxy- group of the aromatic ring is decomposed by 26% 48 h after exudation; however, some amounts of sorgoleone are also extractable after 6 weeks [40, 41]. Nevertheless, constitutive production of the compound allows a continuous supply and accumulation in the soil around 1.5 cm of root zone [42].

Inhibition of H+ -ATPase in plant roots makes sorgoleone an effective growth inhibitor and potential post-emergence herbicide [43]. Decreased activity of that enzyme affects ion uptake and water balance by decreasing water uptake and affecting plant growth. Re‐ droot pigweed, Jimson weed (*Datura stramonium* L.) and tef grown in hydroponic culture with 10 μM sorgoleone were characterized by lower H+ -ATPase activity in roots. Presence of sorgoleone in nutrient solution significantly suppressed growth and evoked brown col‐ oration and necrosis [43, 44].

Sorgoleone may be taken up by roots but cannot be translocated acropetally by xylem due to high lipophilic properties. Therefore, its application as a post-emergence herbicide may be limited. However, as a spray (0.6 kg ha-1), it has inhibited growth by 12% of green foxtail (*Setaria faberi* Herrm.), by 40-50% purslane, hairy crabgrass and velvetleaf, and up to 80-90% of common ragweed, redroot pigweed, and black nightshade [40].

Due to the structural similarity of sorgoleone to plastoquinon, it acts as a photosystem II (PSII) inhibitor [11, 43]. It binds to the niche of the D1 protein in PSII, gathers electrons and does not allow reoxidation of plastoquinon A by the secondary electron acceptor, plastoquinone B. Competition studies under sorgoleone *versus* synthetic herbicides such as atrazine, diuron, metribuzin and bentazon have shown that sorgoleone is an atrazine competitive inhibitor [11, 12]. Moreover, the I50 of sorgoleone is 0.1 μM and similar to other PSII inhibitors. It is worth mentioning that sorgoleone belongs to the His215 family of PSII inhibitors, while atrazine belongs to Ser264. Mutation in Ser264 of the D1 protein is responsible for resistance to triazines as well as other non-triazine herbicides, leading to cross-resistance. However, plants resistant to atrazine, with a QB binding site on PSII mutation (Ser264), are not resistant to sorgoleone. Application of sorgoleone is particular‐ ly justified in the case of triazine-resistant biotypes of redroot pigweed, due to the same physiological effects as applications of atrazine in redroot pigweed-susceptible biotypes [11]. These properties make sorgoleone a potential early post-emergence herbicide when applied as a spray with much less environmental implications than atrazine. Therefore, inhibition of photosynthesis is the main target site of sorgoleone action in young seed‐ lings but its mode of action in older plants may be different [12]. Sorgoleone can be a useful inhibitor of *p*-hydroxyphenylpyruvate dioxygenase (HPPD), which takes part in αtocopherol and plastoquinone synthesis. Inhibition of that enzyme leads to a decreased pool of available plastoquinone and indirectly affects activity of phytoene desaturase, a key enzyme in carotenoid synthesis. Such sequence of events causes declining carotenoid levels and affects photosynthesis [45]. Currently used triketone herbicides (e.g. sulco‐ trione, isoxaflutole) have the same mechanism of action on HPPD as sorgoleone, irreversi‐ ble competitive inhibition, with I50 = 0.4 μM. Triketone herbicides are considered by the U.S. Environmental Protection Agency (EPA) to be a low environmental risk. They are usually utilized as selective herbicides to eliminate broadleaf weeds in corn [10]. It fol‐ lows, due to similar action and chemical structure and environmental friendly profile, sorgoleone might also be useful as a selective herbicide; however, such comparison stud‐ ies have yet to be conducted. Then, its mode of action also cannot explain whether it is more or less active on broadleaf or grass weeds species [44].

#### **5.2. Momilactones**

However, allelochemical sorgoleone has enormous potential as an herbicide due to its high activity against various weed species. Studies conducted under laboratory conditions have shown that low doses of sorgoleone (100 μM) inhibit growth of the following weeds by 80%, black nightshade (*Solanum nigrum* L.), redroot pigweed, common ragweed (*Am‐ brosia atrtemisiflora* L.), and by 40% of sicklepod (*Cassia obtusifolia* L.), hairy crabgrass (*Dig‐ itaria sanguinalis* L.), velvetleaf (*Abutilon theophrasti* Medik.), barnyardgrass and tef

Sorgoleone released into the soil may act as a pre-emergence herbicide. Its persistence in the soil during or after sorghum cultivation inhibits germination and growth of small-seeded weeds, e.g. hairy crabgrass and green bristlegrass (*Setaria viridis* (L.) Beauv.), due to its better absorption and translocation within the small seeds than in large seeds [39]. However, strength and final effect on seeds or seedling physiology is multifactor-dependent. Sorgoleone sorbs strongly to the organic matter. This allows an extended persistence in the soil but unfortu‐ nately, significantly reduces its bioavailability. Moreover, the dynamics of decomposition significantly influences sorgoleone bioactivity, e.g. the methoxy- group of the aromatic ring is decomposed by 26% 48 h after exudation; however, some amounts of sorgoleone are also extractable after 6 weeks [40, 41]. Nevertheless, constitutive production of the compound allows a continuous supply and accumulation in the soil around 1.5 cm of root zone [42].

and potential post-emergence herbicide [43]. Decreased activity of that enzyme affects ion uptake and water balance by decreasing water uptake and affecting plant growth. Re‐ droot pigweed, Jimson weed (*Datura stramonium* L.) and tef grown in hydroponic culture

of sorgoleone in nutrient solution significantly suppressed growth and evoked brown col‐

Sorgoleone may be taken up by roots but cannot be translocated acropetally by xylem due to high lipophilic properties. Therefore, its application as a post-emergence herbicide may be limited. However, as a spray (0.6 kg ha-1), it has inhibited growth by 12% of green foxtail (*Setaria faberi* Herrm.), by 40-50% purslane, hairy crabgrass and velvetleaf, and up to 80-90% of

Due to the structural similarity of sorgoleone to plastoquinon, it acts as a photosystem II (PSII) inhibitor [11, 43]. It binds to the niche of the D1 protein in PSII, gathers electrons and does not allow reoxidation of plastoquinon A by the secondary electron acceptor, plastoquinone B. Competition studies under sorgoleone *versus* synthetic herbicides such as atrazine, diuron, metribuzin and bentazon have shown that sorgoleone is an atrazine competitive inhibitor [11, 12]. Moreover, the I50 of sorgoleone is 0.1 μM and similar to other PSII inhibitors. It is worth mentioning that sorgoleone belongs to the His215 family of PSII inhibitors, while atrazine belongs to Ser264. Mutation in Ser264 of the D1 protein is responsible for resistance to triazines as well as other non-triazine herbicides, leading to cross-resistance. However, plants resistant to atrazine, with a QB binding site on PSII mutation (Ser264), are not resistant to sorgoleone. Application of sorgoleone is particular‐ ly justified in the case of triazine-resistant biotypes of redroot pigweed, due to the same

with 10 μM sorgoleone were characterized by lower H+

common ragweed, redroot pigweed, and black nightshade [40].



(*Eragrostis tef* Zucc., Trotter) [11, 16].

526 Herbicides - Current Research and Case Studies in Use

Inhibition of H+

oration and necrosis [43, 44].

Extracts and residues of rice, the well-known cereal plant, also have allelopathic potential. Among isolated secondary metabolites, phenolic acids, hydroxamic acids, fatty acids, terpenes and indoles were identified [46]. The key role in rice allelopathy plays momilactone A and B isolated from root exudates. High allelopathic rice varieties release up to 2-3 μg of momilactone B per day [3]. These compounds inhibited the growth of typical weeds in rice, e.g. barnyard grass and awnless barnyard grass (*Echinochloa colona* (L.) Link.) at concentrations higher than 1 μM and 10 μM, respectively. Furthermore, phytotoxic abilities of momilacton A and B were also demonstrated on livid pigweed (*Amaranthus lividus* L.), hairy crabgrass and annual bluegrass (*Poa annua* L.) at concentrations higher than 60 μM and 12 μM, respectively [47]. The experiment has shown that momilactone B is secreted by rice roots into the rhizosphere over the entire life cycle [48]. Momilactone A and B belong to the diterpenoid phytoalexins which are known as antimicrobial secondary metabolites generated in response to signal molecules called elicitors (especially biotic elicitors) [49]. Both compounds thought to be unique to rice, recently have been found in the moss (*Hypnum plumaeforme* Wils.), a taxonomically distinct plant [49]. Despite the ability of momilactone A and B to inhibit plant growth, its mode of action in plants is still unknown.

#### **5.3. Artemisinin**

Artemisinin is a sesquiterpenoid lactone of annual wormwood (*Artemisia annua* L.). It is synthesized and sequestered in glandular trichomes located on the leaves and flowers [51]. It can also be excreted by the roots or root hairs, but only at the beginning of the growing season; therefore, dead leaves are the major source of artemisinin in soils [52]. Artemisinin is also lost from annual wormwood by rain runoff but to a minor degree (<0.5%),. This allelopathin is well known as a promising anti-malaric agent but also as a phytotoxin selective mainly to broadleaf weeds. Artemisinin (at 33 μM) significantly reduced shoot and root growth of lettuce, redroot pigweed, pitted morning-glory (*Ipomoea lacunose* L.) common purslane and annual wormwood [53]. However, the same treatment had no effect on sorghum or velvetleaf. Several studies have been aimed at identifying the molecular target site of this compound as well as the structural requirements for herbicidal activity [53-55].The effect of artemisinin is most evident on root growth and chlorophyll content. In onion root tips, artemisinin (10 - 100 μM) decreased the mitotic index, provoked abnormal mitotic figures and caused structural modifications of chromosomes [55]. However, no definite target site has yet been identified. The most recent studies on rice sprayed with 1.86 μM artemisinin indicated its inhibiting abilities on photo‐ synthetic electron transport [56]. Artemisinin site of action is probably plastoquinone B in photosystem II. Interestingly, as authors suggest, this effect is caused not directly by artemi‐ sinin itself, but rather by an unidentified artemisinin-metabolite occured in the plant after artemisinin application [56].

to give acceptable weed control. Such high doses excluded leptospermone from commercial development. The structure of this allelochemical was used as a basis for development of synthetic analogues including mesotrione (trade name Callisto), an herbicide produced by Syngenta AG. Mesotrione is applied for control of broadleaved weeds in maize. The rates of mesotrione are in the range from 75 to 225 g a.s. ha-1 (around 100 times more potent than

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However, leptospermone has lately been found as the main herbicidal component of manuka oil (*Leptospermum scoparium* J.R., G. Forst) [61]. Manuka oil (1%) applied as post-emergence spray, significantly decreased growth and dry weight of redroot pigweed, barnyardgrass, velvetleaf and hairy crabgrass. Though, hairy crabgrass seedlings that emerged after manuka oil application were totally blanched. Pre-emergence application of 0.17% manuka oil which corresponds to 0.2 L ha-1 of leptospermone inhibited hairy crabgrass growth by 50%. The preemergence effects are mainly dependent on its persistence in soil. Average time of leptosper‐ mone half-life in soil was calculated at 15 days while applied as a compound of manuka oil time extended by 3 days. This clearly shows that half-life of active compounds may be longer in mixture than applied alone due to additive or synergistic action. This type of leptospermone application poses another possibility of usage for this compound in its natural form without

Lately, there has been a growing interest for using essential oils as allelopathins with bioher‐ bicide potential. Some of them have already been commercialized and successfully launched in organic agriculture. They disrupt the cuticle and contribute to desiccation or burn down young tissues. Examples of this are the commercially available bioherbicide with the trade name of GreenMatch EX which consists of lemongrass (*Cymbopogon* sp.) oils or InterceptorTM with 10% pine (*Pinus sylvestris* L.) oil [3]. Essential oils are complex mixtures of monoterpenes, sesquiterpenes, and aromatic phenols, oxides, ethers, alcohols, esters, aldehydes and ketones [62]. The main terpenoids of volatile essential oils are monoterpenes (C10) and sesquiterpenes (C15). It has been well documented that essential oils found in foliage of eucalyptus (*Eucalyp‐ tus* sp.) show phytotoxic potential. During field experiments it has been reported that common weeds such as coffee senna (*Cassia occidentalis* L.) and barnyardgrass sprayed with different concentrations of eucalyptus oil (from 5 % to 10 % v/v with 0.05 % v/v Tween-80) exhibited dose-dependent and species-dependent levels of injury. Coffee senna plants were more sensitive to the eucalyptus oil than barnyardgrass [62]. Phytotoxicity of eucalyptus oil is due to the components such as 1,8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, linalool, α-pinene, γ-terpinene, α-terpineol, alloocimene, and aroma‐ dendrene [62]. Pre-emergence herbicidal activity of 1,8-cineole 3, and 1,4-cineole 4 were tested against rigid ryegrass and garden radish var. Long Scarlet in laboratory-based bioassays. 1,8 cineole and its derivatives showed a dose-dependent herbicidal activity against both weed species [64]. Laboratory studies [64, 65] also have shown that soil-applied 1,8-cineole sup‐ pressed the growth of several weeds. However, field reports demonstrated that 1,8-cineole alone has poor herbicidal activity [67, 68]. The commercial herbicide cinmethylin is a 2-benzyl

leptospermone) [60].

**5.5. Essential oils**

chemical modification of the structure [61].

Other controversies around the phytotoxic potential of artemisinin arose when the dichloro‐ methane extracts of annual wormwood leaves containing artemisinin showed a stronger phytotoxic effect on redroot pigweed seed germination and seedling growth than pure artemisinin [57]. Moreover, aqueous extract with disposed artemisinin had equal inhibitory effects on both physiological processes as allelopathin alone. This experiment suggests a marginal role of artemisinin in plant extract and joint action of other allelochemicals. Although, most studies analyzing allelopathic weed–crop interferences using annual wormwood were conducted under laboratory and greenhouse conditions [58].

Toxic studies on duckweed (*Lemna minor* L.) and the fresh water algae *Pseudokirchneriella subcapitata* (Korshikov) had EC50 values 0.24 and 0.19 mg L−1 respectively, with growth rate as endpoint corresponding to those of the herbicide atrazine [59]. These profiles questioned environmental safety of artemisinin for the purpose as a bioherbicide. It may be a result of its complex chemical structure, but this compound may be used as the ba‐ sis for a new herbicide, based on artemisinin chemical structure. Such attempts have al‐ ready been made using artemisinin's analogues [55]. Four of the tested 12 analogues inhibited germination and root growth of lettuce, *Arabidopsis thaliana* (L.) and duckweed at extremely low concentrations (3 μM).

#### **5.4. Leptospermone**

Leptospermone (1-hydroxy-2-isovaloryl-4,4,6,6-tetramethyl cyclohexen-3,5-dione) is a natural triketone produced by the roots of the bottlebrush (*Callistemon citrinus* Curtis) [60]. In its pure form, it was tested both pre- and post-emergence on a range of plant species including: hairy crabgrass, yellow foxtail *(Setaria glauca* (L.) P. Beauv.), barnyard grass, California red oat (*Avena sativa* L.), redroot pigweed, Indian mustard (*Brassica juncea* L.) and curly dock (*Rumex crispus* L.). Leptospermone is a strong p- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor with *I50* values 3 μg mL-1[61]. Inhibition of this enzyme leads to disruption in carotenoid biosynthesis and loss of chlorophyll. Unfortunately, a pure compound rate of 9000 g a.s. ha-1 was required to give acceptable weed control. Such high doses excluded leptospermone from commercial development. The structure of this allelochemical was used as a basis for development of synthetic analogues including mesotrione (trade name Callisto), an herbicide produced by Syngenta AG. Mesotrione is applied for control of broadleaved weeds in maize. The rates of mesotrione are in the range from 75 to 225 g a.s. ha-1 (around 100 times more potent than leptospermone) [60].

However, leptospermone has lately been found as the main herbicidal component of manuka oil (*Leptospermum scoparium* J.R., G. Forst) [61]. Manuka oil (1%) applied as post-emergence spray, significantly decreased growth and dry weight of redroot pigweed, barnyardgrass, velvetleaf and hairy crabgrass. Though, hairy crabgrass seedlings that emerged after manuka oil application were totally blanched. Pre-emergence application of 0.17% manuka oil which corresponds to 0.2 L ha-1 of leptospermone inhibited hairy crabgrass growth by 50%. The preemergence effects are mainly dependent on its persistence in soil. Average time of leptosper‐ mone half-life in soil was calculated at 15 days while applied as a compound of manuka oil time extended by 3 days. This clearly shows that half-life of active compounds may be longer in mixture than applied alone due to additive or synergistic action. This type of leptospermone application poses another possibility of usage for this compound in its natural form without chemical modification of the structure [61].

#### **5.5. Essential oils**

from annual wormwood by rain runoff but to a minor degree (<0.5%),. This allelopathin is well known as a promising anti-malaric agent but also as a phytotoxin selective mainly to broadleaf weeds. Artemisinin (at 33 μM) significantly reduced shoot and root growth of lettuce, redroot pigweed, pitted morning-glory (*Ipomoea lacunose* L.) common purslane and annual wormwood [53]. However, the same treatment had no effect on sorghum or velvetleaf. Several studies have been aimed at identifying the molecular target site of this compound as well as the structural requirements for herbicidal activity [53-55].The effect of artemisinin is most evident on root growth and chlorophyll content. In onion root tips, artemisinin (10 - 100 μM) decreased the mitotic index, provoked abnormal mitotic figures and caused structural modifications of chromosomes [55]. However, no definite target site has yet been identified. The most recent studies on rice sprayed with 1.86 μM artemisinin indicated its inhibiting abilities on photo‐ synthetic electron transport [56]. Artemisinin site of action is probably plastoquinone B in photosystem II. Interestingly, as authors suggest, this effect is caused not directly by artemi‐ sinin itself, but rather by an unidentified artemisinin-metabolite occured in the plant after

Other controversies around the phytotoxic potential of artemisinin arose when the dichloro‐ methane extracts of annual wormwood leaves containing artemisinin showed a stronger phytotoxic effect on redroot pigweed seed germination and seedling growth than pure artemisinin [57]. Moreover, aqueous extract with disposed artemisinin had equal inhibitory effects on both physiological processes as allelopathin alone. This experiment suggests a marginal role of artemisinin in plant extract and joint action of other allelochemicals. Although, most studies analyzing allelopathic weed–crop interferences using annual wormwood were

Toxic studies on duckweed (*Lemna minor* L.) and the fresh water algae *Pseudokirchneriella subcapitata* (Korshikov) had EC50 values 0.24 and 0.19 mg L−1 respectively, with growth rate as endpoint corresponding to those of the herbicide atrazine [59]. These profiles questioned environmental safety of artemisinin for the purpose as a bioherbicide. It may be a result of its complex chemical structure, but this compound may be used as the ba‐ sis for a new herbicide, based on artemisinin chemical structure. Such attempts have al‐ ready been made using artemisinin's analogues [55]. Four of the tested 12 analogues inhibited germination and root growth of lettuce, *Arabidopsis thaliana* (L.) and duckweed

Leptospermone (1-hydroxy-2-isovaloryl-4,4,6,6-tetramethyl cyclohexen-3,5-dione) is a natural triketone produced by the roots of the bottlebrush (*Callistemon citrinus* Curtis) [60]. In its pure form, it was tested both pre- and post-emergence on a range of plant species including: hairy crabgrass, yellow foxtail *(Setaria glauca* (L.) P. Beauv.), barnyard grass, California red oat (*Avena sativa* L.), redroot pigweed, Indian mustard (*Brassica juncea* L.) and curly dock (*Rumex crispus* L.). Leptospermone is a strong p- hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor with *I50* values 3 μg mL-1[61]. Inhibition of this enzyme leads to disruption in carotenoid biosynthesis and loss of chlorophyll. Unfortunately, a pure compound rate of 9000 g a.s. ha-1 was required

conducted under laboratory and greenhouse conditions [58].

at extremely low concentrations (3 μM).

**5.4. Leptospermone**

artemisinin application [56].

528 Herbicides - Current Research and Case Studies in Use

Lately, there has been a growing interest for using essential oils as allelopathins with bioher‐ bicide potential. Some of them have already been commercialized and successfully launched in organic agriculture. They disrupt the cuticle and contribute to desiccation or burn down young tissues. Examples of this are the commercially available bioherbicide with the trade name of GreenMatch EX which consists of lemongrass (*Cymbopogon* sp.) oils or InterceptorTM with 10% pine (*Pinus sylvestris* L.) oil [3]. Essential oils are complex mixtures of monoterpenes, sesquiterpenes, and aromatic phenols, oxides, ethers, alcohols, esters, aldehydes and ketones [62]. The main terpenoids of volatile essential oils are monoterpenes (C10) and sesquiterpenes (C15). It has been well documented that essential oils found in foliage of eucalyptus (*Eucalyp‐ tus* sp.) show phytotoxic potential. During field experiments it has been reported that common weeds such as coffee senna (*Cassia occidentalis* L.) and barnyardgrass sprayed with different concentrations of eucalyptus oil (from 5 % to 10 % v/v with 0.05 % v/v Tween-80) exhibited dose-dependent and species-dependent levels of injury. Coffee senna plants were more sensitive to the eucalyptus oil than barnyardgrass [62]. Phytotoxicity of eucalyptus oil is due to the components such as 1,8-cineole, citronellal, citronellol, citronellyl acetate, p-cymene, eucamalol, limonene, linalool, α-pinene, γ-terpinene, α-terpineol, alloocimene, and aroma‐ dendrene [62]. Pre-emergence herbicidal activity of 1,8-cineole 3, and 1,4-cineole 4 were tested against rigid ryegrass and garden radish var. Long Scarlet in laboratory-based bioassays. 1,8 cineole and its derivatives showed a dose-dependent herbicidal activity against both weed species [64]. Laboratory studies [64, 65] also have shown that soil-applied 1,8-cineole sup‐ pressed the growth of several weeds. However, field reports demonstrated that 1,8-cineole alone has poor herbicidal activity [67, 68]. The commercial herbicide cinmethylin is a 2-benzyl ether substituted analog of the monoterpene 1,4-cineole (1-methyl-4-(1-methylethyl)-7 oxabicyclo heptane). This compound was discovered and partially developed by Shell Chemicals as a derivative of the allelopathic natural monoterpene, 1,8-cineole [69]. The benzyl ether substitution appears to decrease the volatility of the cineole ring by several orders of magnitude thereby rendering it more suitable for herbicide use [70]. Cinmethylin is a moder‐ ately effective growth inhibitor used for monocot weed control [71]. Despite the fact that it has been used commercially in both Europe and Japan and has been studied experimentally for several decades, the mechanism of action of this herbicide is still unknown [54, 72]. Cinme‐ thylin was commercialized outside the United States in 1982 under the trade names of Cinch and Argold. Cinmethylin is active on several important grasses in rice; Echinochloa sp., Cyperus sp. and heartshape false pickerelweed (*Monochoria viginalis* Burm.f.) at rates from 25 to 100 g a.s. ha-1 [73].

opportunity to further the structural modification that makes the compound more stable without any disadvantages on bioherbicide action in plants. It is worth noting that sar‐ mentine has already been patented as an herbicide but not commercialized yet [75].

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A lot of effort has been done to explore the nature of allelopathic interactions. Studies on allelopathic compounds greatly increased thanks to chemical and biochemical techniques, which improved identification and knowledge about its mode of action. Since then, the crucial role of secondary metabolites synthesized and released by plants became better understood. It has been clearly demonstrated that allelopathins may take part in very complex inter- and intra-specific ecological interactions including soil microorganisms. However, despite the extensive research carried out under laboratory conditions, the higher level of such interactions at the ecosystem level has not been sufficiently explored. Structure, chemical properties, and mode of action in plants of multitude allelochemicals are already known but, unfortunately, only a part-per thousand of them have been successfully introduced in agricultural practices. This is mainly due to limitations of compounds as plant protection agents but also due to extended field experiments. A very important aspect that allows the introduction of allelop‐ athy to natural weed management is knowledge about biology of donor and target plants and the exact chemicals responsible for the interaction [78]. All formerly described limitations of natural compounds as bioherbicides decreasing in case of plant extracts as herbicides due to simple and low cost of application. However, separation of one, specific compound that is the most interesting for us among hundreds synthesized by plants often required information

One of the problems is to obtain adequate amounts of the compound, when its chemical synthesis is impossible or collection of plants, unprofitable. Increased synthesis of an allelo‐ pathin gives triple profits. First of all, enhanced allelopathic potential of a plant makes it more competitive against weeds. Second of all, increased concentration of a compound makes plant extract more active. Thirdly, this allows collection of the compound at a sufficient amount and makes it more profitable. However, it is much easier to obtain active compounds from the crop species than wild living ones. Difficulties in introducing plants to cultivation are due to the

Cells and organ cultures provide opportunities to circumvent these limitations. Abilities of undifferentiated and differentiated cells to produce allelochemicals may be commercialized in bioreactors using cell suspension cultures [79]. Such attempts have been made on Artemisia suspension culture for artemisinin production; however, obtained amounts of that compound were insufficient. The addition of β-cyclodextrins to the growing medium has increased artemisinin synthesis up to 300% [80]. Allelochemicals produced by roots may be obtained from hairy root cultures, both *via* callogenesis or infection with *Agrobacterium tumefaciens*. Transgenic hairy roots are characterized by high genetic stability and facility to accumulate metabolites. The hairy root system already has been applied to increased production of

**6. Biotechnology in bioherbicide investigation**

low ability to grow outside their natural ecosystem [79].

about its synthesis *in vivo*.

#### **5.6. Sarmentine**

Sarmentine was first isolated from long pepper (*Piper longum* L.) fruits [74] but is also present in varied organs of other *Piper* species (Huang and Asolkan patent). It has been known as a medicinal plant with many beneficial multidirectional properties on human health. However, methanol extract of long pepper dry fruits has been shown to be suppressive to lettuce [75]. Purification and fractioning of long pepper crude extract allows the dissection of the active compound – sarmentine, a molecule with a long unsaturated fatty acid chain and pyrrolidine. Due to the hydrophobic properties, sarmentine is suspended with surfactants, 0.2 % glycospere O-20, 2% ethanol and 0.1% sodium lauryl sulfate. As a foliar spray, it is active at 2.5 mg mL-1, but its high phytotoxicity is manifested at 5 mg mL-1. Higher concentrations of sarmentine caused almost 100% mortality of redroot pigweed, barnyardgrass, bindweed (*Convonvulus* sp.), hairy crabgrass, sprangletop (*Leptochloa* sp.), annual bluegrass, wild mustard (*Sinapis arvensis* L.), curly dock with impaired effects on horseweed (*Conyza canadensis* (L.) Cronquist) and sedge (*Carex* sp.) growth under laboratory conditions. First phytotoxic symptoms such as bent stems and contact necrosis, have been visible 30 minutes after application; however fullblown implications were seen 7 h after spraying. The most likely mechanism of sarmentine action on plants is disruption of the plant cuticle which leads to disruption of cell membranes and lipid peroxidation followed by formation of radicals [76, 77].

As an herbicide, sarmentine and its derivatives may be both obtained from fruits of long pepper and successfully chemically synthesized [75]. Despite the fact that the compound is active under laboratory conditions, its chemical and biological instability under field conditions may limit its application as an herbicide. However, it has been shown that crucial for sarmentine herbicidal activity is the presence of an amine bond with a secon‐ dary amine. Replacement of the acid moiety with structurally similar fatty acids has not changed its phytotoxic potential. Moreover, natural herbicides based on sarmentine may also contain other derivatives with similar modes of action on plants but higher environ‐ mental stability [75]. Sarmentine may be successfully applied in combination with syn‐ thetic herbicides, e.g. aryloxyphenoxypropionic, benzoic acid, dicarboximide, organophosphorus, triazine, sulfonamide herbicides and with many others. This gives an opportunity to further the structural modification that makes the compound more stable without any disadvantages on bioherbicide action in plants. It is worth noting that sar‐ mentine has already been patented as an herbicide but not commercialized yet [75].

### **6. Biotechnology in bioherbicide investigation**

ether substituted analog of the monoterpene 1,4-cineole (1-methyl-4-(1-methylethyl)-7 oxabicyclo heptane). This compound was discovered and partially developed by Shell Chemicals as a derivative of the allelopathic natural monoterpene, 1,8-cineole [69]. The benzyl ether substitution appears to decrease the volatility of the cineole ring by several orders of magnitude thereby rendering it more suitable for herbicide use [70]. Cinmethylin is a moder‐ ately effective growth inhibitor used for monocot weed control [71]. Despite the fact that it has been used commercially in both Europe and Japan and has been studied experimentally for several decades, the mechanism of action of this herbicide is still unknown [54, 72]. Cinme‐ thylin was commercialized outside the United States in 1982 under the trade names of Cinch and Argold. Cinmethylin is active on several important grasses in rice; Echinochloa sp., Cyperus sp. and heartshape false pickerelweed (*Monochoria viginalis* Burm.f.) at rates from 25

Sarmentine was first isolated from long pepper (*Piper longum* L.) fruits [74] but is also present in varied organs of other *Piper* species (Huang and Asolkan patent). It has been known as a medicinal plant with many beneficial multidirectional properties on human health. However, methanol extract of long pepper dry fruits has been shown to be suppressive to lettuce [75]. Purification and fractioning of long pepper crude extract allows the dissection of the active compound – sarmentine, a molecule with a long unsaturated fatty acid chain and pyrrolidine. Due to the hydrophobic properties, sarmentine is suspended with surfactants, 0.2 % glycospere O-20, 2% ethanol and 0.1% sodium lauryl sulfate. As a foliar spray, it is active at 2.5 mg mL-1, but its high phytotoxicity is manifested at 5 mg mL-1. Higher concentrations of sarmentine caused almost 100% mortality of redroot pigweed, barnyardgrass, bindweed (*Convonvulus* sp.), hairy crabgrass, sprangletop (*Leptochloa* sp.), annual bluegrass, wild mustard (*Sinapis arvensis* L.), curly dock with impaired effects on horseweed (*Conyza canadensis* (L.) Cronquist) and sedge (*Carex* sp.) growth under laboratory conditions. First phytotoxic symptoms such as bent stems and contact necrosis, have been visible 30 minutes after application; however fullblown implications were seen 7 h after spraying. The most likely mechanism of sarmentine action on plants is disruption of the plant cuticle which leads to disruption of cell membranes

As an herbicide, sarmentine and its derivatives may be both obtained from fruits of long pepper and successfully chemically synthesized [75]. Despite the fact that the compound is active under laboratory conditions, its chemical and biological instability under field conditions may limit its application as an herbicide. However, it has been shown that crucial for sarmentine herbicidal activity is the presence of an amine bond with a secon‐ dary amine. Replacement of the acid moiety with structurally similar fatty acids has not changed its phytotoxic potential. Moreover, natural herbicides based on sarmentine may also contain other derivatives with similar modes of action on plants but higher environ‐ mental stability [75]. Sarmentine may be successfully applied in combination with syn‐ thetic herbicides, e.g. aryloxyphenoxypropionic, benzoic acid, dicarboximide, organophosphorus, triazine, sulfonamide herbicides and with many others. This gives an

and lipid peroxidation followed by formation of radicals [76, 77].

to 100 g a.s. ha-1 [73].

530 Herbicides - Current Research and Case Studies in Use

**5.6. Sarmentine**

A lot of effort has been done to explore the nature of allelopathic interactions. Studies on allelopathic compounds greatly increased thanks to chemical and biochemical techniques, which improved identification and knowledge about its mode of action. Since then, the crucial role of secondary metabolites synthesized and released by plants became better understood. It has been clearly demonstrated that allelopathins may take part in very complex inter- and intra-specific ecological interactions including soil microorganisms. However, despite the extensive research carried out under laboratory conditions, the higher level of such interactions at the ecosystem level has not been sufficiently explored. Structure, chemical properties, and mode of action in plants of multitude allelochemicals are already known but, unfortunately, only a part-per thousand of them have been successfully introduced in agricultural practices. This is mainly due to limitations of compounds as plant protection agents but also due to extended field experiments. A very important aspect that allows the introduction of allelop‐ athy to natural weed management is knowledge about biology of donor and target plants and the exact chemicals responsible for the interaction [78]. All formerly described limitations of natural compounds as bioherbicides decreasing in case of plant extracts as herbicides due to simple and low cost of application. However, separation of one, specific compound that is the most interesting for us among hundreds synthesized by plants often required information about its synthesis *in vivo*.

One of the problems is to obtain adequate amounts of the compound, when its chemical synthesis is impossible or collection of plants, unprofitable. Increased synthesis of an allelo‐ pathin gives triple profits. First of all, enhanced allelopathic potential of a plant makes it more competitive against weeds. Second of all, increased concentration of a compound makes plant extract more active. Thirdly, this allows collection of the compound at a sufficient amount and makes it more profitable. However, it is much easier to obtain active compounds from the crop species than wild living ones. Difficulties in introducing plants to cultivation are due to the low ability to grow outside their natural ecosystem [79].

Cells and organ cultures provide opportunities to circumvent these limitations. Abilities of undifferentiated and differentiated cells to produce allelochemicals may be commercialized in bioreactors using cell suspension cultures [79]. Such attempts have been made on Artemisia suspension culture for artemisinin production; however, obtained amounts of that compound were insufficient. The addition of β-cyclodextrins to the growing medium has increased artemisinin synthesis up to 300% [80]. Allelochemicals produced by roots may be obtained from hairy root cultures, both *via* callogenesis or infection with *Agrobacterium tumefaciens*. Transgenic hairy roots are characterized by high genetic stability and facility to accumulate metabolites. The hairy root system already has been applied to increased production of phenolic compounds of nettleleaf goosefoot (*Chenopodium murale* Linn.) [81] and gossypol of cotton [82]. Active growth of roots and rapid colonization of the bioreactor allows rapidly reaching target weight, necessary to obtain an adequate quantity of the compound extracted from plants or growing medium.

easier collection of the compound. Moreover, the well-known pathway of sorgoleone synthesis and characteristic of candidate genes may be a promising source of introducing sorgoleone

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The situation becomes more complicated when more than one gene encoding special enzymes is required to increase synthesis of a plant compound. Such difficulties have been encountered for DIBOA, synthesized by various grass species [95]. In maize, biosynthesis of this compound is determined by five genes (*Bx1* to *Bx5*) encoding three enzymes: tryptophan synthase α

Monoterpenes are a large family of compounds produced by a varied family of aromatic plants. Some of the monoterpenes also take part in allelopathic interactions, e.g. linalool, cineole camphene, pinene, limonene, etc. Currently, metabolic engineering allows im‐ proved production of specific compounds in heterologous systems [96]. The most interest‐ ing are monoterpene synthases which catalyzed geranyl diphosphate (GPP) into output structure of numerous monoterpenes family, e.g. enhanced expression of limonene syn‐ thase in transgenic peppermint (*Mentha piperita* L.) has increased yield of monoterpenes. An alternative approach is to change the density of secretory structures by both plant hormone and transcriptional factors manipulation. Such attempts already have been made in annual wormwood and *A*. *thaliana*. It was recently found that the number of glandular trichomes increased in response to jasmonic acid. Spraying of annual worm‐ wood with this hormone significantly increased density of these structures on leaves what was accompanied with higher artemisinin content [51]. This was an effect of en‐ hanced expression of gene encoding enzymes taking part in artemisinin biosynthesis. On the other hand, in Arabidopsis, co-expression of two positive transcriptional factors (GL1,

and R protein of maize) has significantly improved the number of trichomes [96].

Hook.), has produced linalool but in its glycosylated, non-volatile form [96].

However, we have to bear in mind that biosynthesis of natural compounds can be limited to organs, tissues or even cells. Specific locations of compound synthesis, accumulation or secretion often make that compound toxic to other tissues within the same plant organism. Moreover, even successful transformation of a plant does not guarantee successful and sufficient production of a desirable compound. The gene of (S)-linalool synthase (*Lis*) of fairy fans (*Clarkia breweri* Gray), constitutively expressed in transgenic petunia (*Petunia hybrida*

All presented techniques provide greater knowledge on allelopathy. However, better under‐ standing of such complex interactions among this phenomenon bring us one step forward to development of new strategies in weed management and finding new herbicides and new

The phenomena of allelopathy and phytotoxic interactions between plants are strongly expanding branches of biological science. Allelochemicals, as a group of substances also called

homolog, cytochrome P-450-dependent monooxygenase [95].

production to grass crops [94].

herbicidal target sites.

**7. Conclusions**

The recombinant DNA technology can be useful to improve allelochemical production. Enhancing or suppression of gene expression, metabolic engineering and genetic transforma‐ tion are promising new tools for allelochemical synthesis [79]. This approach is based on elucidation of the metabolic pathway, enzyme activities and identification of genes encoding crucial enzymes, associated with metabolite (allelochemical) synthesis.

Allelopathy is a quantitative trait. A genetic analysis of quantitative trait loci (QTL) is a promising approach to identify genes underlying this trait. Only a few crops are under genetic screening for its allelopathic properties including: rice, wheat, barley and oat [83, 84]. The first QTL map associated with allelopathic properties was developed in rice. A segregating population derived from a cross of two cultivars varying with allelopathic potential against barnyardgrass. The map contained 140 DNA markers with four main-effects QTL located on chromosome 2, 3 and 8 [85]. Proteomic studies on allelopathy of rice against barnyardgrass confirmed the crucial role of three enzymes: phenylalanine ammonia-lyse (PAL), thioredoxin and 3-hydroxy-3-methilglutarilcoenzyme A reductase 3 (HMGR) is highly involved in phenols biosynthesis [86]. Such a genetic approach may allow the location of the gene in the genome and better understanding of its function in plant allelopathy and create the chance of applying marker assisted selection (MAS) to enhance allelopathic abilities.

Just like breeding programs allow improved crop production, they may also improve pro‐ duction of allelopathic compounds increasing allelopathic potential.

Scopoletin has been known as allelopathic root exudates of oats (*Avena* sp.) that affects growth of neighboring plants. Screening of 3000 of Avena accessions has shown varying ability to scopoletin production. Twenty five of them have exuded higher amounts of scopoletin than control cultivar Garry, of which 4 were threefold more than the control [87]. Variation in allelopathin production was also discovered for sorgoleone of seven sorghum accessions [38] nomilacton A and B of 8 rice accessions [88] DIBOA and DIMBOA of 14 rye cultivars [88], gramine of 43 lines of modern cultivar of barley (*Hordeum vulgare* L.) and wild progenitor *H. spontaneum* (C. Koch) [90]. Enhanced production of active compounds from allelopathic plants can be developed by efficient breeding - selection of individuals with high allelopathic ability. Identification of a single gene, arranged in synthesis of allelopathin already has been per‐ formed for sorgoleone. *SOR1* (or compatible *SbDES3*) expression is specific for root hairs of two species of sorghum (*S. bicolor* and *S. halepense*) and associated with sorgoleone synthesis, while it is not expressed in other organs of sorghum *SOR1* encodes novel fatty acid desaturase (FAD), involved in the formation of a specific bond at 16:3Δ9,12,15 pattern [91, 92]. Comparative studies of FAD derived from sorghum with other desaturases showed high similarity to omega-3 fatty acid desaturases (FAD3) [93]. However, none of the hitherto known desaturases can synthesize double bonding at this unique pattern along the aliphathic chain of the sorgoleone molecule. Characterization of this gene allows an overexpression of *SOR1* and increased sorgoleone synthesis and improved allelopathic potential of sorghum, as well as easier collection of the compound. Moreover, the well-known pathway of sorgoleone synthesis and characteristic of candidate genes may be a promising source of introducing sorgoleone production to grass crops [94].

The situation becomes more complicated when more than one gene encoding special enzymes is required to increase synthesis of a plant compound. Such difficulties have been encountered for DIBOA, synthesized by various grass species [95]. In maize, biosynthesis of this compound is determined by five genes (*Bx1* to *Bx5*) encoding three enzymes: tryptophan synthase α homolog, cytochrome P-450-dependent monooxygenase [95].

Monoterpenes are a large family of compounds produced by a varied family of aromatic plants. Some of the monoterpenes also take part in allelopathic interactions, e.g. linalool, cineole camphene, pinene, limonene, etc. Currently, metabolic engineering allows im‐ proved production of specific compounds in heterologous systems [96]. The most interest‐ ing are monoterpene synthases which catalyzed geranyl diphosphate (GPP) into output structure of numerous monoterpenes family, e.g. enhanced expression of limonene syn‐ thase in transgenic peppermint (*Mentha piperita* L.) has increased yield of monoterpenes. An alternative approach is to change the density of secretory structures by both plant hormone and transcriptional factors manipulation. Such attempts already have been made in annual wormwood and *A*. *thaliana*. It was recently found that the number of glandular trichomes increased in response to jasmonic acid. Spraying of annual worm‐ wood with this hormone significantly increased density of these structures on leaves what was accompanied with higher artemisinin content [51]. This was an effect of en‐ hanced expression of gene encoding enzymes taking part in artemisinin biosynthesis. On the other hand, in Arabidopsis, co-expression of two positive transcriptional factors (GL1, and R protein of maize) has significantly improved the number of trichomes [96].

However, we have to bear in mind that biosynthesis of natural compounds can be limited to organs, tissues or even cells. Specific locations of compound synthesis, accumulation or secretion often make that compound toxic to other tissues within the same plant organism. Moreover, even successful transformation of a plant does not guarantee successful and sufficient production of a desirable compound. The gene of (S)-linalool synthase (*Lis*) of fairy fans (*Clarkia breweri* Gray), constitutively expressed in transgenic petunia (*Petunia hybrida* Hook.), has produced linalool but in its glycosylated, non-volatile form [96].

All presented techniques provide greater knowledge on allelopathy. However, better under‐ standing of such complex interactions among this phenomenon bring us one step forward to development of new strategies in weed management and finding new herbicides and new herbicidal target sites.

### **7. Conclusions**

phenolic compounds of nettleleaf goosefoot (*Chenopodium murale* Linn.) [81] and gossypol of cotton [82]. Active growth of roots and rapid colonization of the bioreactor allows rapidly reaching target weight, necessary to obtain an adequate quantity of the compound extracted

The recombinant DNA technology can be useful to improve allelochemical production. Enhancing or suppression of gene expression, metabolic engineering and genetic transforma‐ tion are promising new tools for allelochemical synthesis [79]. This approach is based on elucidation of the metabolic pathway, enzyme activities and identification of genes encoding

Allelopathy is a quantitative trait. A genetic analysis of quantitative trait loci (QTL) is a promising approach to identify genes underlying this trait. Only a few crops are under genetic screening for its allelopathic properties including: rice, wheat, barley and oat [83, 84]. The first QTL map associated with allelopathic properties was developed in rice. A segregating population derived from a cross of two cultivars varying with allelopathic potential against barnyardgrass. The map contained 140 DNA markers with four main-effects QTL located on chromosome 2, 3 and 8 [85]. Proteomic studies on allelopathy of rice against barnyardgrass confirmed the crucial role of three enzymes: phenylalanine ammonia-lyse (PAL), thioredoxin and 3-hydroxy-3-methilglutarilcoenzyme A reductase 3 (HMGR) is highly involved in phenols biosynthesis [86]. Such a genetic approach may allow the location of the gene in the genome and better understanding of its function in plant allelopathy and create the chance of applying

Just like breeding programs allow improved crop production, they may also improve pro‐

Scopoletin has been known as allelopathic root exudates of oats (*Avena* sp.) that affects growth of neighboring plants. Screening of 3000 of Avena accessions has shown varying ability to scopoletin production. Twenty five of them have exuded higher amounts of scopoletin than control cultivar Garry, of which 4 were threefold more than the control [87]. Variation in allelopathin production was also discovered for sorgoleone of seven sorghum accessions [38] nomilacton A and B of 8 rice accessions [88] DIBOA and DIMBOA of 14 rye cultivars [88], gramine of 43 lines of modern cultivar of barley (*Hordeum vulgare* L.) and wild progenitor *H. spontaneum* (C. Koch) [90]. Enhanced production of active compounds from allelopathic plants can be developed by efficient breeding - selection of individuals with high allelopathic ability. Identification of a single gene, arranged in synthesis of allelopathin already has been per‐ formed for sorgoleone. *SOR1* (or compatible *SbDES3*) expression is specific for root hairs of two species of sorghum (*S. bicolor* and *S. halepense*) and associated with sorgoleone synthesis, while it is not expressed in other organs of sorghum *SOR1* encodes novel fatty acid desaturase (FAD), involved in the formation of a specific bond at 16:3Δ9,12,15 pattern [91, 92]. Comparative studies of FAD derived from sorghum with other desaturases showed high similarity to omega-3 fatty acid desaturases (FAD3) [93]. However, none of the hitherto known desaturases can synthesize double bonding at this unique pattern along the aliphathic chain of the sorgoleone molecule. Characterization of this gene allows an overexpression of *SOR1* and increased sorgoleone synthesis and improved allelopathic potential of sorghum, as well as

crucial enzymes, associated with metabolite (allelochemical) synthesis.

marker assisted selection (MAS) to enhance allelopathic abilities.

duction of allelopathic compounds increasing allelopathic potential.

from plants or growing medium.

532 Herbicides - Current Research and Case Studies in Use

The phenomena of allelopathy and phytotoxic interactions between plants are strongly expanding branches of biological science. Allelochemicals, as a group of substances also called biocommunicators, seem to be a fruitful challenge for combining traditional agricultural practices and new approaches in pest management strategies. Allelochemicals have already been used to defend crops against pathogens, insects or nematodes, parallel to some attempts to use them for weed control. Crop rotation, cover crops, dead and living mulches are being employed in agriculture. Both in natural and agricultural ecosystems allelopathic interactions are involved in practically every aspect of plant growth, as they can play the role of stimulants and suppressants. Complex plant-plant and plant-microbe interactions in ecosystems and currently developing studies on molecular, cytological and physiological levels bring us to a better understanding of processes occurring around us. The ancient knowledge of well-known toxic properties of water extracts of a variety of allelopathic plants give us a basis that could be used in the creation of a novel approach in weed control.

**References**

[1] Rola, H, Marczewska, K, & Kucharski, M. Zjawisko odporności chwastów na herbi‐

Allelochemicals as Bioherbicides — Present and Perspectives

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[2] International Survey of Herbicide Resistance Weedshttp://www.weedscience.org/

[3] Dayan, F. E, Cantrell, C. L, & Duke, S. O. Natural products in crop protection. Bioor‐

[4] Gniazdowska, A, & Bogatek, R. Alleopathic interaction between plants. Multiside ac‐

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[8] Hussain, M. I, & Reigosa, M. J. Allelochemical stress inhibits growth, leaf water rela‐ tions, PSII photochemistry, non-photochemical fluorescence quenching, and heat en‐ ergy dissipation in three C3 perennial species. Journal of Experimental Botany

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Some allelochemicals, mainly these that are mentioned in the text above, may act as a starting point for production of new bioherbicides with novel target sites, not previously exploited, as the understanding of their mode of action is still growing. Creation of bio‐ herbicides based on allelochemicals generates the opportunity to exploit natural com‐ pounds in plant protection and shows the possibility to cope with evolved weed resistance to herbicides. Despite the fact that we have extensive knowledge about the chemical nature of natural compounds, we can synthesize its analogues, and we have ba‐ sically explored its phytotoxic potential, we still have insufficient data. Until recently, most studies on phytotoxicity have been conducted under laboratory conditions due to the ability to eliminate other environmental factors such us temperature, soil texture and its chemical and physical properties. Such approach allows the recognition of only direct effects of allelochemical action. There is still a great need to transfer laboratory data into field conditions. Such experiments are not willing to be taken on due to troublesome field experiments dependent on environmental conditions and a few year repetitions. New tools of molecular genetics, proteomics and metabolomics profiling as well as modern and sophisticated methods of chemistry and biochemistry will lead to the creation of sub‐ stances, maybe based on the structure of particular compounds occurring in nature, which could be used without any risks as selective and eco-friendly herbicides.

### **Author details**

Dorota Soltys1\*, Urszula Krasuska1 , Renata Bogatek2 and Agnieszka Gniazdowska2

\*Address all correspondence to: d.soltys@ihar.edu.pl

1 Laboratory of Biotechnology, Plant Breeding and Acclimatization Institute - National Re‐ search Institute, Mlochow, Poland

2 Department of Plant Physiology, Warsaw University of Life Sciences – SGGW, Warsaw, Poland

### **References**

biocommunicators, seem to be a fruitful challenge for combining traditional agricultural practices and new approaches in pest management strategies. Allelochemicals have already been used to defend crops against pathogens, insects or nematodes, parallel to some attempts to use them for weed control. Crop rotation, cover crops, dead and living mulches are being employed in agriculture. Both in natural and agricultural ecosystems allelopathic interactions are involved in practically every aspect of plant growth, as they can play the role of stimulants and suppressants. Complex plant-plant and plant-microbe interactions in ecosystems and currently developing studies on molecular, cytological and physiological levels bring us to a better understanding of processes occurring around us. The ancient knowledge of well-known toxic properties of water extracts of a variety of allelopathic plants give us a basis that could

Some allelochemicals, mainly these that are mentioned in the text above, may act as a starting point for production of new bioherbicides with novel target sites, not previously exploited, as the understanding of their mode of action is still growing. Creation of bio‐ herbicides based on allelochemicals generates the opportunity to exploit natural com‐ pounds in plant protection and shows the possibility to cope with evolved weed resistance to herbicides. Despite the fact that we have extensive knowledge about the chemical nature of natural compounds, we can synthesize its analogues, and we have ba‐ sically explored its phytotoxic potential, we still have insufficient data. Until recently, most studies on phytotoxicity have been conducted under laboratory conditions due to the ability to eliminate other environmental factors such us temperature, soil texture and its chemical and physical properties. Such approach allows the recognition of only direct effects of allelochemical action. There is still a great need to transfer laboratory data into field conditions. Such experiments are not willing to be taken on due to troublesome field experiments dependent on environmental conditions and a few year repetitions. New tools of molecular genetics, proteomics and metabolomics profiling as well as modern and sophisticated methods of chemistry and biochemistry will lead to the creation of sub‐ stances, maybe based on the structure of particular compounds occurring in nature,

which could be used without any risks as selective and eco-friendly herbicides.

, Renata Bogatek2

1 Laboratory of Biotechnology, Plant Breeding and Acclimatization Institute - National Re‐

2 Department of Plant Physiology, Warsaw University of Life Sciences – SGGW, Warsaw,

and Agnieszka Gniazdowska2

**Author details**

Poland

Dorota Soltys1\*, Urszula Krasuska1

search Institute, Mlochow, Poland

\*Address all correspondence to: d.soltys@ihar.edu.pl

be used in the creation of a novel approach in weed control.

534 Herbicides - Current Research and Case Studies in Use


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[51] Nguyen, K. T, Arseault, P. R, & Wethers, P. J. Trichomes + roots + ROS = artemisinin: regulating artemisinin biosynthesis in *Artemisia annua* L. In Vitro Cellular and Devel‐

[52] Jessing, K. K, Cedergreen, N, Mayer, P, Libous-bailey, L, Strobel, B. W, Rimando, A, & Duke, S. O. Loss of artemisinin produced by *Artemisia annua* L. to the soil environ‐

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[43] Hejl, A. M, & Koster, K. L. The allelochemical sorgoleone inhibits root H+

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[82] Triplett, B. A, Moss, S. C, Bland, J. M, & Dowd, M. K. Induction of hairy root cultures from *Gossypium hirsutum* and *Gossypium barbadense* to produce gossypol and related compounds. In Vitro Cellular & Developmental Biology- Plant (2008). , 44(6),

[83] Olofsdotter, M, Jensen, L. B, & Courtois, B. Improving crop competitive ability using

[84] Belz, R. G. Allelopathy in crop/weed interactions- an update. Pest Management Sci‐

[85] Jensen, L. B, Courtois, B, Shen, L, Li, Z, Olofsdotter, M, & Mauleon, R. P. Locating genes controlling allelopathic effects against *Echinochloa crus-galli* (L.) in upland rice.

[86] Lin, W-X, He, H-Q, Shen, L-H, Chen, X-X, Ke, Y, Guo, Y-C, & He, H-B. A proteomic approach to analysing rice allelopathy on barnyard grass (*Echinochloa crus-galli* L.). 4th International Crop Science Congress 26.(2004). Queensland, Australia. http://

[87] Fay, P. K, & Duke, W. B. An assessment of allelopathic potential in *Avena* germ‐

[88] Kato-noguchi, H. Allelopathic substance in rice root exudates: rediscovery of momi‐ lactone B as an allelochemical. Journal of Plant Physiology (2004). , 161(3), 271-276.

[89] Copaja, S. V, Villarroel, E, Bravo, H. R, Pizarro, L, & Argandona, V. H. Hydroxamic acids in *Secale cereale* L. and the relationship with their antifeedant and allelopathic properties. Zeitschrift fuer Naturforschung Section C Journal of Biosciences (2006). ,

[90] Lovett, J. V. Hoult AHC. (1992). Gramine: the occurrence of a self defence chemical in barley, *Hordeum vulgare* L. In: Hutchinson KJ, Vickery PJ. (eds) Looking Back- Plan‐ ning Ahead conference proceedings, February Australian Agronomy Conference. "". Edited by Proceedings of the 6th Australian Agronomy Conference, 1992, The Uni‐ versity of New England, Armidale, New South Wales. http:// www.regional.org.au/au/asa/1992/concurrent/alternative-practices-plant-protection/

[91] Pan, Z, Rimando, A. M, Baerson, S. R, Fishbein, M, & Duke, S. O. Functional charac‐ terization of desaturases involved in the formation of the terminal double bond of an unusual 16:3Δ 9,12,15 fatty acid isolated from *Sorghum bicolor* root hairs. Journal of

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allelopathy- an example from rice. Plant Breeding (2002). , 121(1), 1-9.

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kovic, S. Use of *Chenopodium murale* L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Journal of Plant Physiology (2012). , 169(12), 1203-1211.

[82] Triplett, B. A, Moss, S. C, Bland, J. M, & Dowd, M. K. Induction of hairy root cultures from *Gossypium hirsutum* and *Gossypium barbadense* to produce gossypol and related compounds. In Vitro Cellular & Developmental Biology- Plant (2008). , 44(6), 508-517.

[67] Halligan, J. P. Toxic terpenes from *Artemisia californica*. Ecology (1975). , 56(4),

[68] Heisey, R. M, & Delwiche, C. C. Phytotoxic volatiles from *Trichostema lanceolatum*.

[69] Grayson, B. T, Williams, K. S, Freehauf, P. A, Pease, R. R, Ziesel, W. T, Sereno, R. L, & Reinsfelder, R. E. The physical and chemical properties of the herbicide cinmethylin.

[70] Vaughn, S. F, & Spencer, G. F. Synthesis and herbicidal activity of modified monoter‐ penes structurally similar to cinmethylin. Weed Science (1996). , 44(1), 7-11.

[71] Russell, S. G, Monaco, T. J, & Weber, J. B. Influence of soil moisture on phytotoxicity

[72] Baum, S. F, Karanastasis, L, & Rost, T. L. Morphogenetic effect of the herbicide Cinch on Arabidopsis thaliana root development. Journal of Plant Growth Regulation

[73] Duke, S. O. Allelopathy: Current status of research and the future of the discipline: A

[74] Huang, H, Morgan, C. M, Asolkar, N. R, Koivunen, M. E, & Marrone, P. G. Phytotox‐ icity of sarmentine isolated from long pepper (*Piper longum*) fruit. Journal of Agricul‐

[75] Huang, H, & Asolkar, N. R. (2011). Use of sarmentine and its analogs for controlling plant pests. Patent Patentdocs: http://www.faqs.org/patents/accessed 27 January

[76] Fukuda, M, Tsujino, Y, Fujimori, T, Wakabayashi, K, & Böger, P. Phytotoxic activity of middle-chain fatty acids I: effects on cell constituents. Pesticide Biochemistry and

[77] Lederer, B, Fujimori, T, Tsujino, Y, Wakabayashi, K, & Böger, P. Phytotoxic activity of middle-chain fatty acids II: peroxidation and membrane effects. Pesticide Bio‐

[78] Macías, F. A, Molinillo, J. M, Varela, R. M, & Galindo, J. C. Allelopathy--a natural al‐

[79] Bourgaud, F, Gravot, A, Milesi, S, & Gontier, E. Production of plant secondary me‐

[80] Durante, M, Caretto, S, Quarta, A, De Paolis, A, Nisi, R, & Mita, G. Cyclodextrins en‐ hance artemisinin production in *Artemisia annua* suspension cell cultures. Applied

[81] Mitic, N, Damitrovic, S, Djordjevic, M, Zdravkovic-korac, S, Nikolic, R, Raspora, M, Djordjevic, T, Maksimovic, V, Živkovic, S, Krstic-miloševic, D, Stanišic, M, & Nin‐

ternative for weed control. *Pest Management Science* (2007). , 63(4), 327-48.

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of cinmethylin to various crops. Weed Science (1991). , 39(3), 402-407.

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[92] Yang, X, Scheffler, B. E, & Weston, L. A. SOR1, a gene associated with bioherbicide production in sorghum root hairs. Journal of Experimental Botany (2004). , 55-2251.

**Chapter 21**

**Managing Commelina Species: Prospects and**

Commelina species, notably *C. communis* L, *C. diffusa* Burm, *C. elegans* Kunth. and *C. bengha‐ lensis* L. as well as their biotypes, are perennial herbs of Neotropical origin which now have a pantropical distribution. Members of this family (Commelindeae: Commelinaceae) are common throughout the Caribbean, North and Latin America, Africa, Asia, the Middle East and parts of Oceania [18, 27, 28, 63, 64]. There are 500 - 600 species reported in the family Commelinaceae [50]. Recent data indicates that the Commelinaceae family contains 23 genera and at least 225 species native to or naturalized in the New World and 23 genera and about 200 species in the Neotropics [41] and also website reports of 50 genera and 700 species [16, 31]. There are 170 species of Commelina in the warmer regions of the world and 50 species of Murdannia occurring in the tropics and warm temperate regions worldwide with Tropical

Wilson [84] presented a comprehensive review on Commelina species and its management with emphasis on chemical weed control in 1981. Since Wilson's review much has been written about the weedy members of this family, notably Commelina species [84]. Indeed, the CAB ABSTRACTS Database contains well over 1200 references on Commelinaceae from 1981 to the present. *Commelina benghalensis* in particular has been the most reported species with several reports of research conducted on its control in southern states of the United States of America (USA) including Alabama, Florida, Georgia, Louisiana and North Carolina [18, 74, 75, 78-81]. Many of these studies should be consulted for basic details of the biology and ecology. The National American Plant Protection Organization (NAPPO) offers a comprehensive global

The current review is an attempt to provide an update on the status of the weedy Commelina species in agricultural production systems. This review is based on world literature over the

> © 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.

Wendy-Ann Isaac, Zongjun Gao and Mei Li

Additional information is available at the end of the chapter

**Limitations**

**1. Introduction**

http://dx.doi.org/10.5772/55842

Asia having the greatest diversity [17].

distribution list of this weed species [47].


## **Managing Commelina Species: Prospects and Limitations**

Wendy-Ann Isaac, Zongjun Gao and Mei Li

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55842

### **1. Introduction**

[92] Yang, X, Scheffler, B. E, & Weston, L. A. SOR1, a gene associated with bioherbicide production in sorghum root hairs. Journal of Experimental Botany (2004). , 55-2251.

[93] Yang, X, Owens, T. G, Scheffler, B. E, & Weston, L. A. Manipulation of root hair de‐ velopment and sorgoleone production in sorghum seedlings. Journal of Chemical

[94] Weston, L. A, & Duke, S. O. Weed and crop allelopathy. Critical Reviews in Plant Sci‐

[95] Frey, M, Chomet, P, Glawischnig, E, Stettner, C, Grün, S, Winklmair, A, Wolfgang, E, Bacher, A, Meeley, R. B, Briggs, S. P, Simcox, K, & Gierl, A. Analysis of a chemical

[96] Mahmoud, S. S, & Croteau, R. B. Strategies for transgenic manipulation of monoter‐ pene biosynthesis in plants. Trends in Plant Science (2002). , 7(8), 366-373.

plant defense mechanism in grasses. Science (1997). , 277(3526), 696-699.

Ecology (2004). , 30(1), 199-213.

542 Herbicides - Current Research and Case Studies in Use

ences (2003).

Commelina species, notably *C. communis* L, *C. diffusa* Burm, *C. elegans* Kunth. and *C. bengha‐ lensis* L. as well as their biotypes, are perennial herbs of Neotropical origin which now have a pantropical distribution. Members of this family (Commelindeae: Commelinaceae) are common throughout the Caribbean, North and Latin America, Africa, Asia, the Middle East and parts of Oceania [18, 27, 28, 63, 64]. There are 500 - 600 species reported in the family Commelinaceae [50]. Recent data indicates that the Commelinaceae family contains 23 genera and at least 225 species native to or naturalized in the New World and 23 genera and about 200 species in the Neotropics [41] and also website reports of 50 genera and 700 species [16, 31]. There are 170 species of Commelina in the warmer regions of the world and 50 species of Murdannia occurring in the tropics and warm temperate regions worldwide with Tropical Asia having the greatest diversity [17].

Wilson [84] presented a comprehensive review on Commelina species and its management with emphasis on chemical weed control in 1981. Since Wilson's review much has been written about the weedy members of this family, notably Commelina species [84]. Indeed, the CAB ABSTRACTS Database contains well over 1200 references on Commelinaceae from 1981 to the present. *Commelina benghalensis* in particular has been the most reported species with several reports of research conducted on its control in southern states of the United States of America (USA) including Alabama, Florida, Georgia, Louisiana and North Carolina [18, 74, 75, 78-81]. Many of these studies should be consulted for basic details of the biology and ecology. The National American Plant Protection Organization (NAPPO) offers a comprehensive global distribution list of this weed species [47].

The current review is an attempt to provide an update on the status of the weedy Commelina species in agricultural production systems. This review is based on world literature over the

© 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.

last 45 years and considers major Commelina species found in the tropics and warm temperate regions in relation to their status, distribution, biology and spread and management.

persistent. They are both annuals and perennials and therefore dominate the fallow vegetation because they are most competitive due to their growth and regeneration characteristics [72].

The plant is propagated mainly by seeds, stem cuttings and rooting from nodes and pieces [19, 46, 74, 75]. Plants may arise asexually when buds grow into autonomous, adventitiously erect leafy shoots, which later become separated from each other [12]. Occasionally the buds may sprout and grow into erect shoots directly without undergoing a period of inactivity [12]. The plant produces roots readily at the nodes of the creeping stems and will do so especially when broken or cut [27, 28]. Farmers in the Windward Islands report that Commelina species may

The stems of Commelina species have a high moisture content and once it is well rooted the plant can survive for long periods without moisture [84]. This fact is evident in young banana plantations in the Windward Islands where stems become dried and shrivelled due to the direct contact with solar radiation particularly in the dry season. However, at the onset of rains and when the canopy of the banana closes, stems regain moisture, re-establish and rapidly

The mature aerial seeds of *C. benghalensis* are produced within 14 to 22 days after flower opening [74] and in some instances, e.g., the rice paddies of the Philippines, can produce in excess of 1,600 seeds/plant [53] or even 12,000 seeds/m2 [74], whereas seeds grown from

Commelina species has gained noxious weed status in the Windward Islands because of several factors. Firstly, the fact that the weed was encouraged as a groundcover was com‐ pounded by inappropriate agricultural practices, notably irrational herbicide use which farmers have relied on for decades. The non-judicious use of herbicides has created imbalances and disturbances within the ecosystem in these Islands causing resistant biotypes. Secondly, the move within recent years by banana growers to adopt a Fairtrade system which uses no herbicides has catapulted the spread to an all-time high in the Windward Islands. Farmers have been forced to rely on the use of the cutlass or weed whacker as the only alternative strategies which have further intensified the problem by spreading plant propagules [30]. Most importantly these Islands which are characterized by hilly landscapes have ideal moist conditions for the proliferation of Commelina species. Finally, many of the banana plantations have been farmed for several years with virtually no crop rotations or tillage practices and this

[74]. In cultivated areas the plant

Managing Commelina Species: Prospects and Limitations

http://dx.doi.org/10.5772/55842

545

be intensified when cut with a weed whacker as stolons spread more extensively.

is spread by irrigation water and waterways. Animals may also spread the seeds.

has further contributed to the stabilization of Commelina species populations.

In the USA, its sudden emergence as a noxious weed is attributed to crop production practices which are well suited for prolific weed growth such as minimum – tillage production (which is undertaken in conjunction with the use of glyphosate – resistant crops) and extreme tolerance to glyphosate [79-81]. The weed appears to be well-suited for high input agricultural produc‐ tion where high levels of fertilizers, irrigation and herbicides are used [79, 80]. The spread of *C. benghalensis* is attributed, in part, to the adoption of weed management programmes that lack the use of residual herbicides along with the adoption of reduced-tillage production practices [54]. Additionally, after introduction, invasive species often go long periods of time

begin to spread by runners which root at the nodes.

underground seeds are capable of producing 8,000 seeds/m2

### **2. Weed Status**

*Commelina benghalensis* (Tropical spiderwort or Benghal dayflower) has become increasingly important, gaining pest significance in agronomic production systems in the southeastern coastal plain of the United States of America (USA) in crops such as cotton (*Gossypium* spp.) and peanut (*Arachis hypogea*) [70, 71] and in the North China Plain in crops such as potato (*Solanum tuberosum*) and summer corn (*Zea mays*) [37, 71, 72, Li et al. unpublished data 2007). It is commonly associated with wet locations. This weed was in fact listed as a Federal Noxious weed in Florida and Georgia where it is the most troublesome weed in cotton and a pest in peanut, corn (*Zea mays*), soybean (*Glycine max*), nursery stock and orchards [81]. This species which was first observed in USA in 1928 [18] gained noxious weed status in 1983 [81]. Between 1998 to 2001 and then to 2004 this weed which was ranked among the top 39 most troublesome weeds across all crops by Georgia extension agents (in 1998) moved to the 9th most trouble‐ some (in 2001) to the most troublesome cotton weed in Georgia (in 2003) [77] and Florida (2004) and the 3rd most troublesome weed of peanut in several south Georgia counties [54, 80]. In Georgia alone the weed is estimated to infest more than 80,000 ha [80-82] with a confirmed presence in 29 Georgia counties [54]. It is also observed throughout the panhandle and central Florida and listed by the United States Department of Agriculture (USDA) as appearing in more than 12 Florida counties [82].

*Commelina communis* has become one of the three most troublesome weeds in soybean fields in the Northeast China, and has caused significant reduction in production and quality of soybean [42]. Commelina species, namely *C. diffusa* and *elegans,* were reported as the 3rd most troublesome weed in the Caribbean where they are a serious problem of banana and other crops in the Windward Islands of Dominica, Grenada, St. Lucia and St. Vincent and the Grenadines [24]. Presently, Commelina species, commonly called watergrass, caner grass, pond grass, spiderwort, spreading dayflower, wandering Jew or French weed in these Islands, are by far the most serious in these countries. *Commelina diffusa* was once encouraged as a ground cover to reduce soil erosion [13] and has been identified as the host of the reniformis nematode *Rotylenchulus reniformis* [57], the banana lesion nematode *Pratylenchus goodeyi* [87] and recent data have confirmed its association with the burrowing nematode *Radopholus similis* [55]. These nematodes all contribute to significant reductions in banana production particularly *R. similis*, which may reduce banana production by more than 50 % and decrease the production duration of banana fields [55].

### **3. Biology and spread**

Commelina species are C-3, monocotyledonous plants and therefore have a high efficiency of CO2 uptake at low irradiance [34]; therefore, they tolerate shade very well and could become persistent. They are both annuals and perennials and therefore dominate the fallow vegetation because they are most competitive due to their growth and regeneration characteristics [72].

last 45 years and considers major Commelina species found in the tropics and warm temperate

*Commelina benghalensis* (Tropical spiderwort or Benghal dayflower) has become increasingly important, gaining pest significance in agronomic production systems in the southeastern coastal plain of the United States of America (USA) in crops such as cotton (*Gossypium* spp.) and peanut (*Arachis hypogea*) [70, 71] and in the North China Plain in crops such as potato (*Solanum tuberosum*) and summer corn (*Zea mays*) [37, 71, 72, Li et al. unpublished data 2007). It is commonly associated with wet locations. This weed was in fact listed as a Federal Noxious weed in Florida and Georgia where it is the most troublesome weed in cotton and a pest in peanut, corn (*Zea mays*), soybean (*Glycine max*), nursery stock and orchards [81]. This species which was first observed in USA in 1928 [18] gained noxious weed status in 1983 [81]. Between 1998 to 2001 and then to 2004 this weed which was ranked among the top 39 most troublesome weeds across all crops by Georgia extension agents (in 1998) moved to the 9th most trouble‐ some (in 2001) to the most troublesome cotton weed in Georgia (in 2003) [77] and Florida (2004) and the 3rd most troublesome weed of peanut in several south Georgia counties [54, 80]. In Georgia alone the weed is estimated to infest more than 80,000 ha [80-82] with a confirmed presence in 29 Georgia counties [54]. It is also observed throughout the panhandle and central Florida and listed by the United States Department of Agriculture (USDA) as appearing in

*Commelina communis* has become one of the three most troublesome weeds in soybean fields in the Northeast China, and has caused significant reduction in production and quality of soybean [42]. Commelina species, namely *C. diffusa* and *elegans,* were reported as the 3rd most troublesome weed in the Caribbean where they are a serious problem of banana and other crops in the Windward Islands of Dominica, Grenada, St. Lucia and St. Vincent and the Grenadines [24]. Presently, Commelina species, commonly called watergrass, caner grass, pond grass, spiderwort, spreading dayflower, wandering Jew or French weed in these Islands, are by far the most serious in these countries. *Commelina diffusa* was once encouraged as a ground cover to reduce soil erosion [13] and has been identified as the host of the reniformis nematode *Rotylenchulus reniformis* [57], the banana lesion nematode *Pratylenchus goodeyi* [87] and recent data have confirmed its association with the burrowing nematode *Radopholus similis* [55]. These nematodes all contribute to significant reductions in banana production particularly *R. similis*, which may reduce banana production by more than 50 % and decrease

Commelina species are C-3, monocotyledonous plants and therefore have a high efficiency of CO2 uptake at low irradiance [34]; therefore, they tolerate shade very well and could become

regions in relation to their status, distribution, biology and spread and management.

**2. Weed Status**

544 Herbicides - Current Research and Case Studies in Use

more than 12 Florida counties [82].

the production duration of banana fields [55].

**3. Biology and spread**

The plant is propagated mainly by seeds, stem cuttings and rooting from nodes and pieces [19, 46, 74, 75]. Plants may arise asexually when buds grow into autonomous, adventitiously erect leafy shoots, which later become separated from each other [12]. Occasionally the buds may sprout and grow into erect shoots directly without undergoing a period of inactivity [12]. The plant produces roots readily at the nodes of the creeping stems and will do so especially when broken or cut [27, 28]. Farmers in the Windward Islands report that Commelina species may be intensified when cut with a weed whacker as stolons spread more extensively.

The stems of Commelina species have a high moisture content and once it is well rooted the plant can survive for long periods without moisture [84]. This fact is evident in young banana plantations in the Windward Islands where stems become dried and shrivelled due to the direct contact with solar radiation particularly in the dry season. However, at the onset of rains and when the canopy of the banana closes, stems regain moisture, re-establish and rapidly begin to spread by runners which root at the nodes.

The mature aerial seeds of *C. benghalensis* are produced within 14 to 22 days after flower opening [74] and in some instances, e.g., the rice paddies of the Philippines, can produce in excess of 1,600 seeds/plant [53] or even 12,000 seeds/m2 [74], whereas seeds grown from underground seeds are capable of producing 8,000 seeds/m2 [74]. In cultivated areas the plant is spread by irrigation water and waterways. Animals may also spread the seeds.

Commelina species has gained noxious weed status in the Windward Islands because of several factors. Firstly, the fact that the weed was encouraged as a groundcover was com‐ pounded by inappropriate agricultural practices, notably irrational herbicide use which farmers have relied on for decades. The non-judicious use of herbicides has created imbalances and disturbances within the ecosystem in these Islands causing resistant biotypes. Secondly, the move within recent years by banana growers to adopt a Fairtrade system which uses no herbicides has catapulted the spread to an all-time high in the Windward Islands. Farmers have been forced to rely on the use of the cutlass or weed whacker as the only alternative strategies which have further intensified the problem by spreading plant propagules [30]. Most importantly these Islands which are characterized by hilly landscapes have ideal moist conditions for the proliferation of Commelina species. Finally, many of the banana plantations have been farmed for several years with virtually no crop rotations or tillage practices and this has further contributed to the stabilization of Commelina species populations.

In the USA, its sudden emergence as a noxious weed is attributed to crop production practices which are well suited for prolific weed growth such as minimum – tillage production (which is undertaken in conjunction with the use of glyphosate – resistant crops) and extreme tolerance to glyphosate [79-81]. The weed appears to be well-suited for high input agricultural produc‐ tion where high levels of fertilizers, irrigation and herbicides are used [79, 80]. The spread of *C. benghalensis* is attributed, in part, to the adoption of weed management programmes that lack the use of residual herbicides along with the adoption of reduced-tillage production practices [54]. Additionally, after introduction, invasive species often go long periods of time (lag period) during which the pest increases in distribution or density without being noticed as an obvious pest [54].

periods of 15 to 88 and 22 to 38 days after coffee seedling sowing under winter and summer conditions, respectively [11]. In cotton it was found that yield loss from *C. benghalensis* can be minimized by planting cotton early in the growing season, prior to substantial emergence of

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547

*Commelina diffusa* is an alternate host plant for the nematodes *Rotylenchulus reniformis*, *Helicotylenchus* spp., *Pratylenchus* spp., *Meloidogyne* sp. and *Radopholus similis* in banana [13, 27, 29, 44, 55, 57, 60, 87] and coffee [58]. The plant is also a collateral host of *Helicotylenchus dihystera* infecting guava fields [35]. *Commelina benghalensis* has also been identified as an alternate host of the southern root-knot nematode (*Meloidogyne incognita*) [55]. The southern root-knot nematode is widely distributed across cotton regions in Georgia [54]. Snails and slugs

Five viruses have been found naturally infecting species of Commelinaceae. Aneilema a potyvirus has also been found infecting 15 species of the Commelinaceae family including 4 of Commelina. There have been reports of *Commelina diffusa* potyvirus, which causes a mosaic in *Commelina diffusa* and *C. benghalensis* [2]. The virus is transmitted by two insect vectors, *Aphis gossypi* and *Myzus persicae*; Aphididae. It is transmitted in a non – persistent manner. The virus is transmitted by mechanical inoculation and not by grafting or contact between plants or by seeds. The isolate for cucumber mosaic virus (CMV) is originally from *Comme‐ lina elegans* but it is transmitted by Aphis gossypi, and not *Myzus persicae. Commelina diffusa* is susceptible to Commelina X potexvirus, Commelina yellow mottle badnavirus, Spring beauty latent bromovirus, Tradescantia – *Zebrina potyvirus*, spotted wilt and Cherry leaf roll nepovirus [2]. However, *Commelina elegans* is insusceptible to Tradescantia – *Zebrina potyvirus*. U2- tobacco mosaic virus has also been found infecting *C. communis* and *Z. pendula*. Brome mosaic virus isolates have been identified [70] infecting *C. diffusa* and *C. communis* in

Wilson's review on the control of these weed species was directed towards finding suitable chemicals for their control in the early stages of growth, summarizing results of trials from difference parts of the world [84]. However, he suggested that since dense mats of plant material make chemical weed control of older plants difficult, removal by hand is the only

Currently, chemical control is still generally considered the only practical means of controlling large infestations of Commelina species [78-82]. However, no single method of control seems to be effective for control of Commelina spp. in any crop. The difficulty lies in its ability for regeneration after attempted management even by cultural, mechanical or chemical control.

feed on *C. diffusa* plants and these affect crops such as pineapple and soybean [84].

**5. Pests and diseases associated with commelina species**

the weed [81].

Fayetteveille, Arkansas, USA.

effective control at that stage [84].

**6. Methods of management in selected crops**

### **4. Economic impact in crop production**

Three species of the Commelinaceae family are considered to be major problem weeds in cropping systems where they have become persistent and difficult to manage [27]. *Commelina benghalensis* is the most important of the three and it occurs as a weed in 25 different crops in 28 countries [27]. This weed has gained high importance in peanut and cotton in the southern United States [78, 79]. *Commelina diffusa* occurs as a weed in 17 crops in 26 countries and *Murdannia nudiflora* occurs as a weed in 16 crops in 23 countries [27].

*Commelina diffusa* thrives on cultivated soils of cocoa (*Theobroma cacao*), citrus, root crops such as dasheen (*Colocasia esculenta*) that tolerate water, and it is also a major weed in sugarcane (*Saccharum officinarum*), upland rice (*Oryza sativa*), soybean (*Glycine max*), cassava (*Manihot esculenta*), corn (*Zea mays*), banana and plantain (*Musa* spp.) [27]. *Commelina benghalensis* has been reported as a principal weed in upland rice in India and the Philippines, tea (*Camellia sinensis*) in India, coffee (*Coffee arabica*) in Tanzania and Kenya, soybean in the Philippines and cotton and maize in Kenya [27, 47]. This species is common in rice in Sri Lanka, sugarcane in India, the Philippines and Mozambique, cassava in Taiwan and maize in Zimbabwe [9]. *Commelina benghalensis* was reported as a weed of jute (*Corchorus olitorius*), sisal (*Agave sisalana*), beans (*Phaseolus* spp.), pastures, sweet potatoes (*Ipomoea batatas*), vineyards and barley (*Hordeum vulgare*) and other cereals in many countries [7].

Because of Commelina's vigorous growth habit, which allows the plant to form dense pure stands, they may compete easily with low growing crops such as vegetables, pulses and cereals as well as pasture grasses and legumes by smothering them [27]. Because Commelina species is a broadleaved weed it is generally not considered highly competitive for nutrients however this fact is not well researched and its allelopathic potential also needs to be ascertained. Invasive species such as *C. benghalensis* had higher plant growth rate at high nutrient availa‐ bility and across water availability compared to a related non – invasive, but alien, congener, *C. bracteosa* Hassk. [6]. Interestingly, severe stunting has been reported in *C. diffusa* caused by high nitrogen [59] and altered growth and physiological characteristics for different *C. erecta* clones with increased phosphorus supply [71]. Results from systematic studies on the influence of *C. benghalensis* populations on crop yield are limited [54]. Increased reduction in aboveground and root dry matter as well as a 100% reduction in the number of leaves in lettuce (*Lactuca sativa*) plants were recorded with 1% and 3% hydro – alcoholic extracts of *C.bengha‐ lensis* suggesting its allelopathic potential [68].

Studies on the critical periods of interference in Commelina species are limited. Generally crops are affected most severely during the first 2 – 5 weeds of crop growth although mature plants can also be affected [7]. *Commelina benghalensis* in particular may affect crop growth and yield but this varies with environmental conditions [47]. Research aimed at evaluating the periods of interference of *C. benghalensis* in the initial growth of coffee seedlings reported prevention periods of 15 to 88 and 22 to 38 days after coffee seedling sowing under winter and summer conditions, respectively [11]. In cotton it was found that yield loss from *C. benghalensis* can be minimized by planting cotton early in the growing season, prior to substantial emergence of the weed [81].

### **5. Pests and diseases associated with commelina species**

(lag period) during which the pest increases in distribution or density without being noticed

Three species of the Commelinaceae family are considered to be major problem weeds in cropping systems where they have become persistent and difficult to manage [27]. *Commelina benghalensis* is the most important of the three and it occurs as a weed in 25 different crops in 28 countries [27]. This weed has gained high importance in peanut and cotton in the southern United States [78, 79]. *Commelina diffusa* occurs as a weed in 17 crops in 26 countries and

*Commelina diffusa* thrives on cultivated soils of cocoa (*Theobroma cacao*), citrus, root crops such as dasheen (*Colocasia esculenta*) that tolerate water, and it is also a major weed in sugarcane (*Saccharum officinarum*), upland rice (*Oryza sativa*), soybean (*Glycine max*), cassava (*Manihot esculenta*), corn (*Zea mays*), banana and plantain (*Musa* spp.) [27]. *Commelina benghalensis* has been reported as a principal weed in upland rice in India and the Philippines, tea (*Camellia sinensis*) in India, coffee (*Coffee arabica*) in Tanzania and Kenya, soybean in the Philippines and cotton and maize in Kenya [27, 47]. This species is common in rice in Sri Lanka, sugarcane in India, the Philippines and Mozambique, cassava in Taiwan and maize in Zimbabwe [9]. *Commelina benghalensis* was reported as a weed of jute (*Corchorus olitorius*), sisal (*Agave sisalana*), beans (*Phaseolus* spp.), pastures, sweet potatoes (*Ipomoea batatas*), vineyards and

Because of Commelina's vigorous growth habit, which allows the plant to form dense pure stands, they may compete easily with low growing crops such as vegetables, pulses and cereals as well as pasture grasses and legumes by smothering them [27]. Because Commelina species is a broadleaved weed it is generally not considered highly competitive for nutrients however this fact is not well researched and its allelopathic potential also needs to be ascertained. Invasive species such as *C. benghalensis* had higher plant growth rate at high nutrient availa‐ bility and across water availability compared to a related non – invasive, but alien, congener, *C. bracteosa* Hassk. [6]. Interestingly, severe stunting has been reported in *C. diffusa* caused by high nitrogen [59] and altered growth and physiological characteristics for different *C. erecta* clones with increased phosphorus supply [71]. Results from systematic studies on the influence of *C. benghalensis* populations on crop yield are limited [54]. Increased reduction in aboveground and root dry matter as well as a 100% reduction in the number of leaves in lettuce (*Lactuca sativa*) plants were recorded with 1% and 3% hydro – alcoholic extracts of *C.bengha‐*

Studies on the critical periods of interference in Commelina species are limited. Generally crops are affected most severely during the first 2 – 5 weeds of crop growth although mature plants can also be affected [7]. *Commelina benghalensis* in particular may affect crop growth and yield but this varies with environmental conditions [47]. Research aimed at evaluating the periods of interference of *C. benghalensis* in the initial growth of coffee seedlings reported prevention

as an obvious pest [54].

546 Herbicides - Current Research and Case Studies in Use

**4. Economic impact in crop production**

*Murdannia nudiflora* occurs as a weed in 16 crops in 23 countries [27].

barley (*Hordeum vulgare*) and other cereals in many countries [7].

*lensis* suggesting its allelopathic potential [68].

*Commelina diffusa* is an alternate host plant for the nematodes *Rotylenchulus reniformis*, *Helicotylenchus* spp., *Pratylenchus* spp., *Meloidogyne* sp. and *Radopholus similis* in banana [13, 27, 29, 44, 55, 57, 60, 87] and coffee [58]. The plant is also a collateral host of *Helicotylenchus dihystera* infecting guava fields [35]. *Commelina benghalensis* has also been identified as an alternate host of the southern root-knot nematode (*Meloidogyne incognita*) [55]. The southern root-knot nematode is widely distributed across cotton regions in Georgia [54]. Snails and slugs feed on *C. diffusa* plants and these affect crops such as pineapple and soybean [84].

Five viruses have been found naturally infecting species of Commelinaceae. Aneilema a potyvirus has also been found infecting 15 species of the Commelinaceae family including 4 of Commelina. There have been reports of *Commelina diffusa* potyvirus, which causes a mosaic in *Commelina diffusa* and *C. benghalensis* [2]. The virus is transmitted by two insect vectors, *Aphis gossypi* and *Myzus persicae*; Aphididae. It is transmitted in a non – persistent manner. The virus is transmitted by mechanical inoculation and not by grafting or contact between plants or by seeds. The isolate for cucumber mosaic virus (CMV) is originally from *Comme‐ lina elegans* but it is transmitted by Aphis gossypi, and not *Myzus persicae. Commelina diffusa* is susceptible to Commelina X potexvirus, Commelina yellow mottle badnavirus, Spring beauty latent bromovirus, Tradescantia – *Zebrina potyvirus*, spotted wilt and Cherry leaf roll nepovirus [2]. However, *Commelina elegans* is insusceptible to Tradescantia – *Zebrina potyvirus*. U2- tobacco mosaic virus has also been found infecting *C. communis* and *Z. pendula*. Brome mosaic virus isolates have been identified [70] infecting *C. diffusa* and *C. communis* in Fayetteveille, Arkansas, USA.

### **6. Methods of management in selected crops**

Wilson's review on the control of these weed species was directed towards finding suitable chemicals for their control in the early stages of growth, summarizing results of trials from difference parts of the world [84]. However, he suggested that since dense mats of plant material make chemical weed control of older plants difficult, removal by hand is the only effective control at that stage [84].

Currently, chemical control is still generally considered the only practical means of controlling large infestations of Commelina species [78-82]. However, no single method of control seems to be effective for control of Commelina spp. in any crop. The difficulty lies in its ability for regeneration after attempted management even by cultural, mechanical or chemical control. An Integrated Management Strategy (IWM) is therefore suggested for the best control of this weed species. A multi-component approach including an effective herbicide for successful management has been suggested [80-82].

Canopy SP® (metribuzin + chlorimuron) and Sencor® (metribuzin) and postemergence herbicides with fair to good activity such as Basagran®, Classic® (acetochlor) and Pursuit® (Imazethapyr). Gramoxone Max® and Aim® (acetochlor) can be used post-directed. In evaluating the effectiveness of several pre-emergence herbicides in suppressing *C. benghalen‐ sis* emergence, it was reported that s-metolachlor (at 1.07 and 1.60 kg a.i./ha), clomazone (at 0.42 and 1.05 kg a.i./ha) and flumetron (at 1.68 kg a.i./ha) provided ≥ 80% control at 6 weeks after treatment (WAT) in cotton [80]. It was stressed that the application of herbicides with soil

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In the Windward Islands, farmers started using paraquat around 1989 and noticed that it was ineffective. In an interview on August 10, 2002, Paddy Thomas, an experienced banana grower and pesticide salesman in St. Vincent and the Grenadines revealed that farmers started using gramocil (paraquat + diuron) at high doses for example and this too was not effective and resistance in Commelina spp. began to show. He also stated that Reglone, Round – up and Talent (paraquat + asulam) have also been used with little success for the control of Commelina species in the Windward Islands. Glufosinate has since been promoted as an environmentally-

friendly option for the control of broad-leaved weeds including Commelina species.

1-2 leaf stage of *C. communis* [41].

Studies were conducted into the efficacy of glufosinate for weed control in coffee plantations and it was found that it did not effectively control Commelina spp. at a rate of 0.3 – 0.6 kg a.i. / ha, however, paracol and gardoprim suppressed this perennial weed better [50]. Fomasefen and lactofen have shown good potential for control of this broadleaf weed [10]. Glufosinate (240 g a.i./ha) and fomasefen (WIP 276 g a.i./ha) were used in St. Vincent and the Grenadines in Fairtrade banana fields to compare their efficacy in controlling *C. diffusa* [30]. They were both applied at the early post-emergence, 3-5 leaf stage with a backpack sprayer using a TJ-8002 fan-nozzle. Regrowth of *C. diffusa* and other weeds were observed 6 weeks after application with glufosinate, however, no regrowth was observed for up to 3 months with fomasefen. Fomasefen, however, caused damage by burning banana suckers and leaves (about 30%) of established banana plants [30]. Studies were conducted to evaluate the efficacy of several postemergence herbicides in controlling *C. communis* in soybean, the results showed that imaze‐ thapyr (150 g a.i./ha), cloransulam-methyl (31.5 g a.i./ha), fomesafen (375 g a.i./ha) and mixture (756 g a.i./ha) of fomesafen plus imazethapyr with clomazone provided > 80% control of this weed at 30 days after treatment (DAT) [36, 37, 65, 67]. The efficacy of imazethapyr (90 g a.i./ha) in controlling *C. communis* reduced with increased leaf stage, and the control levels at 15 DAT were 100% (at 1 leaf stage), 89.17% (at 2 leaf stage), 56.45% (at 3 leaf stage) and 52.71% (at 4 leaf stage), respectively [41]. Therefore, the optimal application time of imazethapyr was

To screen more suitable herbicides for control of *C. benghalensis* and *C. communis* and determine the level of weed control provided by a single application of selected post-emergence herbi‐ cides, greenhouse studies on the laboratory toxicity of 23 herbicides to these weeds were conducted in 2010 [21]. The results indicated that, as for *C. benghalensis*, mesotrione, lactofen, oxyfluorfen, clomazone and flumioxazin provide complete control (100%), oxadiazon, fomesafen, metribuzin, acifluorfen, isoproturon, MCPA-sodium, carfentrazone-ethyl, flurox‐ ypyr, fluoroglycofen-ethyl and bentazone are herbicides with excellent activity (90.0 - 100%

residual activity will be crucial for the management of *C. benghalensis* [80].

### **7. Chemical management**

Herbicides are not usually very effective against most Commelina species. The first verified resistance was registered in 1957, when *C. diffusa* biotypes were identified in the United States [26]. *Commelina elegans* has shown resistance to growth – regulator type herbicides [32]. Control using herbicides is, however, variable depending on the herbicide, accuracy of leaf coverage and environmental conditions [7]. Spraying with a selective or non – selective herbicide may work but repeated treatments are required for regrowth. Plants should not be under moisture stress when sprayed. Surfactants will improve penetration into the waxy-coated leaves.

Many standard herbicides have relatively low activity on species of Commelina [84]. These include 2,4-D, propanil, butachlor, trifluralin and pendimethalin. Treatment with 2,4-D or MCPA at the pre-emergent stage has been shown to be ineffective and although a reasonable kill of very young seedlings can be obtained, the plants develop a rapid resistance with age [32]. Particular biotypes are resistant to 2,4-D and they may be cross resistant to other Group O / 4 herbicides [83]. It has been found that one biotype of *C. diffusa* could withstand five times the dosage of a susceptible species [83].

In rice, bentazone, molinate, oxyfluorfen and bifenox are herbicides with good activity [7]. Post-emergent sequential treatments of propanil followed by nitrogen or of molinate followed by KN3 controlled *C. diffusa* in rice [61]. In soybean, bentazone and metribuzin are effective [7]. In corn, combination of bromoxynil and 2,4-D butylate produced a synergistic effect in postemergent control of 3-4 leaf stage *C. communis* [85]. In plantation crops such as banana, paraquat is not always effective but mixture with diuron is recommended [7]. Dinoseb has been found to kill seedlings as well as dalapon but paraquat is reported to be relatively ineffective [32]. Prodiamine has been reported to be effective in ornamental fern beds [62]. Extreme tolerance to glyphosate has been documented [54]. Glyphosate has been shown to be effective but additives or mixtures may be needed for good results at moderate doses [7]. However, *C. diffusa* has been reported to have larger possibilities of recovery after glyphosate application because of its larger starch reservation [71].

Resistance to residual herbicides has also been reported and relatively high doses of simazine and diuron appear to be necessary to achieve control [32]. Recent studies on use of residual herbicides have identified Dual Magnum® (s-metolachlor) (applied as a preplant incorporat‐ ed, pre-emergent and post-emergent) as providing excellent residual control (>80%) of *C. benghalensis* in peanut [54]. Atrazine and Dual Magnum®, two commonly used corn herbicides used in the USA, also gave good to excellent residual activity on *C. benghalensis* [3]. The most effective herbicide control strategies for *C. benghalensis* involve combinations of both preemergence and postemergence conventional herbicides [54]. These include preemergence herbicides with residual activity such as Axiom® (flufenacet + metribuzin), Dual Magnum® Canopy SP® (metribuzin + chlorimuron) and Sencor® (metribuzin) and postemergence herbicides with fair to good activity such as Basagran®, Classic® (acetochlor) and Pursuit® (Imazethapyr). Gramoxone Max® and Aim® (acetochlor) can be used post-directed. In evaluating the effectiveness of several pre-emergence herbicides in suppressing *C. benghalen‐ sis* emergence, it was reported that s-metolachlor (at 1.07 and 1.60 kg a.i./ha), clomazone (at 0.42 and 1.05 kg a.i./ha) and flumetron (at 1.68 kg a.i./ha) provided ≥ 80% control at 6 weeks after treatment (WAT) in cotton [80]. It was stressed that the application of herbicides with soil residual activity will be crucial for the management of *C. benghalensis* [80].

An Integrated Management Strategy (IWM) is therefore suggested for the best control of this weed species. A multi-component approach including an effective herbicide for successful

Herbicides are not usually very effective against most Commelina species. The first verified resistance was registered in 1957, when *C. diffusa* biotypes were identified in the United States [26]. *Commelina elegans* has shown resistance to growth – regulator type herbicides [32]. Control using herbicides is, however, variable depending on the herbicide, accuracy of leaf coverage and environmental conditions [7]. Spraying with a selective or non – selective herbicide may work but repeated treatments are required for regrowth. Plants should not be under moisture stress when sprayed. Surfactants will improve penetration into the waxy-coated leaves.

Many standard herbicides have relatively low activity on species of Commelina [84]. These include 2,4-D, propanil, butachlor, trifluralin and pendimethalin. Treatment with 2,4-D or MCPA at the pre-emergent stage has been shown to be ineffective and although a reasonable kill of very young seedlings can be obtained, the plants develop a rapid resistance with age [32]. Particular biotypes are resistant to 2,4-D and they may be cross resistant to other Group O / 4 herbicides [83]. It has been found that one biotype of *C. diffusa* could withstand five times

In rice, bentazone, molinate, oxyfluorfen and bifenox are herbicides with good activity [7]. Post-emergent sequential treatments of propanil followed by nitrogen or of molinate followed by KN3 controlled *C. diffusa* in rice [61]. In soybean, bentazone and metribuzin are effective [7]. In corn, combination of bromoxynil and 2,4-D butylate produced a synergistic effect in postemergent control of 3-4 leaf stage *C. communis* [85]. In plantation crops such as banana, paraquat is not always effective but mixture with diuron is recommended [7]. Dinoseb has been found to kill seedlings as well as dalapon but paraquat is reported to be relatively ineffective [32]. Prodiamine has been reported to be effective in ornamental fern beds [62]. Extreme tolerance to glyphosate has been documented [54]. Glyphosate has been shown to be effective but additives or mixtures may be needed for good results at moderate doses [7]. However, *C. diffusa* has been reported to have larger possibilities of recovery after glyphosate

Resistance to residual herbicides has also been reported and relatively high doses of simazine and diuron appear to be necessary to achieve control [32]. Recent studies on use of residual herbicides have identified Dual Magnum® (s-metolachlor) (applied as a preplant incorporat‐ ed, pre-emergent and post-emergent) as providing excellent residual control (>80%) of *C. benghalensis* in peanut [54]. Atrazine and Dual Magnum®, two commonly used corn herbicides used in the USA, also gave good to excellent residual activity on *C. benghalensis* [3]. The most effective herbicide control strategies for *C. benghalensis* involve combinations of both preemergence and postemergence conventional herbicides [54]. These include preemergence herbicides with residual activity such as Axiom® (flufenacet + metribuzin), Dual Magnum®

management has been suggested [80-82].

548 Herbicides - Current Research and Case Studies in Use

the dosage of a susceptible species [83].

application because of its larger starch reservation [71].

**7. Chemical management**

In the Windward Islands, farmers started using paraquat around 1989 and noticed that it was ineffective. In an interview on August 10, 2002, Paddy Thomas, an experienced banana grower and pesticide salesman in St. Vincent and the Grenadines revealed that farmers started using gramocil (paraquat + diuron) at high doses for example and this too was not effective and resistance in Commelina spp. began to show. He also stated that Reglone, Round – up and Talent (paraquat + asulam) have also been used with little success for the control of Commelina species in the Windward Islands. Glufosinate has since been promoted as an environmentallyfriendly option for the control of broad-leaved weeds including Commelina species.

Studies were conducted into the efficacy of glufosinate for weed control in coffee plantations and it was found that it did not effectively control Commelina spp. at a rate of 0.3 – 0.6 kg a.i. / ha, however, paracol and gardoprim suppressed this perennial weed better [50]. Fomasefen and lactofen have shown good potential for control of this broadleaf weed [10]. Glufosinate (240 g a.i./ha) and fomasefen (WIP 276 g a.i./ha) were used in St. Vincent and the Grenadines in Fairtrade banana fields to compare their efficacy in controlling *C. diffusa* [30]. They were both applied at the early post-emergence, 3-5 leaf stage with a backpack sprayer using a TJ-8002 fan-nozzle. Regrowth of *C. diffusa* and other weeds were observed 6 weeks after application with glufosinate, however, no regrowth was observed for up to 3 months with fomasefen. Fomasefen, however, caused damage by burning banana suckers and leaves (about 30%) of established banana plants [30]. Studies were conducted to evaluate the efficacy of several postemergence herbicides in controlling *C. communis* in soybean, the results showed that imaze‐ thapyr (150 g a.i./ha), cloransulam-methyl (31.5 g a.i./ha), fomesafen (375 g a.i./ha) and mixture (756 g a.i./ha) of fomesafen plus imazethapyr with clomazone provided > 80% control of this weed at 30 days after treatment (DAT) [36, 37, 65, 67]. The efficacy of imazethapyr (90 g a.i./ha) in controlling *C. communis* reduced with increased leaf stage, and the control levels at 15 DAT were 100% (at 1 leaf stage), 89.17% (at 2 leaf stage), 56.45% (at 3 leaf stage) and 52.71% (at 4 leaf stage), respectively [41]. Therefore, the optimal application time of imazethapyr was 1-2 leaf stage of *C. communis* [41].

To screen more suitable herbicides for control of *C. benghalensis* and *C. communis* and determine the level of weed control provided by a single application of selected post-emergence herbi‐ cides, greenhouse studies on the laboratory toxicity of 23 herbicides to these weeds were conducted in 2010 [21]. The results indicated that, as for *C. benghalensis*, mesotrione, lactofen, oxyfluorfen, clomazone and flumioxazin provide complete control (100%), oxadiazon, fomesafen, metribuzin, acifluorfen, isoproturon, MCPA-sodium, carfentrazone-ethyl, flurox‐ ypyr, fluoroglycofen-ethyl and bentazone are herbicides with excellent activity (90.0 - 100% control), paraquat, 2,4-D butylate, rimsulfuron and thifensulfuron-methyl are herbicides with good activity (80.0 - 90.0% control), and nicosulfuron, bensulfuron-methyl, dicamba and glyphosate-isopropylammonium are relatively ineffective (< 80.0% control) at their own recommended dose, respectively. As for *C. communis*, mesotrione and thifensulfuron-methyl provide complete control (100%); metribuzin, paraquat, carfentrazone-ethyl, 2,4-D butylate, nicosulfuron, MCPA-sodium, fluroxypyr, flumioxazin and acifluorfen are herbicides with excellent activity (90.0 - 100% control); rimsulfuron, lactofen and fomesafen are herbicides with good activity (80.0 - 90.0% control); and glyphosate-isopropylammonium, bensulfuronmethyl, fluoroglycofen-ethyl, bentazone, clomazone, oxadiazon, oxyfluorfen, isoproturon and dicamba are relatively ineffective (< 80.0% control) at their own recommended dose, respec‐ tively. There are 19 and 14 herbicides which provided good to excellent control (> 80%) to *C. benghalensis* and *C. communis* under greenhouse conditions, respectively. However, the performance of those herbicides applied in different crops to control *C. benghalensis* and *C. communis* also needs to be ascertained.

Field studies conducted in St. Vincent and the Grenadines in 2003/2004 compared several treatments including 3 cover crops in suppressing *Commelina diffusa* weed infestations in banana at 63 days after application (DAA) [30]. The cover crops included *Arachis pintoi* (wild peanuts) which was sown by seed and stem cuttings, 16 cm apart, *Mucuna pruriens* (velvet beans) drilled 30 cm apart and *Desmodium heterocarpon* var *ovalifolium* (CIAT 13651) broadcast at a rate of 5 kg/ha. Best results were obtained from *Desmodium heterocarpon* (86.7%) followed by *Arachis pintoi* (52.1%) and *Mucuna pruriens* (43.3%). *Desmodium heterocarpon* was also found to be competitive to *C. diffusa* significantly suppressing its growth in Farmer Participatory

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Mulching is another viable option for management of the weed. Mulching with rice straw, cut bush, grass, coffee hulls, water hyacinth or even the dead or senescent banana leaves, pruned suckers and old stems could significantly suppress weed growth. Black plastic mulch also provides good weed control as it stifles weed seed growth and development when light penetration is reduced. There are no reports of work done on the use of these mulches for suppression of Commelina species. In field studies in St. Vincent and the Grenadines in 2003/2004 three dead mulches were compared using senescent banana leaves (traditional practice of farmers) applied to a depth of 3-5 cm, coffee hulls applied to a depth of 3-5 cm and black plastic polyethylene tarp at 1.0 mils thickness [30]. Results indicate a 94.5% and 95.6% suppression of weeds including *C. diffusa* with coffee hulls and banana mulch treatments

*Commelina diffusa* is particularly difficult to control by cultivation, partly because broken pieces of the stem readily take root and underground stems with pale, reduced leaves and flowers are often produced [32]. The plant is easy to rake up, roll up or hand pull and very small infestations can be dug out. It can be bagged and well baked in the sun, however, follow – up work is essential as any small fragment of the stem remaining will regrow and needs to be removed and destroyed off - site. Mechanical control using the weed whacker may also contribute the spread of stem cuttings in addition to damaging the banana root system as much

To investigate the effect of cutting and depth on the regeneration potential of *C. diffusa* greenhouse studies were conducted in 2004/2005 (Isaac et al. unpublished data 2005) using three cutting types: tip cuttings (2 nodes, 2 leaves), 2 node pieces only and 1 node, 1 leaf piece buried at depths including 0 (control), 2.5, 5.0 and 7.0 cm to demonstrate emergence patterns. These cuttings were intended to simulate cuttings made from a weed whacker and the practice of burying the weed. Regeneration was observed from all cuttings from 0 – 5.0 cm depths but no growth was observed at 7.0 cm. *C. diffusa* dry matter (DM) was highest at surface level (0cm - control) for all cuttings and reduced with increased depth. Results indicate that for effective management of *C. diffusa* by cutting, nodes must be reduced to less than half with no leaves which may starve the plants' photosynthetic ability and hence suppress regeneration. Burial

Research trials also conducted in St. Vincent in 2005/2006 [30].

respectively and 100% suppression with black plastic mulch.

of the plant lies within the top 15 cm of the soil [30].

**9. Mechanical management**

### **8. Cultural management**

This method depends on the crop infested, land size, level of technology available, value of crop, labour availability and costs, availability of draft power and the associated equipment and availability of herbicides [47]. The document further indicates that the methods currently used include proper land preparation, hand hoeing and pulling, removing the plants from the fields and drying, use of ox-drawn and tractor drawn cultivation, slashing and herbicide application. *Commelina diffusa* is very difficult to control manually as the stolons are cut into small pieces which can easily regenerate. Hand weeding and rolling the weed up like a carpet is considered suitable for removal of small infestations [30], if care is taken to remove every last piece. In Uganda, it was reported that heaping of stubborn weeds of Commelina plants is practical during the rainy season to speed up rotting and reduce the frequency of weeding [48]. In the dry season, heaps are then scattered as the dry conditions desiccate Commelina stems rapidly. A small percent of Ugandan farmers (5.9%) dig ditches and bury Commelina species, turning it into manure. Some farmers in St. Vincent have also tried this technique in the field with varying success.

A potential solution to overcoming Commelina weed infestations in banana is by intercrop‐ ping with a fast, low – growing shade tolerant cover crop. This can be done by intercropping with melons, *Mucuna pruriens* (negra and ceniza), tropical alfalfa, *Cajanus cajan, Vigna radiata* (mung bean), *V. unguiculata* (cowpea), *Crotalaria juncea, Indigofera endecaphylla, Phaseolus trinervius,* and *Ipomea batatas* (sweet potato) which have rapid canopy coverage to suppress the establishment of weeds. Melon (*Colocynthis citrullus* L.) planted at a density of 5,000 plants/ha suppressed weed growth of *Commelina diffusa* for five months, enhancing establishment and yield of melon in Nigeria [49]. Use of vigorous healthy planting material and close spacing of the crop may also be used. It has been shown that spacings of 1.2 x 1.2 m (6,944 plants/ha) and 1.5 x 1.2 m (4,444 plants/ha) gave high yields and "natural" control of these weeds [8, 66].

Field studies conducted in St. Vincent and the Grenadines in 2003/2004 compared several treatments including 3 cover crops in suppressing *Commelina diffusa* weed infestations in banana at 63 days after application (DAA) [30]. The cover crops included *Arachis pintoi* (wild peanuts) which was sown by seed and stem cuttings, 16 cm apart, *Mucuna pruriens* (velvet beans) drilled 30 cm apart and *Desmodium heterocarpon* var *ovalifolium* (CIAT 13651) broadcast at a rate of 5 kg/ha. Best results were obtained from *Desmodium heterocarpon* (86.7%) followed by *Arachis pintoi* (52.1%) and *Mucuna pruriens* (43.3%). *Desmodium heterocarpon* was also found to be competitive to *C. diffusa* significantly suppressing its growth in Farmer Participatory Research trials also conducted in St. Vincent in 2005/2006 [30].

Mulching is another viable option for management of the weed. Mulching with rice straw, cut bush, grass, coffee hulls, water hyacinth or even the dead or senescent banana leaves, pruned suckers and old stems could significantly suppress weed growth. Black plastic mulch also provides good weed control as it stifles weed seed growth and development when light penetration is reduced. There are no reports of work done on the use of these mulches for suppression of Commelina species. In field studies in St. Vincent and the Grenadines in 2003/2004 three dead mulches were compared using senescent banana leaves (traditional practice of farmers) applied to a depth of 3-5 cm, coffee hulls applied to a depth of 3-5 cm and black plastic polyethylene tarp at 1.0 mils thickness [30]. Results indicate a 94.5% and 95.6% suppression of weeds including *C. diffusa* with coffee hulls and banana mulch treatments respectively and 100% suppression with black plastic mulch.

### **9. Mechanical management**

control), paraquat, 2,4-D butylate, rimsulfuron and thifensulfuron-methyl are herbicides with good activity (80.0 - 90.0% control), and nicosulfuron, bensulfuron-methyl, dicamba and glyphosate-isopropylammonium are relatively ineffective (< 80.0% control) at their own recommended dose, respectively. As for *C. communis*, mesotrione and thifensulfuron-methyl provide complete control (100%); metribuzin, paraquat, carfentrazone-ethyl, 2,4-D butylate, nicosulfuron, MCPA-sodium, fluroxypyr, flumioxazin and acifluorfen are herbicides with excellent activity (90.0 - 100% control); rimsulfuron, lactofen and fomesafen are herbicides with good activity (80.0 - 90.0% control); and glyphosate-isopropylammonium, bensulfuronmethyl, fluoroglycofen-ethyl, bentazone, clomazone, oxadiazon, oxyfluorfen, isoproturon and dicamba are relatively ineffective (< 80.0% control) at their own recommended dose, respec‐ tively. There are 19 and 14 herbicides which provided good to excellent control (> 80%) to *C. benghalensis* and *C. communis* under greenhouse conditions, respectively. However, the performance of those herbicides applied in different crops to control *C. benghalensis* and *C.*

This method depends on the crop infested, land size, level of technology available, value of crop, labour availability and costs, availability of draft power and the associated equipment and availability of herbicides [47]. The document further indicates that the methods currently used include proper land preparation, hand hoeing and pulling, removing the plants from the fields and drying, use of ox-drawn and tractor drawn cultivation, slashing and herbicide application. *Commelina diffusa* is very difficult to control manually as the stolons are cut into small pieces which can easily regenerate. Hand weeding and rolling the weed up like a carpet is considered suitable for removal of small infestations [30], if care is taken to remove every last piece. In Uganda, it was reported that heaping of stubborn weeds of Commelina plants is practical during the rainy season to speed up rotting and reduce the frequency of weeding [48]. In the dry season, heaps are then scattered as the dry conditions desiccate Commelina stems rapidly. A small percent of Ugandan farmers (5.9%) dig ditches and bury Commelina species, turning it into manure. Some farmers in St. Vincent have also tried this technique in the field

A potential solution to overcoming Commelina weed infestations in banana is by intercrop‐ ping with a fast, low – growing shade tolerant cover crop. This can be done by intercropping with melons, *Mucuna pruriens* (negra and ceniza), tropical alfalfa, *Cajanus cajan, Vigna radiata* (mung bean), *V. unguiculata* (cowpea), *Crotalaria juncea, Indigofera endecaphylla, Phaseolus trinervius,* and *Ipomea batatas* (sweet potato) which have rapid canopy coverage to suppress the establishment of weeds. Melon (*Colocynthis citrullus* L.) planted at a density of 5,000 plants/ha suppressed weed growth of *Commelina diffusa* for five months, enhancing establishment and yield of melon in Nigeria [49]. Use of vigorous healthy planting material and close spacing of the crop may also be used. It has been shown that spacings of 1.2 x 1.2 m (6,944 plants/ha) and 1.5 x 1.2 m (4,444 plants/ha) gave high yields and "natural" control of these weeds [8, 66].

*communis* also needs to be ascertained.

550 Herbicides - Current Research and Case Studies in Use

**8. Cultural management**

with varying success.

*Commelina diffusa* is particularly difficult to control by cultivation, partly because broken pieces of the stem readily take root and underground stems with pale, reduced leaves and flowers are often produced [32]. The plant is easy to rake up, roll up or hand pull and very small infestations can be dug out. It can be bagged and well baked in the sun, however, follow – up work is essential as any small fragment of the stem remaining will regrow and needs to be removed and destroyed off - site. Mechanical control using the weed whacker may also contribute the spread of stem cuttings in addition to damaging the banana root system as much of the plant lies within the top 15 cm of the soil [30].

To investigate the effect of cutting and depth on the regeneration potential of *C. diffusa* greenhouse studies were conducted in 2004/2005 (Isaac et al. unpublished data 2005) using three cutting types: tip cuttings (2 nodes, 2 leaves), 2 node pieces only and 1 node, 1 leaf piece buried at depths including 0 (control), 2.5, 5.0 and 7.0 cm to demonstrate emergence patterns. These cuttings were intended to simulate cuttings made from a weed whacker and the practice of burying the weed. Regeneration was observed from all cuttings from 0 – 5.0 cm depths but no growth was observed at 7.0 cm. *C. diffusa* dry matter (DM) was highest at surface level (0cm - control) for all cuttings and reduced with increased depth. Results indicate that for effective management of *C. diffusa* by cutting, nodes must be reduced to less than half with no leaves which may starve the plants' photosynthetic ability and hence suppress regeneration. Burial should be up to 5.0 cm to ensure that there is no emergence of the weed. Similar studies [5] indicated that cuttings buried deeper than 2 cm failed to regenerate.

germination and root length of *C. benghalensis* [73]. Both AA and FA have the potential for use

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There have not been many reports on biological control of Commelina species. *Commelina diffusa* is grazed by small ruminants, pigs and cows. Because this species is very fleshy and has a high moisture content, it is difficult to use it as fodder for domestic stock [27]. However, recent research has indicated that *C. diffusa* compared well with many commonly used fodder crops and could contribute as a protein source for ruminants on smallholder farms [30]. There

There are no reports of promising insect candidates for biological control reported on Com‐ melina spp. in the USA [63, 64]. In Korea and China there have been reports of *Lema concinn‐ pennis* and *Lema scutellaris* (Coleoptera: Chrysomelidae) two leaf-feeding species on *C. communis* [86]. *Noelema sexpunctata* (Coleoptera: Chrysomelidae) another leaf-feeding species

In Central Virginia, USA, *Pycnodees medius* (Hemiptera: Miridae) was found to cause tissue necrosis on *C. communis* [33]. Various insects were also screened for their potential as biocontrol agents of weeds in rice and it was found that *Necrobis ruficollis* (blue beetle), *Rhaphido‐ palpa africana* (yellow beetle), *Conocephalus* sp., *Tetragrnathidae* spp. and *Paracinema tricolor* (grasshopper) were promising [45]. Feeding and nymphal development (up to 3rd and 4th instar) of *Cornop aquaticaum* (grasshopper) were reported on *C. africana* L., and *Murdannia africana* (Vahl.) [25]. It was also observed that *Rhaphidopalpa africana* beetles fed more than the

There are records of agromyzid leaf miners which may be promising sources of candidate biological control agents [75]. *Liriomyza commelinae*(Diptera: Agromyzidae), a leaf-miner, was however reported on *C. diffusa* in Jamaica [20, 61]. *Commelina diffusa* is the main food plant of *L. commelinae*, however, it is susceptible to predation by the formicid: *Crematogaster brevispi‐ nosa* as well as competition and exposure to the sun (high temperatures) which causes high

There are prospects for the management of invasive alien weeds in Latin America using coevolved fungal pathogens in selected species from the genera Commelina [14]. Pathogens recorded in the native range of Commelina species include: *Cercospora benghalensis* Chidd., *Cylindrosporium kilimandscharium* Allesch. (Hyphomycete), *Kordyana celebensis* Gaum, (Exoba‐ sidiales: Brachybasidiaceae), *Phakopsora tecta* H.S. Jacks and Holw (Uredinales: Phakopsora‐ ceae), *Septoria commelinae* Canonaco (Coelomycete), *Uromyces commelinae* Cooke (Uredinales: Pucciniaceae), *Phoma herbarum* [14, 23, 76]. These mycobiota would appear to be good potential agents for classical biological control (CBC) [14]. Although some of the most promising (e.g. the rusts *Phakopsora tecta* and *Uromyces commelinae*) are already present in the New World, they are restricted to certain regions and could be redistributed [14]. The uredinal state of a rust

have also been reports of foraging of this weed by *Gallus domesticus* (chickens) [30].

as bio-herbicides.

**11. Biological management**

was also reported on *C. communis* [45].

others on the weed, *C. benghalensis* L. [25].

mortality [20].

Research has shown that soil solarization, a hydrothermal process of heating moist soil, can successfully disinfect soil pests and control weeds [1, 4, 15, 56]. Soil solarization by covering with plastic sheeting for 6 weeks in the warmer months will weaken the plant. After removing the plastic any regrowth can be dug out or sprayed, however, this method will not be effective in full shade. Solarization can be used alone or in combination with other chemicals or biological agents as the framework for an IPM programme for soilborne pests in open fields. In field trials in St. Vincent, soil solarization using clear polyethylene plastic at 0.5 mils under Fairtrade banana plants showed variable suppression of *C. diffusa* as the weed emerged under the clear plastic showing chlorotic and suppressed growth symptoms, resuming its full growth potential after removal of the plastic covering 2 months after application (Isaac et al. unpub‐ lished data 2005). Seed germination of *C. benghalensis* was found to increase by soil solarization in studies conducted in Brazil [43].

#### **10. Organic management**

Attempts have also been made to find organic treatments for control of Commelina species in banana in St. Vincent and the Grenadines [30]. DTE corn weed blocker (corn gluten meal) preemergent weed blocker and slow release fertilizer (9-1-0) which controls emerging weeds was applied at a rate of 10 kg/ha. Burnout® (concentrated vinegar and acetic acid) (20%), urea (20%), and fertilizer solution (20%) were also used to evaluate their efficacy on the control of Commelina species and other weed species. All treatments showed varying levels of control for up to 3 weeks. Best results were obtained from Burnout® which caused phytotoxic damage on the leaves of actively growing plants offering 43% control. This was followed by urea (41%), fertilizer solution (34%) and corn weed blocker (20%). Urea, fertilizer and corn weed blocker treatments resulted in the general stunting of plants in addition to the burning of leaves. However, stems and roots remained intact. Similar results using treatments high in nitrogen were obtained in Russia [59] where seed production of *C. benghalensis* and stunted growth under artificial dense competition in cereals resulted. These results indicate that there is no evidence that this Commelina species competes for nitrogen. In fact the species does not pose any threat in competing for nutrients with banana. Repeat applications of these treatments are therefore necessary for the effective management of Commelina species in organic farming systems.

Studies conducted in Brazil in soybean-wheat rotations under no-tillage conditions showed reductions in the seedbank of *C. benghalensis* in areas infested with *Brachiaria plantaginea* [73]. Analysis of the soluble fraction of *B. plantaginea* indicated a predominance of aconitic acid (AA) among the aliphatic acids and ferulic acid (FA) among the phenolic acids. Laboratory bioassays using *C. benghalensis* were carried out to evaluate phytotoxic effects of pure organic acid solutions and dilute extracts of *B. plantaginea* on seeds germination, root development and fungal germination and AA and FA solutions and the extract of *B. plantaginea* extract reduced germination and root length of *C. benghalensis* [73]. Both AA and FA have the potential for use as bio-herbicides.

### **11. Biological management**

should be up to 5.0 cm to ensure that there is no emergence of the weed. Similar studies [5]

Research has shown that soil solarization, a hydrothermal process of heating moist soil, can successfully disinfect soil pests and control weeds [1, 4, 15, 56]. Soil solarization by covering with plastic sheeting for 6 weeks in the warmer months will weaken the plant. After removing the plastic any regrowth can be dug out or sprayed, however, this method will not be effective in full shade. Solarization can be used alone or in combination with other chemicals or biological agents as the framework for an IPM programme for soilborne pests in open fields. In field trials in St. Vincent, soil solarization using clear polyethylene plastic at 0.5 mils under Fairtrade banana plants showed variable suppression of *C. diffusa* as the weed emerged under the clear plastic showing chlorotic and suppressed growth symptoms, resuming its full growth potential after removal of the plastic covering 2 months after application (Isaac et al. unpub‐ lished data 2005). Seed germination of *C. benghalensis* was found to increase by soil solarization

Attempts have also been made to find organic treatments for control of Commelina species in banana in St. Vincent and the Grenadines [30]. DTE corn weed blocker (corn gluten meal) preemergent weed blocker and slow release fertilizer (9-1-0) which controls emerging weeds was applied at a rate of 10 kg/ha. Burnout® (concentrated vinegar and acetic acid) (20%), urea (20%), and fertilizer solution (20%) were also used to evaluate their efficacy on the control of Commelina species and other weed species. All treatments showed varying levels of control for up to 3 weeks. Best results were obtained from Burnout® which caused phytotoxic damage on the leaves of actively growing plants offering 43% control. This was followed by urea (41%), fertilizer solution (34%) and corn weed blocker (20%). Urea, fertilizer and corn weed blocker treatments resulted in the general stunting of plants in addition to the burning of leaves. However, stems and roots remained intact. Similar results using treatments high in nitrogen were obtained in Russia [59] where seed production of *C. benghalensis* and stunted growth under artificial dense competition in cereals resulted. These results indicate that there is no evidence that this Commelina species competes for nitrogen. In fact the species does not pose any threat in competing for nutrients with banana. Repeat applications of these treatments are therefore necessary for the effective management of Commelina species in organic farming

Studies conducted in Brazil in soybean-wheat rotations under no-tillage conditions showed reductions in the seedbank of *C. benghalensis* in areas infested with *Brachiaria plantaginea* [73]. Analysis of the soluble fraction of *B. plantaginea* indicated a predominance of aconitic acid (AA) among the aliphatic acids and ferulic acid (FA) among the phenolic acids. Laboratory bioassays using *C. benghalensis* were carried out to evaluate phytotoxic effects of pure organic acid solutions and dilute extracts of *B. plantaginea* on seeds germination, root development and fungal germination and AA and FA solutions and the extract of *B. plantaginea* extract reduced

indicated that cuttings buried deeper than 2 cm failed to regenerate.

in studies conducted in Brazil [43].

552 Herbicides - Current Research and Case Studies in Use

**10. Organic management**

systems.

There have not been many reports on biological control of Commelina species. *Commelina diffusa* is grazed by small ruminants, pigs and cows. Because this species is very fleshy and has a high moisture content, it is difficult to use it as fodder for domestic stock [27]. However, recent research has indicated that *C. diffusa* compared well with many commonly used fodder crops and could contribute as a protein source for ruminants on smallholder farms [30]. There have also been reports of foraging of this weed by *Gallus domesticus* (chickens) [30].

There are no reports of promising insect candidates for biological control reported on Com‐ melina spp. in the USA [63, 64]. In Korea and China there have been reports of *Lema concinn‐ pennis* and *Lema scutellaris* (Coleoptera: Chrysomelidae) two leaf-feeding species on *C. communis* [86]. *Noelema sexpunctata* (Coleoptera: Chrysomelidae) another leaf-feeding species was also reported on *C. communis* [45].

In Central Virginia, USA, *Pycnodees medius* (Hemiptera: Miridae) was found to cause tissue necrosis on *C. communis* [33]. Various insects were also screened for their potential as biocontrol agents of weeds in rice and it was found that *Necrobis ruficollis* (blue beetle), *Rhaphido‐ palpa africana* (yellow beetle), *Conocephalus* sp., *Tetragrnathidae* spp. and *Paracinema tricolor* (grasshopper) were promising [45]. Feeding and nymphal development (up to 3rd and 4th instar) of *Cornop aquaticaum* (grasshopper) were reported on *C. africana* L., and *Murdannia africana* (Vahl.) [25]. It was also observed that *Rhaphidopalpa africana* beetles fed more than the others on the weed, *C. benghalensis* L. [25].

There are records of agromyzid leaf miners which may be promising sources of candidate biological control agents [75]. *Liriomyza commelinae*(Diptera: Agromyzidae), a leaf-miner, was however reported on *C. diffusa* in Jamaica [20, 61]. *Commelina diffusa* is the main food plant of *L. commelinae*, however, it is susceptible to predation by the formicid: *Crematogaster brevispi‐ nosa* as well as competition and exposure to the sun (high temperatures) which causes high mortality [20].

There are prospects for the management of invasive alien weeds in Latin America using coevolved fungal pathogens in selected species from the genera Commelina [14]. Pathogens recorded in the native range of Commelina species include: *Cercospora benghalensis* Chidd., *Cylindrosporium kilimandscharium* Allesch. (Hyphomycete), *Kordyana celebensis* Gaum, (Exoba‐ sidiales: Brachybasidiaceae), *Phakopsora tecta* H.S. Jacks and Holw (Uredinales: Phakopsora‐ ceae), *Septoria commelinae* Canonaco (Coelomycete), *Uromyces commelinae* Cooke (Uredinales: Pucciniaceae), *Phoma herbarum* [14, 23, 76]. These mycobiota would appear to be good potential agents for classical biological control (CBC) [14]. Although some of the most promising (e.g. the rusts *Phakopsora tecta* and *Uromyces commelinae*) are already present in the New World, they are restricted to certain regions and could be redistributed [14]. The uredinal state of a rust was found widespread on *C. diffusa* in Hawaii [22] sometimes causing death of parts above ground. Studies aimed at identifying mycoherbicidal biocontrol agents have been conducted in Brazil on three endemic pathogens of *C. benghalensis* which were: a bacterium (Erwinia sp.) and two fungi (*Corynespora cassiicola* and *Cercospora* sp.) [38, 39].

**•** Evaluate the seedbank longevity of *C. benghalensis*

**•** Characterize the environmental limits of *C. benghalensis* in the U.S.A. [80].

**•** Determine threshold levels of *C. diffusa* in crops such as banana

commilinae which has been identified in several Caribbean Islands.

and Mei Li2

duction and Protection Paper (Amman, Jordon), (1991).

Commelinaceae. Plant Disease (1988). , 72(6), 513-518.

Primary Industries Publication #QL03056; (2003).

underground) of *C. benghalensis* species in the USA.

, Zongjun Gao2

West Indies, St. Augustine, Trinidad

Surely this list can be expanded to include other Commelina species such as *C. diffusa* which is definitely a problematic weed in the cropping systems in the Windward Islands. The research

Managing Commelina Species: Prospects and Limitations

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555

**•** Evaluate the allelopathic potential of Commelina species by extracting hydro alcoholic compounds which could be used as a possible bioherbicide in controlling other problem

**•** Screen for mycobiota with good potential for CBC such as the rust species Uromyces

**•** Determine the reasons for reduced seed production of *C. diffusa* species found under banana fields in the Windward Islands as compared to higher seed numbers (both aerial and

1 Department of Food Production, Faculty of Food and Agriculture, The University of the

[1] Abu-irmaileh, B. E. Weed control in vegetables by soil solarization, In FAO Plant Pro‐

[2] Baker, C. A, & Zettler, F. W. Viruses infecting wild and cultivated species of the

[3] Barnes, J. Managing hairy wandering Jew. Queensland Government, Department of

[4] Benjamin, A, & Rubin, B. Soil solarization as a means of weed control. Phytoparasiti‐

2 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, China

**•** Determine the primary dispersal mechanism(s)

direction should also:

weeds

**Author details**

Wendy-Ann Isaac1

**References**

ca (1982).

### **12. Conclusion and recommendations**

The Commelina species are very persistent, noxious weeds which must be managed using an integrated approach to weed management. Weed management strategies that are narrowly focused will ultimately cause shifts in weed populations to species that no longer respond to the strategy resulting in adapted species, tolerant species or herbicide-resistant biotypes [51], which is the case with Commelina species in cropping systems. The integrated approach should utilize alternative strategies such as those mentioned in this paper including the most practical options, cultural and mechanical not negating the judicious use of herbicides. Such combinations should provide significant management levels of Commelina species for both conventional as well as organic growers using a pesticide free production PFP approach. Utilization of the useful benefits of Commelina species after uprooting will also serve to check the heavy use of herbicides in cropping systems.

The integrated approach must begin very early as once an infestation is really entrenched it presents several difficulties because of the pernicious growth habit of this weed. Successful management of *C. benghalensis* will require a multi-component approach including an effective herbicide that provides soil residual activity [80]. Recent studies on the management of Commelina species have, however, still focused primarily on effective herbicides and herbi‐ cide mixtures for their control despite hard evidence of the development of herbicide-resistant biotypes. Additionally, the adoption within recent years of GM crops particularly herbicide – resistant crops presents serious issues involving their negative ecological impact as already there are reports of Commelina species prominence in some agroecosystems due to simple and significant selection pressure brought to bear by these herbicide – resistant crops and the concomitant use of the herbicide [52].

The best way to control Commelina species for small holders in developing countries would be by implementing an integrated approach that embraces a variety of options which should be attuned to the individual farmer's agronomic and socio – economic conditions (soil type, climate, costs, local practices and preferences). For example, in banana growing areas in the Windward Islands, the growth of the weed can be suppressed by a single application of a herbicide or weed whacking very early before extensive spread of the weed followed by planting a competitive cover crop like *Desmodium heterocarpon* that would not only prevent reinvasion but improve soil fertility.

Future research in developing effective management strategies for *Commelina benghalensis* should:

**•** Develop an accurate predictive model for *C. benghalensis* germination


Surely this list can be expanded to include other Commelina species such as *C. diffusa* which is definitely a problematic weed in the cropping systems in the Windward Islands. The research direction should also:


### **Author details**

was found widespread on *C. diffusa* in Hawaii [22] sometimes causing death of parts above ground. Studies aimed at identifying mycoherbicidal biocontrol agents have been conducted in Brazil on three endemic pathogens of *C. benghalensis* which were: a bacterium (Erwinia sp.)

The Commelina species are very persistent, noxious weeds which must be managed using an integrated approach to weed management. Weed management strategies that are narrowly focused will ultimately cause shifts in weed populations to species that no longer respond to the strategy resulting in adapted species, tolerant species or herbicide-resistant biotypes [51], which is the case with Commelina species in cropping systems. The integrated approach should utilize alternative strategies such as those mentioned in this paper including the most practical options, cultural and mechanical not negating the judicious use of herbicides. Such combinations should provide significant management levels of Commelina species for both conventional as well as organic growers using a pesticide free production PFP approach. Utilization of the useful benefits of Commelina species after uprooting will also serve to check

The integrated approach must begin very early as once an infestation is really entrenched it presents several difficulties because of the pernicious growth habit of this weed. Successful management of *C. benghalensis* will require a multi-component approach including an effective herbicide that provides soil residual activity [80]. Recent studies on the management of Commelina species have, however, still focused primarily on effective herbicides and herbi‐ cide mixtures for their control despite hard evidence of the development of herbicide-resistant biotypes. Additionally, the adoption within recent years of GM crops particularly herbicide – resistant crops presents serious issues involving their negative ecological impact as already there are reports of Commelina species prominence in some agroecosystems due to simple and significant selection pressure brought to bear by these herbicide – resistant crops and the

The best way to control Commelina species for small holders in developing countries would be by implementing an integrated approach that embraces a variety of options which should be attuned to the individual farmer's agronomic and socio – economic conditions (soil type, climate, costs, local practices and preferences). For example, in banana growing areas in the Windward Islands, the growth of the weed can be suppressed by a single application of a herbicide or weed whacking very early before extensive spread of the weed followed by planting a competitive cover crop like *Desmodium heterocarpon* that would not only prevent re-

Future research in developing effective management strategies for *Commelina benghalensis*

**•** Develop an accurate predictive model for *C. benghalensis* germination

and two fungi (*Corynespora cassiicola* and *Cercospora* sp.) [38, 39].

**12. Conclusion and recommendations**

554 Herbicides - Current Research and Case Studies in Use

the heavy use of herbicides in cropping systems.

concomitant use of the herbicide [52].

invasion but improve soil fertility.

should:

Wendy-Ann Isaac1 , Zongjun Gao2 and Mei Li2

1 Department of Food Production, Faculty of Food and Agriculture, The University of the West Indies, St. Augustine, Trinidad

2 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, China

### **References**


[5] Budd, G. D, Thomas, P. E. L, & Allison, J. C. S. Vegetative regeneration depth of ger‐ mination and seed dormancy in *Commelina benghalensis* L. Rhodesian Journal of Agri‐ cultural Research (1979). , 17, 151-153.

[18] Faden, R. B. The misconstrued and rare species of Commelina (Commelinaceae) in the eastern United States. Annals of Missouri Botanical Gardens (1993). , 80, 208-218.

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557

[19] Fournet, J, & Hammerton, J. L. Weeds of the Lesser Antilles. Institute of National Re‐

[20] Freeman, B. E, & Smith, D. C. Variation of density-dependence with spatial scale in the leaf-mining fly *Liromyza commelinae* (Diptera: Agromyzidae). Ecological Entomol‐

[21] Gao ZongJunLi Mei and Gao XingXiang. Laboratory toxicity of 20 herbicides against Bengal dayflower (*Commelina bengalensis* L.). (Abstract) VI International Weed Sci‐ ence Congress, Hangzhou, China, 17- 22 June, 2012. Published by the International

[22] Gardener, D. E. Rust on *Commelina diffusa* in Hawaii. Plant Disease (1981). , 65(8),

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**Chapter 22**

**Integrating Herbicides in a**

Andrew J. Price and Jessica A. Kelton

http://dx.doi.org/10.5772/56142

**1. Introduction**

qent productivity.

ations.

**High-Residue Cover Crop Setting**

Additional information is available at the end of the chapter

Sustainable agriculture requires the use of multiple, integrated weed management practi‐ ces to ensure long-term viability. A number of cultural, mechanical, and chemical weed control options can be utilized in a production system to reduce weed interference and safeguard crop yield. The dependence on one single weed control strategy may result in short-term success; however, long-term use can lead to multiple setbacks including poor soil health, reduced crop production, and increasing herbicide resistance. In turn, employing multiple weed control tactics simultaneously may prove difficult without previous knowl‐ edge as to how best to implement an integrated weed management system. To that end, this chapter is dedicated to illustrating successful herbicide use in conjunction with cover crops and their residues, practices proven not only to suppress weed germination and growth, but also to reduce soil erosion and water runoff and build soil organic matter and thus subse‐

Use of cover crops, particularly those producing high amounts of biomass (greater than 4,500 kg ha-1), can provide numerous benefits for a cropping system [1]. However, care must be taken when choosing herbicides to apply to these cover crops both prior to and after primary crop planting. This chapter provides an overview of effective herbicide choices for use prior to and within cover crop as well as efficient application methods for use after planting the primary crop(s). We also discuss herbicide interception by cover crop residue and means to control reduced efficacy due to interception. It is hoped that this summary will aid in the adoption of sustainable farming practices to ensure successful agricultural productivity for future gener‐

> © 2013 Price and Kelton; 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 Price and Kelton; 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 22**

## **Integrating Herbicides in a High-Residue Cover Crop Setting**

Andrew J. Price and Jessica A. Kelton

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56142

### **1. Introduction**

[82] Webster, T, Flanders, J, & Culpepper, A. Critical period of tropical spiderwort (*Com‐ melina benghalensis*) control in cotton. Weed Science Society of America Abstracts

[83] WeedScience org. Group O/4 resistant spreading dayflower (*Commelina diffusa*), USA: Hawaii. (2005). http://www.weedscience.org/Case/Case.asp?ResistID=394accessed 15

[84] Wilson, A. K. Commelinacea- review of the distribution, biology and control of the important weeds belonging to this family. Tropical Pest Management (1981). , 27(3),

[85] Yang YuTing, Lin ChangFu, Geng HeLi, Sun BaoXiang and William H. Ahrens.Stud‐ ies on the action of bromoxynil and 2,4-D butyl ester herbicides combinations against dayflower (Commelina communis L.). Pesticides (2001). in Chinese with English ab‐

[86] Zhang XiuRong, Ma Shu, Ying, Dai BingLi, X.R. Zhang, S.Y. Ma and B.L. Dai.Mono‐ phagy of *Lema scutellaris* on *Commelina communis*. Acta Entomologica Sinica (1996). ,

[87] Zimmerman, A. De nematoden der koffiewortels. Deel I Mededeel's. Lands Planten‐

(2006). c , 80.

562 Herbicides - Current Research and Case Studies in Use

March 2007)

405-418.

stract)., 40(7), 37-38.

tium (Buitenzorg) (1898).

39, 281-285.

Sustainable agriculture requires the use of multiple, integrated weed management practi‐ ces to ensure long-term viability. A number of cultural, mechanical, and chemical weed control options can be utilized in a production system to reduce weed interference and safeguard crop yield. The dependence on one single weed control strategy may result in short-term success; however, long-term use can lead to multiple setbacks including poor soil health, reduced crop production, and increasing herbicide resistance. In turn, employing multiple weed control tactics simultaneously may prove difficult without previous knowl‐ edge as to how best to implement an integrated weed management system. To that end, this chapter is dedicated to illustrating successful herbicide use in conjunction with cover crops and their residues, practices proven not only to suppress weed germination and growth, but also to reduce soil erosion and water runoff and build soil organic matter and thus subse‐ qent productivity.

Use of cover crops, particularly those producing high amounts of biomass (greater than 4,500 kg ha-1), can provide numerous benefits for a cropping system [1]. However, care must be taken when choosing herbicides to apply to these cover crops both prior to and after primary crop planting. This chapter provides an overview of effective herbicide choices for use prior to and within cover crop as well as efficient application methods for use after planting the primary crop(s). We also discuss herbicide interception by cover crop residue and means to control reduced efficacy due to interception. It is hoped that this summary will aid in the adoption of sustainable farming practices to ensure successful agricultural productivity for future gener‐ ations.

© 2013 Price and Kelton; 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 Price and Kelton; 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.

### **2. Conservation agriculture**

As demands are placed on agriculture to produce increasing yields for a growing global population, the need to implement systems with high productivity and sound environmental standards is key to ensuring agricultural sustainability for future generations. To this end, conservation agriculture is a systems-based approach for food, feed, and fiber production that utilizes a number of practices aimed at maintaining yields while limiting energy and chemical inputs, minimizing soil degradation and erosion, and reducing long-term, detrimental impacts to the environment [2]. Conservation agriculture is comprised of many different management practices, particularly cultural techniques such as crop rotation, planting date, and seeding rate, that can reduce dependence on chemical inputs for successful yield production. More‐ over, limited tillage practices, or conservation-tillage, is essential to conservation agriculture systems to ensure soil quality, reduce runoff, and lessen energy consumption on agricultural lands.

**2.3. Herbicide use**

cover crop species.

biotypes [13].

*2.3.1. Cover crop establishment and termination*

of these herbicides for cover crop termination [11].

*2.3.2. Cash crop establishment and management*

To produce substantial cover crop biomass, it is imperative to adequately manage cover crop production. Besides using correct seeding rates, early planting dates, and sufficient fertilizer applications, it is important to be aware of herbicide applications made prior to cover crop establishment. Often times, postemergent (POST) herbicides applied late season or postharvest can have residual carryover than may be detrimental to cover crops. Rotation restric‐ tions listed on herbicide labels should be referred to when planning POST applications and

Integrating Herbicides in a High-Residue Cover Crop Setting

http://dx.doi.org/10.5772/56142

565

To manage cover crops before cash crop planting, herbicides are typically utilized for cover crop termination. Most often, these herbicides, such as glyphosate and glufosinate, are nonselective with little to no carryover risk. However, consideration should be given to in-season chemical weed control regimes in order to limit repeated applications of a single herbicide mode of action. Moreover, care should be taken to avoid reduced herbicide rates applied for cover termination to reduce the risk of herbicide resistance [10]. Recent research has focused on mechanical termination with a roller or crimper which may reduce or eliminate the need

Although use of in-season herbicides can be substantially reduced when using high-residue cover crops, some chemical applications are generally required to achieve the most effective weed suppression and minimize crop loss due to weed competition. While an ideal agricultural system would require no chemical inputs for sufficient weed control, practicality dictates the use of herbicides to guarantee crop yield since no system, as of yet, exists that can successfully suppress weed populations without intensive labor or mechanical requirements. To this end, cover crops are a means to minimize, rather than eliminate, herbicide inputs in crop systems. In recognizing the fact that the majority of agricultural systems will require chemical weed control measures for optimum crop production even when utilizing cover crops, it is essential

to understand how cover crops affect herbicide selection and efficacy for each crop.

Primarily, the use of reduced-tillage and cover crops eliminates the ability to utilize preplant incorporated herbicides which offer residual soil activity [11]. Furthermore, cover crop residue can impede preemergent (PRE) herbicide applications from reaching the soil surface, reducing herbicide efficacy [12]. While postemergent chemical weed control can be effective alternatives in these settings, many weed species can prove to be difficult to control if not killed early in the season. Moreover, resistance concerns essentially necessitate the use of preemergent herbicides with differing mechanisms of action to avoid selection pressure for resistant weed

Along with many cultural pracitces, production of crops under reduced-tillage with cover crops requires development of specific herbicide regimes to ensure minimal chemical inputs while achieving sufficient weed control to allow for successful crop production. The following

#### **2.1. Conservation tillage**

Conservation-tillage, or reduced-tillage, has been proven to provide multiple benefits in agricultural settings. In addition to erosion and runoff control, soil health improvement, and reduced energy demands, reduced-tillage practices can produce crops yields similar to that of conventional systems [3-5]. The use of reduced-tillage, however, can alter weed communities. Seed production by annual weed species remains, in most part, on the soil surface where it is subject to increased decomposition and predation. With reduced competition and minimized soil disruption, perennial weed species can become established and dominate the weed community in conservation-tillage [6]. To aid in the control of both annual and perennial weeds, the use of cover crops for ground cover can reduce herbicide requirements in conser‐ vation-tillage settings.

#### **2.2. Cover crops**

A number of cereal and legume cover crops are utilized in various crop productions for several purposes. Currently, a large portion of cover crops are planted as a green manure which are turned under prior to sowing the primary crop [7,8]. In reduced-tillage, however, cover crops are grown as a ground cover and remain on the soil surface after cover crop termination. In addition to further reducing soil erosion, increasing soil organic matter, and improving water infiltration, cover crops can provide a level of weed suppression both prior to and during the primary growing season [9]. When compared to fallow conservation-tillage systems, cover crops offer increased weed control through direct resource competition while actively growing as well as through shading and/or allelopathy after termination. Covers grown to produce high levels of biomass, in particular, can increase shading of germinating weed species and provide greater ground cover for an extended period during the growing season. When employing cover crops, however, knowledge concerning herbicide use both during cover crop production and primary crop growth is essential.

#### **2.3. Herbicide use**

**2. Conservation agriculture**

564 Herbicides - Current Research and Case Studies in Use

lands.

**2.1. Conservation tillage**

vation-tillage settings.

production and primary crop growth is essential.

**2.2. Cover crops**

As demands are placed on agriculture to produce increasing yields for a growing global population, the need to implement systems with high productivity and sound environmental standards is key to ensuring agricultural sustainability for future generations. To this end, conservation agriculture is a systems-based approach for food, feed, and fiber production that utilizes a number of practices aimed at maintaining yields while limiting energy and chemical inputs, minimizing soil degradation and erosion, and reducing long-term, detrimental impacts to the environment [2]. Conservation agriculture is comprised of many different management practices, particularly cultural techniques such as crop rotation, planting date, and seeding rate, that can reduce dependence on chemical inputs for successful yield production. More‐ over, limited tillage practices, or conservation-tillage, is essential to conservation agriculture systems to ensure soil quality, reduce runoff, and lessen energy consumption on agricultural

Conservation-tillage, or reduced-tillage, has been proven to provide multiple benefits in agricultural settings. In addition to erosion and runoff control, soil health improvement, and reduced energy demands, reduced-tillage practices can produce crops yields similar to that of conventional systems [3-5]. The use of reduced-tillage, however, can alter weed communities. Seed production by annual weed species remains, in most part, on the soil surface where it is subject to increased decomposition and predation. With reduced competition and minimized soil disruption, perennial weed species can become established and dominate the weed community in conservation-tillage [6]. To aid in the control of both annual and perennial weeds, the use of cover crops for ground cover can reduce herbicide requirements in conser‐

A number of cereal and legume cover crops are utilized in various crop productions for several purposes. Currently, a large portion of cover crops are planted as a green manure which are turned under prior to sowing the primary crop [7,8]. In reduced-tillage, however, cover crops are grown as a ground cover and remain on the soil surface after cover crop termination. In addition to further reducing soil erosion, increasing soil organic matter, and improving water infiltration, cover crops can provide a level of weed suppression both prior to and during the primary growing season [9]. When compared to fallow conservation-tillage systems, cover crops offer increased weed control through direct resource competition while actively growing as well as through shading and/or allelopathy after termination. Covers grown to produce high levels of biomass, in particular, can increase shading of germinating weed species and provide greater ground cover for an extended period during the growing season. When employing cover crops, however, knowledge concerning herbicide use both during cover crop

#### *2.3.1. Cover crop establishment and termination*

To produce substantial cover crop biomass, it is imperative to adequately manage cover crop production. Besides using correct seeding rates, early planting dates, and sufficient fertilizer applications, it is important to be aware of herbicide applications made prior to cover crop establishment. Often times, postemergent (POST) herbicides applied late season or postharvest can have residual carryover than may be detrimental to cover crops. Rotation restric‐ tions listed on herbicide labels should be referred to when planning POST applications and cover crop species.

To manage cover crops before cash crop planting, herbicides are typically utilized for cover crop termination. Most often, these herbicides, such as glyphosate and glufosinate, are nonselective with little to no carryover risk. However, consideration should be given to in-season chemical weed control regimes in order to limit repeated applications of a single herbicide mode of action. Moreover, care should be taken to avoid reduced herbicide rates applied for cover termination to reduce the risk of herbicide resistance [10]. Recent research has focused on mechanical termination with a roller or crimper which may reduce or eliminate the need of these herbicides for cover crop termination [11].

#### *2.3.2. Cash crop establishment and management*

Although use of in-season herbicides can be substantially reduced when using high-residue cover crops, some chemical applications are generally required to achieve the most effective weed suppression and minimize crop loss due to weed competition. While an ideal agricultural system would require no chemical inputs for sufficient weed control, practicality dictates the use of herbicides to guarantee crop yield since no system, as of yet, exists that can successfully suppress weed populations without intensive labor or mechanical requirements. To this end, cover crops are a means to minimize, rather than eliminate, herbicide inputs in crop systems. In recognizing the fact that the majority of agricultural systems will require chemical weed control measures for optimum crop production even when utilizing cover crops, it is essential to understand how cover crops affect herbicide selection and efficacy for each crop.

Primarily, the use of reduced-tillage and cover crops eliminates the ability to utilize preplant incorporated herbicides which offer residual soil activity [11]. Furthermore, cover crop residue can impede preemergent (PRE) herbicide applications from reaching the soil surface, reducing herbicide efficacy [12]. While postemergent chemical weed control can be effective alternatives in these settings, many weed species can prove to be difficult to control if not killed early in the season. Moreover, resistance concerns essentially necessitate the use of preemergent herbicides with differing mechanisms of action to avoid selection pressure for resistant weed biotypes [13].

Along with many cultural pracitces, production of crops under reduced-tillage with cover crops requires development of specific herbicide regimes to ensure minimal chemical inputs while achieving sufficient weed control to allow for successful crop production. The following sections review major crops produced globally, describe research conducted in respect to reduced-tillage production, as well as list available herbicides for use when using reducedtillage and cover crops. These reviews are designed to provide information that can be beneficial for producers implementing conservation-tillage.

diets in many countries, corn is also used for bioethanol production and the manufacturing of many non-food products. Consumption of corn and products derived from corn continues to increase. Given the demand, it is imperative for sustainable production systems that produce

Conservation-tillage practices have been researched and utilized for several decades in some regions such as the Midwest in the United States. As with many other crops, some variability has been noted for corn yield in no-tillage systems compared to conventional tillage methods. However, many reports show at least equal corn yields can be achieved when tillage practices are reduced [3]. Adequate yield potential, coupled with the reduction of on-farm expenses,

**Timing**

Burndown

Pyrasulfotole + Bromoxynil HuskieTM [28] Early POST Emerged broadleaf seedlings such as

**Weed Species Controlled**

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[27] PRE or POSTb *Bromus* species, annual ryegrass (*Lolium*

*amplexicaule*)

POST Actively growing broadleaves, wild garlic

(*Cirsium arvense*)

[30] POST Broadleaves henbit and chickweed (*Stellaria*

types)

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author

Non-selective control of emerged broadleaves

*multiflorum*), kochia (*Kochia scoparia*)

dandelion (*Taraxacum officinale*); suppression of established dandelion and henbit (*Lamium*

(*Allium vineale*); suppression of Canada thistle

*media*), grasses barnyardgrass (*Echinochloa crus-galli*), jointed goatgrass (*Aegilops cylindrica*), volunteer cereals (non-Clearfield

high yields while preserving long-term productivity of the land to be implemented.

have made conservation-tillage systems a good fit for corn production.

[23] Preplant

and grasses Glufosinate Liberty® [24]

[25]

[26]

WeatherMax®

Extra [29]

**Common Name Trade Namea Application**

**Herbicide**

Carfentrazone Aim®

Glyphosate Roundup

Paraquat Gramoxone®

Chlorsulfuron + Metsulfuron Finesse®

Thifensulfuron + Tribenuron Harmony®

Imazamox Beyond®

bPRE, preemergence; POST, postemergence.

**Table 1.** Herbicides for use in reduced-tillage wheat production.

*Clearfield wheat*

endorsement.

a

### **3. Wheat**

Global production of wheat (*Triticum aestivum* L.) was estimated at approximately 217 million hectares in 2010 [14] representing the largest single crop, in area grown, and providing approximately 19% of the caloric intake of the world's diet [15]. In recent years, concerns have been noted over stagnant wheat yields due to drought and rising temperatures attributed to global warming [16]. Efforts to maintain current wheat production levels and identify potential measures to aid in increasing yield have led researchers to explore conservation practices in wheat systems. In addition to preserving high crop yields, long-term conservation systems are intended to protect environmental quality and reduce chemical and energy inputs necessary for crop production. Components of conservation systems such as reduced- or no-tillage can produce crop yields equal to or exceeding conventional tillage practices while reducing erosion, water runoff, and increasing water infiltration.

Much research has been conducted to evaluate wheat productivity in conservation-tillage practices. Reports reveal similar or increased grain yield for reduced-tillage compared to conventional tillage systems [17-19]. With little or no tillage operations, some chemical applications are required to achieve successful levels of weed control; however, with herbicide applications, weed species have been effectively controlled below levels that could reduce yield [20]. To offset the herbicide needs in conservation-tillage, evaluations of cover crops as ground cover have been conducted. Crops such as mustard (*Sinapis alba* L.), pea (*Pisum sativum* L.), and lentil (*Lens culinaris* Medik.) have proven to be good choices with little yield differences [21]. However, other reports show negative impacts on wheat production when implementing cover crops prior to wheat production for reasons such as increased weed competition, primarily *Bromus* spp., and reduced fertilizer uptake [22].

Like most crops produced in conservation-tillage, herbicide options may be limited to a degree whether utilizing a cover crop or not. With reduced-tillage, preplant incorporation of residual herbicides cannot be utilized. Moreover, when planting into cover crops, soil-applied pre‐ emergent herbicides may be less effective due to interception by crop residue. When planting wheat, preplant burndown herbicides may be necessary to control early weeds. POST herbicides are also necessary to control weeds that germinate after planting. Table 1 lists many of the herbicide options for use in conservation-tillage systems for wheat production.

### **4. Maize**

Maize, or corn (*Zea mays* L.), is one of the most economically important grain crops worldwide with 162 million ha produced in 2010 [2]. In addition to being a staple in human and livestock diets in many countries, corn is also used for bioethanol production and the manufacturing of many non-food products. Consumption of corn and products derived from corn continues to increase. Given the demand, it is imperative for sustainable production systems that produce high yields while preserving long-term productivity of the land to be implemented.

Conservation-tillage practices have been researched and utilized for several decades in some regions such as the Midwest in the United States. As with many other crops, some variability has been noted for corn yield in no-tillage systems compared to conventional tillage methods. However, many reports show at least equal corn yields can be achieved when tillage practices are reduced [3]. Adequate yield potential, coupled with the reduction of on-farm expenses, have made conservation-tillage systems a good fit for corn production.


a Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

bPRE, preemergence; POST, postemergence.

sections review major crops produced globally, describe research conducted in respect to reduced-tillage production, as well as list available herbicides for use when using reducedtillage and cover crops. These reviews are designed to provide information that can be

Global production of wheat (*Triticum aestivum* L.) was estimated at approximately 217 million hectares in 2010 [14] representing the largest single crop, in area grown, and providing approximately 19% of the caloric intake of the world's diet [15]. In recent years, concerns have been noted over stagnant wheat yields due to drought and rising temperatures attributed to global warming [16]. Efforts to maintain current wheat production levels and identify potential measures to aid in increasing yield have led researchers to explore conservation practices in wheat systems. In addition to preserving high crop yields, long-term conservation systems are intended to protect environmental quality and reduce chemical and energy inputs necessary for crop production. Components of conservation systems such as reduced- or no-tillage can produce crop yields equal to or exceeding conventional tillage practices while reducing

Much research has been conducted to evaluate wheat productivity in conservation-tillage practices. Reports reveal similar or increased grain yield for reduced-tillage compared to conventional tillage systems [17-19]. With little or no tillage operations, some chemical applications are required to achieve successful levels of weed control; however, with herbicide applications, weed species have been effectively controlled below levels that could reduce yield [20]. To offset the herbicide needs in conservation-tillage, evaluations of cover crops as ground cover have been conducted. Crops such as mustard (*Sinapis alba* L.), pea (*Pisum sativum* L.), and lentil (*Lens culinaris* Medik.) have proven to be good choices with little yield differences [21]. However, other reports show negative impacts on wheat production when implementing cover crops prior to wheat production for reasons such as increased weed

Like most crops produced in conservation-tillage, herbicide options may be limited to a degree whether utilizing a cover crop or not. With reduced-tillage, preplant incorporation of residual herbicides cannot be utilized. Moreover, when planting into cover crops, soil-applied pre‐ emergent herbicides may be less effective due to interception by crop residue. When planting wheat, preplant burndown herbicides may be necessary to control early weeds. POST herbicides are also necessary to control weeds that germinate after planting. Table 1 lists many

Maize, or corn (*Zea mays* L.), is one of the most economically important grain crops worldwide with 162 million ha produced in 2010 [2]. In addition to being a staple in human and livestock

of the herbicide options for use in conservation-tillage systems for wheat production.

beneficial for producers implementing conservation-tillage.

566 Herbicides - Current Research and Case Studies in Use

erosion, water runoff, and increasing water infiltration.

competition, primarily *Bromus* spp., and reduced fertilizer uptake [22].

**3. Wheat**

**4. Maize**

**Table 1.** Herbicides for use in reduced-tillage wheat production.

A major limiting factor to adopting reduced-tillage in corn production is the concern of less effective weed control. Tillage has long been used as a means for weed seed burial which reduces the number of seeds in the upper portion of the soil, the area most favorable for germination for most species. In addition to weed seed remaining in the upper layer of soil, shifts in weed species have also been noted. With the implementation of conservation-tillage, most crop systems experience a shift in weed species from annuals to perennials dominating the weed community.

**Herbicide**

Flumioxazin Valor®

Pendimethalin Prowl®

Carfentrazone Aim®

Bromoxynil Buctril®

Dicamba Banvel®

Mesotrione Callisto®

Tembotrione Laudis®

Ametryn Evik®

Linuron Lorox®

Lightning®

*Clearfield Corn* Imazethapyr + Imazapyr

*LibertyLink Corn*

*Roundup Ready Corn*

*S*-metolachlor Dual Magnum®

**Common Name Trade Namea Application Timing Weed Species Controlled**

[39] Broadleaf species such as horseweed (*Conyza*

*indica*)

[40] Germinating, small-seeded grass and broadleaf

[23] POSTc Certain broadleaf weed control; tank mix with

[42] Broadleaf weeds such as burcucumber (*Sicyos*

[43] Annual broadleaf species as well as certain

[44] POST Broadleaf species such as wild mustard (*Sinapis*

[45] Broadleaf and grass species such as common

*sanguinalis*)

 [46] POST-directed spray Grass species such as Texas panicum, goosegrass, and foxtail

[47] Broadleaf and grass species such as dog fennel,

[48] POST Broadleaves, grasses, and sedges such as kochia,

Glufosinate Liberty® POST Broadleaf and grass species; ragweed, horseweed,

nutsedge (*Cyperus* spp.)

johnsongrass seedlings

 [41] Grass and broadleaf species such as foxtail and *Amaranthus* spp.

atrazine or dicamba

wild onion (*Allium* sp.)

thistle (*Cirsium arvense*)

*canadensis*); suppression of grass species such as panicum (*Panicum* spp.) and goosegrass (*Eleusine*

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species such as crabgrass (*Digitaria* spp.) and common lambsquarters (*Chenopodium alba*)

*angulatus*), giant ragweed (*Ambrosia trifida*)

perennial species such as dock (*Rumex* spp.) and

*arvensis*), nightshade (*Solanum* spp.), and Canada

chickweed, purple deadnettle (*Lamium purpureum*), *Amaranthus* spp., and large crabgrass (*Digitaria*

common ragweed (*Ambrosia artemisiifolia*), velvetleaf, and annual ryegrass (*Lolium multiflorum*)

ragweed, quackgrass (*Elytrigia repens*), and

Perennial weed species, largely controlled with tillage practices, can thrive on less distur‐ bed crop land. For effective weed control, producers implementing reduced-tillage have relied on increased herbicide applications. To curb herbicide use, cover crops have been adopted in conjunction with reduced-tillage corn systems. Research has shown that utiliz‐ ing a legume or grain cover crop can reduce weed density and growth while not affecting corn yield [31,32]. For corn in particular, cover crops offer a potential benefit in addition to weed suppression. Adequate nitrogen availability is essential for corn development. The use of legume cover crops, such as hairy vetch (*Vicia villosa* Roth), red clover (*Trifolium pratense* L.), or medics (*Medicago* spp.), may provide a portion of corn nitrogen requirements and reduce fertilizer inputs into the system [33]. Some research indicates that legume covers do not reduce fertilizer requirements but improves grain production with standard fertilizer applications [34]. Other research shows that legume covers can provide some nitrogen required for successful corn production[35,36]. Selecting the right legume cover crop for maximum nitrogen contribution with timely availability for corn uptake is key for utilizing these crops as nitrogen sources.

Use of burndown herbicides prior to corn planting is critical for early season weed control when using cover crops. A residual herbicide applied in conjunction with the herbicide used for cover crop termination can broaden weed species controlled as well as extend control into the season. A number of PRE herbicides are available that can be applied without incorporation into the soil and are effective even with plant residue on the soil surface. These herbicides and POST herbicide choices that can be successfully utilized in conservation-tillage corn with cover crops are listed in Table 2.



A major limiting factor to adopting reduced-tillage in corn production is the concern of less effective weed control. Tillage has long been used as a means for weed seed burial which reduces the number of seeds in the upper portion of the soil, the area most favorable for germination for most species. In addition to weed seed remaining in the upper layer of soil, shifts in weed species have also been noted. With the implementation of conservation-tillage, most crop systems experience a shift in weed species from annuals to perennials dominating

Perennial weed species, largely controlled with tillage practices, can thrive on less distur‐ bed crop land. For effective weed control, producers implementing reduced-tillage have relied on increased herbicide applications. To curb herbicide use, cover crops have been adopted in conjunction with reduced-tillage corn systems. Research has shown that utiliz‐ ing a legume or grain cover crop can reduce weed density and growth while not affecting corn yield [31,32]. For corn in particular, cover crops offer a potential benefit in addition to weed suppression. Adequate nitrogen availability is essential for corn development. The use of legume cover crops, such as hairy vetch (*Vicia villosa* Roth), red clover (*Trifolium pratense* L.), or medics (*Medicago* spp.), may provide a portion of corn nitrogen requirements and reduce fertilizer inputs into the system [33]. Some research indicates that legume covers do not reduce fertilizer requirements but improves grain production with standard fertilizer applications [34]. Other research shows that legume covers can provide some nitrogen required for successful corn production[35,36]. Selecting the right legume cover crop for maximum nitrogen contribution with timely availability for corn uptake is key for utilizing

Use of burndown herbicides prior to corn planting is critical for early season weed control when using cover crops. A residual herbicide applied in conjunction with the herbicide used for cover crop termination can broaden weed species controlled as well as extend control into the season. A number of PRE herbicides are available that can be applied without incorporation into the soil and are effective even with plant residue on the soil surface. These herbicides and POST herbicide choices that can be successfully utilized in conservation-tillage corn with cover

[24] Preplant burndown Emerged weed species

Broadleaves such as kochia (*Kochia scoparia*); suppression of foxtail (*Setaria* spp.), velvetleaf (*Abutilon theophrasti*). Can also be applied POST

**Common Name Trade Namea Application Timing Weed Species Controlled**

[25]

[38] Preplant

or PREb

[26]

the weed community.

568 Herbicides - Current Research and Case Studies in Use

these crops as nitrogen sources.

crops are listed in Table 2.

Glufosinate Liberty®

Glyphosate Roundup

Paraquat Gramoxone®

Atrazine Aatrex®

2,4-D Agri Star® 2,4-D [37]

WeatherMax®

**Herbicide**


reduced weed biomass in upland rice [55]. Future research needs include addressing the effects

Due to challenging weed issues in rice systems, particularly dry-seeded rice, herbicide use will continue to be necessary for effective weed suppression in both conventional and reducedtillage systems. The implementation of cover crops into these systems may lessen the herbicide requirements but will not eliminate the use of chemicals altogether. Currently there are a number of preemergent and postemergent herbicides available for use in rice production (Table 3); however, as dry-seeded, conservation-tillage rice systems increase in popularity,

[56] PREb Grass species such as barnyardgrass

[57] Broadleaf species such as dayflower (*Commelina*

application.

[40] Germinating, small-seeded grass and broadleaf

*alba*)

[58] Broadleaf and grass species such as

[59] Grass and broadleaf species such as

(*Echinochloa crus-galli*), crabgrass (*Digitaria* spp.), and panicum (*Panicum* spp.)

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*erecta*) and kochia (*Kochia scoparia*). Broadleaf and grass species may be controlled with a POST

species such as crabgrass (*Digitaria* spp.), foxtail, and common lambsquarters (*Chenopodium*

morningglory (*Ipomoea* spp.), and barnyardgrass. Can also be applied POST

barnyardgrass, dayflower (*Commelina communis*), and eclipta (*Eclipta alba*)

eclipta, and yellow nutsedge (*Cyperus*

(*Xanthium strumarium*), dayflower, and

weeds such as ducksalad (*Heteranthera limosa*) and ricefield bulrush (*Scirpus mucronatus*)

POSTc Grasses and broadleaves such as foxtail (*Setaria* spp.), panicum, and eclipta

[61] Broadleaf and sedge species, particularly aquatic

[62] POST Broadleaf and sedge species such as dayflower,

*esculentus*)

*Amaranthus* spp.

[23] Broadleaf species such as common cocklebur

of cover crops on rice production in dry-seeded rice systems.

**Common Name Trade Namea Application Timing Weed Species Controlled**

more herbicide options may become available.

**Herbicide**

Clomazone Command®

Halosulfuron Permit®

Pendimethalin Prowl®

Quinclorac Facet®

Thiobencarb Bolero®

Acifluorfen Ultra

Bensulfuron Londax®

Bentazon Basagran®

Carfentrazone Aim®

Blazer® [60]

a Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

bPRE, preemergence.

c POST, postemergence.

**Table 2.** Herbicides for use in reduced-tillage maize production.

#### **5. Rice**

Production of rice (*Oryza sativa* L.) in 2010 was near 154 million ha worldwide [2]. In many regions, rice provides nearly half or more of calories consumed by humans [50] and is the most important grain crop grown. Rice yield has steadily grown in the past several decades due to breeding and fertilizer advancements; however, it is necessary for rice yield to continue to improve in order to meet increased demands by a growing world population. Given that little land exists in rice-producing countries to expand production, it is necessary for methods to be established that can continue yield improvement without depleting future soil productivity.

Wetland, transplant rice production is the dominant and highest yielding rice system in most regions [50, 51]. However, the water and energy requirements may limit rice production as competition for resources increases [52]. To reduce strain on environmental and economic resources and to ensure sustainable rice systems in the future, dry-seeded rice production has been implemented in some areas [53]. Dry-seeded rice production can be initiated in conjunc‐ tion with conservation-tillage with fewer water demands, lower energy and labor require‐ ments, and reduced soil erosion. Research has reported that dry-seeded rice in no-tillage can be a successful alternative to conventional systems [52].

A limiting factor to widespread adoption of dry-seeded, reduced-tillage rice, however, is reduced weed control. For rice, transitioning from wetland, conventional systems to a dry system with reduced-tillage can affect weed compositions in multiple ways. Standing water can reduce germinating weed seeds while the transplanted rice becomes established; removing this water barrier can increase weed numbers [54]. Additionally, reduced-tillage practices can result in an increase of weed seed germination due to less seed burial.

In dry-seeded rice, mulches have been suggested as a means to combat weed increases [51]. Little research has been conducted to fully understand the benefits of cover crops for weed control in rice; however, legume covers have been associated with increased rice yield and reduced weed biomass in upland rice [55]. Future research needs include addressing the effects of cover crops on rice production in dry-seeded rice systems.

**Herbicide**

Glyphosate + *s*-metolachlor + atrazine

bPRE, preemergence.

**5. Rice**

POST, postemergence.

a

c

Glyphosate Roundup

Weathermax®

**Table 2.** Herbicides for use in reduced-tillage maize production.

be a successful alternative to conventional systems [52].

result in an increase of weed seed germination due to less seed burial.

Expert®

570 Herbicides - Current Research and Case Studies in Use

**Common Name Trade Namea Application Timing Weed Species Controlled**

POST Nonselective control of some broadleaf and grass

quackgrass, dandelion (*Taraxacum officinale*), and

species

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

Production of rice (*Oryza sativa* L.) in 2010 was near 154 million ha worldwide [2]. In many regions, rice provides nearly half or more of calories consumed by humans [50] and is the most important grain crop grown. Rice yield has steadily grown in the past several decades due to breeding and fertilizer advancements; however, it is necessary for rice yield to continue to improve in order to meet increased demands by a growing world population. Given that little land exists in rice-producing countries to expand production, it is necessary for methods to be established that can continue yield improvement without depleting future soil productivity. Wetland, transplant rice production is the dominant and highest yielding rice system in most regions [50, 51]. However, the water and energy requirements may limit rice production as competition for resources increases [52]. To reduce strain on environmental and economic resources and to ensure sustainable rice systems in the future, dry-seeded rice production has been implemented in some areas [53]. Dry-seeded rice production can be initiated in conjunc‐ tion with conservation-tillage with fewer water demands, lower energy and labor require‐ ments, and reduced soil erosion. Research has reported that dry-seeded rice in no-tillage can

A limiting factor to widespread adoption of dry-seeded, reduced-tillage rice, however, is reduced weed control. For rice, transitioning from wetland, conventional systems to a dry system with reduced-tillage can affect weed compositions in multiple ways. Standing water can reduce germinating weed seeds while the transplanted rice becomes established; removing this water barrier can increase weed numbers [54]. Additionally, reduced-tillage practices can

In dry-seeded rice, mulches have been suggested as a means to combat weed increases [51]. Little research has been conducted to fully understand the benefits of cover crops for weed control in rice; however, legume covers have been associated with increased rice yield and

[49] PRE or POST Annual broadleaves and grasses; perennials such as

Canada thistle

Due to challenging weed issues in rice systems, particularly dry-seeded rice, herbicide use will continue to be necessary for effective weed suppression in both conventional and reducedtillage systems. The implementation of cover crops into these systems may lessen the herbicide requirements but will not eliminate the use of chemicals altogether. Currently there are a number of preemergent and postemergent herbicides available for use in rice production (Table 3); however, as dry-seeded, conservation-tillage rice systems increase in popularity, more herbicide options may become available.



Early work in conservation-tillage soybean have reported equal or improved yield in soybean with reduced-tillage compared to conventional systems [68, 69]. Previous research has also examined soybean systems planted behind wheat or a cover crop such as rye with improved weed control being noted when compared to a fallow system [70] and greater yield with a cover crop than with just the previous crop's stubble [71]. The inclusion of plant residue, either from a cover crop or a previous crop, provides a level of weed control by acting as a physical barrier for germinating weed seed or through allelopathic inhibition released by some cover crop species. The weed control provided by ground cover is crucial in a no-till practice due to the loss of control from tillage reduction and the shift towards more difficult to control

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While cover crops and plant residue have been identified as means to reduce weed emergence when implemented in reduced-tillage practices further measures are required to keep the weed population below an acceptable level [70]. Many cultural practices, such as crop rotation, row spacing, and planting date, can be manipulated in such a way as to reduce weed populations;

As with most field crops grown in conservation-tillage systems, soybean production with reduced-tillage has heavily relied on postemergent herbicide applications. Use of cover crops in these systems may also contribute to the tendency for fewer PRE herbicides due to inter‐ ception concerns. However, the increase in herbicide-resistant weed species such as Palmer amaranth (*Amaranthus palmeri* S. Wats) and horsweed [*Conyza canadensis* (L.) Cronq.] in herbicide resistant crops, like soybean, necessitates the use of multiple herbicides to slow the development of weed resitance and safeguard the effectiveness of current herbicide options for the future. Table 4 provides a partial list of herbicides that can be utilized in reduced-tillage

[24] Preplant Burndown Emerged weed species

[56] PREb Grasses and broadleaves such as crabgrass (*Digitaria*

[72] Grass and broadleaf species such as foxtail (*Setaria*

[39] Broadleaf species such as horseweed (*Conyza*

*tortuosum*)

spp.), panicum (*Panicum* spp.), velvetleaf (*Abutilon theophrasti*), and Florida beggarweed (*Desmodium*

*canadensis*); suppression of grass species such as panicum and goosegrass (*Eleusine indica*)

spp.), panicum, and *Amaranthus* spp.

**Common Name Trade Namea Application Timing Weed Species Controlled**

[25]

[26]

2,4-D [37]

however, herbicide use is still necessary in many systems.

perennial weed species.

soybean with cover crops.

Glufosinate Liberty®

Paraquat Gramoxone®

2,4-D Agri Star®

Clomazone Command®

Dimethenamid Outlook®

Flumioxazin Valor®

Glyphosate Roundup WeatherMax®

**Herbicide**

a Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

bPRE, preemergence.

c POST, postemergence.

**Table 3.** Herbicides for use in reduced-tillage rice production.

### **6. Soybean**

Production of soybean [*Glycine max* (L.) Merr.], estimated at 102 million ha in 2010 [2], meets a number of livestock and human food needs as well as industrial demands for use in products such as paints, lubricants, and biofuel. Due to its diversity of uses, the soybean is an important field crop for much of the world. In light of the value of soybeans, it is essential to establish sustainable growing practices to ensure global demand continues to be met.

Implementation of conservation practices, such as reduced-tillage, can be utilized as compo‐ nents of alternative management systems replacing conventional systems to provide erosion and runoff control while reducing labor and cost inputs. In the United States, in fact, approx‐ imately 80% of soybeans were produced with some form of conservation-tillage by 2006 [67]. This increase in conservation-tillage can be attributed to the environmental and economic benefits achieved with reduced-tillage as well as the commercial availability of herbicidetolerant soybeans, which have made successful chemical weed control achievable with the use of fewer herbicides.

Early work in conservation-tillage soybean have reported equal or improved yield in soybean with reduced-tillage compared to conventional systems [68, 69]. Previous research has also examined soybean systems planted behind wheat or a cover crop such as rye with improved weed control being noted when compared to a fallow system [70] and greater yield with a cover crop than with just the previous crop's stubble [71]. The inclusion of plant residue, either from a cover crop or a previous crop, provides a level of weed control by acting as a physical barrier for germinating weed seed or through allelopathic inhibition released by some cover crop species. The weed control provided by ground cover is crucial in a no-till practice due to the loss of control from tillage reduction and the shift towards more difficult to control perennial weed species.

**Herbicide**

*Clearfield Rice*

Imazethapyr + Quinclorac

endorsement.

bPRE, preemergence.

**6. Soybean**

of fewer herbicides.

POST, postemergence.

a

c

Propanil Stam®

572 Herbicides - Current Research and Case Studies in Use

Cyhalofop Clincher®

2,4-D Agri Star®

Imazamox Beyond®

Imazethapyr Newpath®

2,4-D [37]

Clearpath®

**Table 3.** Herbicides for use in reduced-tillage rice production.

**Common Name Trade Namea Application Timing Weed Species Controlled**

[63] Grass, rush, and broadleaf species such as

[30] POST Grass and broadleaf species such as

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author

Production of soybean [*Glycine max* (L.) Merr.], estimated at 102 million ha in 2010 [2], meets a number of livestock and human food needs as well as industrial demands for use in products such as paints, lubricants, and biofuel. Due to its diversity of uses, the soybean is an important field crop for much of the world. In light of the value of soybeans, it is essential to establish

Implementation of conservation practices, such as reduced-tillage, can be utilized as compo‐ nents of alternative management systems replacing conventional systems to provide erosion and runoff control while reducing labor and cost inputs. In the United States, in fact, approx‐ imately 80% of soybeans were produced with some form of conservation-tillage by 2006 [67]. This increase in conservation-tillage can be attributed to the environmental and economic benefits achieved with reduced-tillage as well as the commercial availability of herbicidetolerant soybeans, which have made successful chemical weed control achievable with the use

sustainable growing practices to ensure global demand continues to be met.

[65] Grass, sedge, and broadleaf species such as

[66] Grass, sedge, and broadleaf species such as

[64] After Flooding Grass species such as barnyardgrass, broadleaf

barnyardgrass, spikerush (*Eleocharis* spp.), and

signalgrass (*Brachiaria platyphylla*), and junglerice (*Echnochloa colona*)

Annual and perennial weed species such as cocklebur, morningglory, and dock

morningglory, barnyardgrass, and panicum

barnyardgrass, morningglory, and nutsedge

junglerice, eclipta, morningglory, and nutsedge

curly dock (*Rumex crispus*)

While cover crops and plant residue have been identified as means to reduce weed emergence when implemented in reduced-tillage practices further measures are required to keep the weed population below an acceptable level [70]. Many cultural practices, such as crop rotation, row spacing, and planting date, can be manipulated in such a way as to reduce weed populations; however, herbicide use is still necessary in many systems.

As with most field crops grown in conservation-tillage systems, soybean production with reduced-tillage has heavily relied on postemergent herbicide applications. Use of cover crops in these systems may also contribute to the tendency for fewer PRE herbicides due to inter‐ ception concerns. However, the increase in herbicide-resistant weed species such as Palmer amaranth (*Amaranthus palmeri* S. Wats) and horsweed [*Conyza canadensis* (L.) Cronq.] in herbicide resistant crops, like soybean, necessitates the use of multiple herbicides to slow the development of weed resitance and safeguard the effectiveness of current herbicide options for the future. Table 4 provides a partial list of herbicides that can be utilized in reduced-tillage soybean with cover crops.



**7. Cotton**

Cotton production around the world is estimated at approximately 23 million tonnes (lint production) [2] with China, India, and the United States being the top producers [82]. Efforts to adopt sustainable cotton practices have led producers to utilize conservation-tillage systems in cotton production. Besides environmental benefits achieved with reduced-tillage, major economic advantages can be realized due to reduced time, labor, and fuel requirements when operating with less tillage. Prior to the introduction of herbicide-resistant crops, adoption of reduced-tillage was difficult due to control of weed species required multiple and costly herbicide inputs [13]. In some instances, effective herbicides were not available to control problematic weed species such as perennials that can thrive in reduced-tillage. When glyph‐ osate-resistant cotton was made available, reduced-tillage became practical since a broad

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Extensive research has been carried out in conservation-tillage cotton with positive benefits seen for cotton yield [84-86]. Moreover, with herbicide-resistant cotton varieties, weed control has been as successful as conventional tillage cotton. Because of this success, conservationtillage practices have been widely adopted in areas such as the southeastern United States. This dependence on a single herbicide, however, has led to the appearance of herbicideresistant weed species and now threatens the feasibility of reduced-tillage cotton production. Currently, research efforts are focused on identifying ways to ensure the long-term viability of conservation-tillage while controlling established populations of herbicide-resistant weed

Multiple weed management tactics are necessary to control weed resistance development with cover crops playing an important role in resistance management. The use of cover crops, particularly high-residue crops such as rye and black oat, can reduce herbicide inputs through shading and allelopathy. The use of high-residue crops allows for maximum shading of the soil surface during the beginning of the season while also providing a ground cover for a longer period into the growing season. Cover crops, along with multiple herbicide modes of action and rotation, have been shown to effectively control weeds in reduced-tillage cotton [87, 88]. A number of herbicide choices are available for use with conservation-tillage cotton (Table 5). PRE herbicides are especially important in early-season weed control to ensure manage‐ ment of weed species that are difficult to control later in the season. Although concerns have been raised as to whether cover crops reduce the efficacy of PRE herbicides, it has been suggested that any loss in weed control due to herbicide interception is offset by the control

spectrum of weed species could be controlled with a single herbicide [83].

species and reducing the risk of future development of resistant weeds.

**Common Name Trade Namea Application Timing Weed Species Controlled**

[43] Preplant Burndown Emerged weed species

provided by cover crop residue [89-91].

[39]

[24]

**Herbicide**

Dicamba Banvel®

Flumioxazin Valor®

Glufosinate Liberty®

a Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

bPRE, preemergence.

c POST, postemergence.

**Table 4.** Herbicides for use in reduced-tillage soybean production.

### **7. Cotton**

**Herbicide**

Imazaquin Scepter®

574 Herbicides - Current Research and Case Studies in Use

Pendimethalin Prowl®

*S-*metolachlor Dual Magnum®

Bentazon Basagran®

Chlorimuron Classic®

Cloransulam FirstRate®

Fluazifop Fusilade®

Imazethapyr Pursuit®

Lactofen Cobra®

Sethoxydim Poast®

Flexstar®

**Table 4.** Herbicides for use in reduced-tillage soybean production.

*LibertyLink Soybean*

*Roundup Ready Soybean* Fomesafen + Glyphosate

endorsement.

bPRE, preemergence.

POST, postemergence.

a

c

**Common Name Trade Namea Application Timing Weed Species Controlled**

Metribuzin Metribuzin [74] Broadleaf and grass species such as *Amaranthus*

[73] Broadleaf and grass species such as morningglory

[40] Grass and broadleaf species such as panicum and

[62] POSTc Broadleaf weeds such as coffee senna (*Senna*

[75] Broadleaf weeds such as Florida beggarweed and

[76] Broadleaf weeds such as common cocklebur

[77] Annual and perennial grass species such as crabgrass

[78] Broadleaf and grass species such as morningglory

[79] Broadleaf species such as croton (*Croton* spp.) and

[80] Grass species such as foxtail, crabgrass, and panicum

[81] POST Broadleaf and grass species such as morningglory,

Glufosinate Liberty® POST Broadleaf and grass species such as *Amaranthus* spp.,

Glyphosate Roundup WeatherMax® POST Grass and broadleaf species such as Florida

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author

[41] Grass and broadleaves such as barnyardgrass

*Amaranthus* spp.

(*Richardia scabra*)

morningglory

and crabgrass

Florida beggarweed

morningglory, and goosegrass

velvetleaf, and broadleaf signalgrass

beggarweed, crabgrass and groundcherry

*occidentalis*) and velvetleaf

(*Xanthium strumarium*) and velvetleaf

and bermudagrass (*Cynadon dactylon*)

(*Ipomoea* spp.), velvetleaf, and foxtail

spp.and broadleaf signalgrass (*Brachiaria platyphylla*)

(*Echinochloa crus-galli*), crabgrass, and Florida pusley

Cotton production around the world is estimated at approximately 23 million tonnes (lint production) [2] with China, India, and the United States being the top producers [82]. Efforts to adopt sustainable cotton practices have led producers to utilize conservation-tillage systems in cotton production. Besides environmental benefits achieved with reduced-tillage, major economic advantages can be realized due to reduced time, labor, and fuel requirements when operating with less tillage. Prior to the introduction of herbicide-resistant crops, adoption of reduced-tillage was difficult due to control of weed species required multiple and costly herbicide inputs [13]. In some instances, effective herbicides were not available to control problematic weed species such as perennials that can thrive in reduced-tillage. When glyph‐ osate-resistant cotton was made available, reduced-tillage became practical since a broad spectrum of weed species could be controlled with a single herbicide [83].

Extensive research has been carried out in conservation-tillage cotton with positive benefits seen for cotton yield [84-86]. Moreover, with herbicide-resistant cotton varieties, weed control has been as successful as conventional tillage cotton. Because of this success, conservationtillage practices have been widely adopted in areas such as the southeastern United States. This dependence on a single herbicide, however, has led to the appearance of herbicideresistant weed species and now threatens the feasibility of reduced-tillage cotton production. Currently, research efforts are focused on identifying ways to ensure the long-term viability of conservation-tillage while controlling established populations of herbicide-resistant weed species and reducing the risk of future development of resistant weeds.

Multiple weed management tactics are necessary to control weed resistance development with cover crops playing an important role in resistance management. The use of cover crops, particularly high-residue crops such as rye and black oat, can reduce herbicide inputs through shading and allelopathy. The use of high-residue crops allows for maximum shading of the soil surface during the beginning of the season while also providing a ground cover for a longer period into the growing season. Cover crops, along with multiple herbicide modes of action and rotation, have been shown to effectively control weeds in reduced-tillage cotton [87, 88].

A number of herbicide choices are available for use with conservation-tillage cotton (Table 5). PRE herbicides are especially important in early-season weed control to ensure manage‐ ment of weed species that are difficult to control later in the season. Although concerns have been raised as to whether cover crops reduce the efficacy of PRE herbicides, it has been suggested that any loss in weed control due to herbicide interception is offset by the control provided by cover crop residue [89-91].



**Herbicide**

a

c

endorsement. bPRE, preemergence.

**8. Peanut**

*LibertyLink Cotton*

*Roundup Ready Cotton*

POST, postemergence.

**Table 5.** Herbicides for use in reduced-tillage cotton.

**Common Name Trade Namea Application Timing Weed Species Controlled**

Glufosinate Liberty® POST Broadleaf and grass species such as

Glyphosate Roundup WeatherMax® POST Grass and broadleaf species such as Florida

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author

Groundnut, or peanut (*Arachis hypogaea* L.), was planted on approximately 21 million ha between 2011 and 2012 wordwide with top production occurring in China, India, Indonesia, the United States, and some African countries such as Nigeria, Senegal, and Sudan [100]. Besides being a nutrient rich food source, the peanut is utilized for its oil in cooking and manufacturing as well as a livestock feed. In the United States, peanuts offer an exceptional rotational crop with cotton to replenish soil nitrogen. The benefits of peanuts to a cotton system, which have been shifting toward long-term, reduced-tillage practices, have necessitated the

The increased farming costs of conventional tillage systems have spurred producers to implement conservation-tillage to reduce expenses; however, peanut growers face unique difficulties when using these systems [101,102]. Particularly, concerns over peanut response to reduced-tillage due to peanut growth habits have required research in order to identify successful means of conservation-tillage integration into peanut production [102, 103].

Peanut yield variability under reduced-tillage compared to conventional tillage has been noted as one of the greatest concerns when adopting conservation-tillage practices [101,102]. Inconsistent yield response by peanut has been noted in previous studies investigating conservation-tillage. Research has reported yields of peanut to be reduced or equal to con‐ ventionally tilled peanut [101, 104]; other studies have shown reduced-tillage peanuts to produce equally or greater than conventional tillage peanuts [103,105]. Research efforts continue to recognize the contributing factors that affect peanut response to tillage systems.

adoption of minimum tillage practices in peanut production as well.

*Amaranthus* spp., morningglory, and

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beggarweed, crabgrass, foxtail, groundcherry,

goosegrass

and velvetleaf


**Herbicide**

Glyphosate Roundup

576 Herbicides - Current Research and Case Studies in Use

Paraquat Gramoxone®

Clomazone Command®

Fluometuron Cotoran®

Pendimethalin Prowl®

Prometryn Caparol®

Clethodim Select®

Quizalofop Assure®

Sethoxydim Poast®

Trifloxysulfuron Envoke®

Diuron Direx®

Linuron Linex®

Herbicide

*S*-metolachlor Dual Magnum®

WeatherMax®

[25]

[26]

**Common Name Trade Namea Application Timing Weed Species Controlled**

Common Name Trade Name Application Timing Weed Species Controlled

[56] Preplant or PREb Grasses and broadleaves such as crabgrass

[92] Grasses and broadleaves such as signalgrass

[40] Grass and broadleaf species such as foxtail

[93] Annual grass and broadleaves such as

[41] Grass and broadleaves such as barnyardgrass

pusley

 [94] POSTc Grass species such as crabgrass, panicum, and foxtail

[95] Annual and perennial grasses such as foxtail,

 [80] POST Grass species such as foxtail, crabgrass, and panicum

[96] Broadleaf and grass species such as coffee

[97] POST-directed spray Broadleaf and grass species such as sicklepod,

[98] Broadleaves and grasses such as

MSMA MSMA [99] Grass and broadleaf species such as crabgrass,

(*Digitaria* spp.), panicum (*Panicum*spp.), velvetleaf (*Abutilon theophrasti*), and Florida beggarweed (*Desmodium tortuosum*)

(*Brachiaria sp.),* horseweed (*Conyza*

*canadensis*) and sicklepod (*Senna obtusifolia*)

(*Setaria* spp.), panicum, and *Amaranthus* spp.

groundcherry (*Physalis* sp.), Florida pusley

(*Echinochloa crus-galli*), crabgrass, and Florida

(*Richardia scabra*), and panicum

goosegrass (*Eleusine indica*), and bermudagrass (*Cynodon dactylon*)

and Florida beggarweed

velvetleaf, and crabgrass

senna (*Senna occidentalis*), barnyardgrass,

morningglory, Florida pusley, and panicum

Florida beggarweed, and *Amaranthus* spp.


#### bPRE, preemergence.

c POST, postemergence.

**Table 5.** Herbicides for use in reduced-tillage cotton.

#### **8. Peanut**

Groundnut, or peanut (*Arachis hypogaea* L.), was planted on approximately 21 million ha between 2011 and 2012 wordwide with top production occurring in China, India, Indonesia, the United States, and some African countries such as Nigeria, Senegal, and Sudan [100]. Besides being a nutrient rich food source, the peanut is utilized for its oil in cooking and manufacturing as well as a livestock feed. In the United States, peanuts offer an exceptional rotational crop with cotton to replenish soil nitrogen. The benefits of peanuts to a cotton system, which have been shifting toward long-term, reduced-tillage practices, have necessitated the adoption of minimum tillage practices in peanut production as well.

The increased farming costs of conventional tillage systems have spurred producers to implement conservation-tillage to reduce expenses; however, peanut growers face unique difficulties when using these systems [101,102]. Particularly, concerns over peanut response to reduced-tillage due to peanut growth habits have required research in order to identify successful means of conservation-tillage integration into peanut production [102, 103].

Peanut yield variability under reduced-tillage compared to conventional tillage has been noted as one of the greatest concerns when adopting conservation-tillage practices [101,102]. Inconsistent yield response by peanut has been noted in previous studies investigating conservation-tillage. Research has reported yields of peanut to be reduced or equal to con‐ ventionally tilled peanut [101, 104]; other studies have shown reduced-tillage peanuts to produce equally or greater than conventional tillage peanuts [103,105]. Research efforts continue to recognize the contributing factors that affect peanut response to tillage systems.

Weed management in conservation-tillage peanut is also a concern for producers. Weed control in peanut, regardless of tillage system, can be problematic due to the extended growing season and unique growth habits [106,107]. Generally, peanut production requires an incor‐ porated residual as well as a POST herbicide to provide effective weed control under the slowclosing canopy of peanut [107]. Moreover, in-season cultivation for weed management cannot be implemented due to the potential to damage developing peanut pods [106,108].

**Herbicide**

**Common Name Trade Namea Application**

[26]

2,4-D [37]

Glyphosate Roundup WeatherMax® [25]

Paraquat Gramoxone®

2,4-D Agri Star®

Diclosulam Strongarm®

Flumioxazin Valor®

Pendimethalin Prowl®

Acifluorfen Ultra Blazer®

Bentazon Basagran®

Chlorimuron Classic®

Clethodim Select®

Imazapic Cadre®

Imazethapyr Pursuit®

Sethoxydim Poast®

2,4-DB Butyrac®

a

c

endorsement. bPRE, preemergence.

POST, postemergence.

**Table 6.** Herbicides for use in reduced-tillage peanut.

**Timing**

Preplant Burndown **Weed Species Controlled**

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Emerged weed species

 [110] PREb Broadleaf species such as eclipta (*Eclipta prostrata*) and *Amaranthus* spp.

 [39] Broadleaf species such as horseweed (*Conyza canadensis*)

[40] Grass and broadleaf species such as foxtail (*Setaria*

*theophrasti*)

[75] Broadleaf weeds such as Florida beggarweed

[94] Grass species such as panicum, foxtail, and

[111] Broadleaf and grass species such as morningglory,

[78] Broadleaf, grass, and sedge species such as Florida

(*Cyperus* spp.)

 [112] Broadleaf species such as velvetleaf and prickly sida (*Sida spinosa*)

[80] Grass species, foxtail and panicum

Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author

Paraquat Gramoxone® Grass and broadleaf species

 [62] Broadleaf species such as morningglory (*Ipomoea* spp.) and velvetleaf

crabgrass (*Digitaria* spp.)

*Amaranthus* spp. and crabgrass

[60] POSTc Broadleaf and grass species such as coffee senna

spp.) and *Amaranthus* spp.

(*Senna occidentalis*) and velvetleaf (*Abutilon*

(*Desmodium tortuosum*) and morningglory

pusley (*Richardia scabra*), crabgrass, and nutsedge

Weed control in reduced-tillage peanuts can be even more difficult than in conventional tillage due to the loss of weed control through seed burial and the inability to utilize preplant incorporated herbicides [109]. This results in increased dependence on post emergent herbi‐ cides which may or may not control the number of perennial weed species that may predom‐ inate in a reduced-tillage setting; the loss of effective weed management can reduce peanut productivity due to weed competition [102,107].

Utilization of cover crops in peanut systems may offer beneficial weed control while reducing the need for increased postemergent herbicide applications. Research has shown effective weed control with cover crops in strip-tillage peanut systems that use a dinitroaniline pre‐ emergent herbicide over cover crop residue [107]. Other effective herbicides used in conser‐ vation-tillage peanut systems are listed in Table 6.

### **9. Herbicide interception**

Preemergent, residual herbicides must reach the soil surface to be effective. When spraying over cover crop residue, herbicide applications can be intercepted and absorbed prior to reaching the soil surface. Herbicides, such as acetochlor, chlorimuron, and oryzalin have been shown to be impeded by plant stubble [113,114]. While timely rainfall can move herbicides to the soil, some portion of herbicide can be retained in the residue.

Herbicide amounts intercepted by stubble can affect weed control achieved with the herbicide; efficacy can be reduced by cover crops either through physical interception preventing soil contact or through increased microbial activity in the residue speeding herbicide degradation [115]. Increases in soil organic matter from extended conservation-tillage practices may also increase herbicide adsorption within the soil [116]. Additionally, herbicide persistence and carryover risks may be increased when applied to residue [114]. Certain crops may be susceptible to herbicides at low doses that can persist in cover crop residue that would otherwise have dissipated in bare soil. However, little research has been done to determine the extent of persistence for most herbicides.

Methods to reduce herbicide interception are limited when using cover crops. Interception could potentially be managed, particularly in strip-till operations, through banded herbicide applications over the row allowing for in-row weed control while reducing herbicide inputs. Furthermore, a water-based, microencapsulated herbicide formulation, like Prowl H2O® (pendimethalin), may allow more herbicide to reach the soil after a rain event or irrigation.


a Trade names listed are representative of available herbicides. Inclusion of particular trade names does not suggest author endorsement.

bPRE, preemergence.

Weed management in conservation-tillage peanut is also a concern for producers. Weed control in peanut, regardless of tillage system, can be problematic due to the extended growing season and unique growth habits [106,107]. Generally, peanut production requires an incor‐ porated residual as well as a POST herbicide to provide effective weed control under the slowclosing canopy of peanut [107]. Moreover, in-season cultivation for weed management cannot

Weed control in reduced-tillage peanuts can be even more difficult than in conventional tillage due to the loss of weed control through seed burial and the inability to utilize preplant incorporated herbicides [109]. This results in increased dependence on post emergent herbi‐ cides which may or may not control the number of perennial weed species that may predom‐ inate in a reduced-tillage setting; the loss of effective weed management can reduce peanut

Utilization of cover crops in peanut systems may offer beneficial weed control while reducing the need for increased postemergent herbicide applications. Research has shown effective weed control with cover crops in strip-tillage peanut systems that use a dinitroaniline pre‐ emergent herbicide over cover crop residue [107]. Other effective herbicides used in conser‐

Preemergent, residual herbicides must reach the soil surface to be effective. When spraying over cover crop residue, herbicide applications can be intercepted and absorbed prior to reaching the soil surface. Herbicides, such as acetochlor, chlorimuron, and oryzalin have been shown to be impeded by plant stubble [113,114]. While timely rainfall can move herbicides to

Herbicide amounts intercepted by stubble can affect weed control achieved with the herbicide; efficacy can be reduced by cover crops either through physical interception preventing soil contact or through increased microbial activity in the residue speeding herbicide degradation [115]. Increases in soil organic matter from extended conservation-tillage practices may also increase herbicide adsorption within the soil [116]. Additionally, herbicide persistence and carryover risks may be increased when applied to residue [114]. Certain crops may be susceptible to herbicides at low doses that can persist in cover crop residue that would otherwise have dissipated in bare soil. However, little research has been done to determine

Methods to reduce herbicide interception are limited when using cover crops. Interception could potentially be managed, particularly in strip-till operations, through banded herbicide applications over the row allowing for in-row weed control while reducing herbicide inputs. Furthermore, a water-based, microencapsulated herbicide formulation, like Prowl H2O® (pendimethalin), may allow more herbicide to reach the soil after a rain event or irrigation.

be implemented due to the potential to damage developing peanut pods [106,108].

productivity due to weed competition [102,107].

578 Herbicides - Current Research and Case Studies in Use

vation-tillage peanut systems are listed in Table 6.

the extent of persistence for most herbicides.

the soil, some portion of herbicide can be retained in the residue.

**9. Herbicide interception**

c POST, postemergence.

**Table 6.** Herbicides for use in reduced-tillage peanut.

### **10. Conclusion**

The ever increasing demands on global agriculture dictate the use of intensive, high-yielding production practices. However, the inability to sustain these systems long-term necessitates the implementation of more energy-efficient, environmentally-sound practices that can still produce successful yields. Conservation agriculture practices seek to achieve these goals in order to ensure current and future agricultural production. While components of conservation agriculture, such as reduced-tillage and cover crops, are fundamental practices in these systems, herbicides are still valuable and necessary weed management tools within conser‐ vation systems. Integrating these management practices can be challenging and continue to warrant research to identify the most successful means of utilizes herbicides in conjunction with reduced-tillage and cover crops.

Rhodic Paleudult under simulated rainfall. Journal of Soil and Water Conservation,

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168-172, ISSN 0043-1745.

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3-16.

### **Author details**

Andrew J. Price1\* and Jessica A. Kelton2

\*Address all correspondence to: Andrew.price@ars.usda.gov

1 United States Department of Agriculture, Agricultural Research Service, National Soil Dy‐ namics Laboratory, Auburn, Alabama, USA

2 Auburn University, Auburn, Alabama, USA

### **References**


Rhodic Paleudult under simulated rainfall. Journal of Soil and Water Conservation, 2003; 58,258-267, ISSN 0022-4561.

**10. Conclusion**

**Author details**

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with reduced-tillage and cover crops.

580 Herbicides - Current Research and Case Studies in Use

Andrew J. Price1\* and Jessica A. Kelton2

namics Laboratory, Auburn, Alabama, USA

2 Auburn University, Auburn, Alabama, USA

\*Address all correspondence to: Andrew.price@ars.usda.gov

The ever increasing demands on global agriculture dictate the use of intensive, high-yielding production practices. However, the inability to sustain these systems long-term necessitates the implementation of more energy-efficient, environmentally-sound practices that can still produce successful yields. Conservation agriculture practices seek to achieve these goals in order to ensure current and future agricultural production. While components of conservation agriculture, such as reduced-tillage and cover crops, are fundamental practices in these systems, herbicides are still valuable and necessary weed management tools within conser‐ vation systems. Integrating these management practices can be challenging and continue to warrant research to identify the most successful means of utilizes herbicides in conjunction

1 United States Department of Agriculture, Agricultural Research Service, National Soil Dy‐

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**Chapter 23**

**Herbicide Safeners: Effective Tools to Improve Herbicide**

Herbicide safeners, formerly referred to as herbicide antidotes, are chemical agents that increase the tolerance of monocotyledonous cereal plants to herbicides without affecting the weed control effectiveness. The use of safeners offer several benefits to agricultural weed control. Safeners may allow: (1) the selective chemical control of weeds in botanically related crops; (2) the use of nonselective herbicides for selective weed control; (3) the counteraction of residual activity of soil-applied persistent herbicides such as triazines in crop rotation systems; (4) an increase in the spectrum of herbicides available for weed control in "minor" crops; (5) an expansion and extension of the uses and marketability of generic herbicides; (6) the elucidation of sites and mechanism by serving as useful biochemical tools [1]. The commercial viability of safener concept is indicated by the growing number of herbicide-safener products available on the pesticide market. With the use of safeners, difficult weed control problems can be addressed and without safeners, many herbicidally active substances could have never

The concept to enhance crop tolerance to nonselective herbicide by using chemical agents was introduced by Otto Hoffman. In the late 1940s Hoffmann serendipitiously found that no herbicide injury symptons were developed in tomato plants previously treated with 2,4,6-T, an inactive analogue of herbicide 2,4-D when plant were exposed accidentally to vapors of 2,4- D due to the malfunction of the ventillation system of the greenhouse [3]. Following this observation Hoffmann reported later the antagonistic effects of 2,4-D against herbicidal injury by barban after foliar treatments of wheat plants [4]. Research and development in finding new safeners as well as subsequent commercialization proceeded very intensively in the 1970s. Since the patent application of the safening properties of 1,8-naphthalic anhydride (NA) intensive research on discovery of new safeners resulted in compounds with diverse chemis‐

> © 2013 Jablonkai; 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 Jablonkai; 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.

**Selectivity**

Istvan Jablonkai

**1. Introduction**

http://dx.doi.org/10.5772/55168

been applied for weed control [2].

Additional information is available at the end of the chapter


## **Herbicide Safeners: Effective Tools to Improve Herbicide Selectivity**

Istvan Jablonkai

ces in Peanut Science. Stillwater, OK, USA: American Peanut Research Educational

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p.

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1445-6664.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55168

### **1. Introduction**

Herbicide safeners, formerly referred to as herbicide antidotes, are chemical agents that increase the tolerance of monocotyledonous cereal plants to herbicides without affecting the weed control effectiveness. The use of safeners offer several benefits to agricultural weed control. Safeners may allow: (1) the selective chemical control of weeds in botanically related crops; (2) the use of nonselective herbicides for selective weed control; (3) the counteraction of residual activity of soil-applied persistent herbicides such as triazines in crop rotation systems; (4) an increase in the spectrum of herbicides available for weed control in "minor" crops; (5) an expansion and extension of the uses and marketability of generic herbicides; (6) the elucidation of sites and mechanism by serving as useful biochemical tools [1]. The commercial viability of safener concept is indicated by the growing number of herbicide-safener products available on the pesticide market. With the use of safeners, difficult weed control problems can be addressed and without safeners, many herbicidally active substances could have never been applied for weed control [2].

The concept to enhance crop tolerance to nonselective herbicide by using chemical agents was introduced by Otto Hoffman. In the late 1940s Hoffmann serendipitiously found that no herbicide injury symptons were developed in tomato plants previously treated with 2,4,6-T, an inactive analogue of herbicide 2,4-D when plant were exposed accidentally to vapors of 2,4- D due to the malfunction of the ventillation system of the greenhouse [3]. Following this observation Hoffmann reported later the antagonistic effects of 2,4-D against herbicidal injury by barban after foliar treatments of wheat plants [4]. Research and development in finding new safeners as well as subsequent commercialization proceeded very intensively in the 1970s. Since the patent application of the safening properties of 1,8-naphthalic anhydride (NA) intensive research on discovery of new safeners resulted in compounds with diverse chemis‐

© 2013 Jablonkai; 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 Jablonkai; 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.

tries (Table 1) successfully applied to alleviate injury symptoms by various classes of herbicides in cereal crops.

ethyl as well as mesosulfuron and iodosulfuron in a variety of cereals. The main application of 8-quinolinoxy-acetate cloquintocet-mexyl is against clodinafop-propargyl in wheat. Dihydroisoxazole-carboxylate isoxadifen-ethyl can safen herbicides of various mode of action. First, it was applied in maize in combination with foramsulfuron but mixture with foramsul‐ furon and iodosulfuron-methyl is also in use. In rice, it can be used with fenoxafop-P-ethyl and ethoxysulfuron. The arylsulfonyl-benzamide, cyprosulfamide is the latest achievement in safener research. It protects maize against isoxaflutole pre-emergence and can also be used in maize with isoxaflutole plus thiencarbazone in pre-emergence and early post-emergence

Interestingly, no successful safeners have been developed for broad-leafed crops. Recently, the non-phytotoxic microbial inhibitor dietholate (*O*, *O*-diethyl-*O*-phenyl phosphorothioate) [11] used to inhibit soil microbes that degrade thiocarbamate herbicides was patented as a Table

Despite large amount of information published on the activity, mode of action and uses of safeners during the 50-year history of these herbicide antagonists this overview will focus on several less addressed topics such as a) relationships between the molecular structure and the safening properties; b) basis for differential chemical selectivity; and c) safener effects on

**Chemical class Name Structurea logP Herbicide Crop Appl.**

O O O

O Cl Cl

O

O

O

<sup>O</sup> Cl Cl

R/S

N O Cl Cl

Cl Cl

R/S 2.12

**method**

Seed– treatment

Maize PPI, PRE

Maize PRE

2.54 Thiocarbamates Maize

Herbicide Safeners: Effective Tools to Improve Herbicide Selectivity

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591

Thiocarbamates Chloroacetanilides

Acetochlor Halosulfuronmethyl

2.32b Acetochlor Maize PRE

2.69 Metolachlor Maize PRE

1.84

1 safener for cotton plants against injuries by clomazone [12].

detoxifying enzymes in crop plants and weeds.

anhydride (NA)

Dichlormid <sup>N</sup>

Furilazole <sup>O</sup>

AD–67 NO

Benoxacor <sup>N</sup>

Anhydride 1,8–Naphthalic

Dichloro– acetamide

applications [10].

NA patented by Hoffmann [5] has been considered as the most versatile safener showing less botanical and chemical specificity than other safeners developed later. NA protected cereals as seed treatments against various herbicide chemistries [6]. NA was reported to be mildly phytotoxic to maize (chlorosis and growth inhibition) under some growing conditions. One problem in treating seeds with safeners prior to planting is that phytotoxicity can increase as the time the safener is exposed to the seed increases. With NA, the phytotoxicity to the crop increases with increased time the safener is in contact with the seed during storage. This problem has thus far prevented NA from being introduced to the commercial market [7].

The introduction of dichloroacetamide derivatives developed as safeners against thiocarba‐ mates and chloroacetanilides was a breakthough in the history of the safeners since these compounds can be applied to the soil in preplant incorporated (PPI) or preemergence (PRE) technology in prepackaged tank mixture with the herbicide. Generally, prepackaged herbi‐ cide-safener mixtures offer several advantages over seed safeners. First of all, the manufacturer controls all components of the formulation secondly, the farmers buy and use a single and reliable product which allows a wider selection of crop cultivars. Dichlormid exhibited a remarkable degree of chemical and botanical specificity in protection of maize against thiocarbamates such as EPTC, butylate, vernolate but the safener was less protective to maize against chloroacetanilides. In addition to dichlormid, a number of dichloroacetylated amine derivatives were marketed. Among them AD-67, a spiro-oxazolidine compound was com‐ mercialized to protect maize plants against acetochlor while benoxaxor can be used to safen *S*-metolachlor or racemic metolachlor in maize. Furilazole, in addition to providing protection against acetochlor, has a very good safening effect on sulfonylureas particularly halosulfuron. The dichloromethyl-1,3-dioxolane MG-191 the most active member of dichloroacetal and ketal derivatives, protects maize against thiocarbamate and chloroacetanilide injuries. MG-191, similarly to dichlormid, is more effective against thiocarbamates than chloroacetanilides.

The oxime ethers such as cyometrinil, oxabetrinil, and fluxofenim were marketed as seed treatment safeners to protect sorghum plants against chloroacetanilides, in particular, meto‐ lachlor. Flurazole, a 2,4-disubstituted 5-thiazolecarboxylate is also a seed safener allowing the safe use of alachlor in sorghum. The phenylpyrimidine safener fenclorim was introduced against pretilachlor in rice and can be used in tank mixture formulated together with the chloroacetanilide herbicide.

The urea type dymron and the thiocarbamate dimepiperate are actually herbicidally active compounds that possess safening activity against pretilachlor [8] and bensulfuron [9] in rice.

Trends toward post-emergence herbicide treatments and the use of high-activity herbicide molecules have led to the development of safeners with post-emergence application in winter cereals. A new era in safener research began with the discovery of 1,2,4-triazolcarboxylates and fenchlorazole-ethyl was developed as a post-emergence safener against ACCase inhibitor fenoxaprop-ethyl in wheat in a tank mixture with the herbicide. Similarly, the dihydropyrazol dicarboxylate mefenpyr-diethyl was used against ACCase inhibitors including fenoxapropethyl as well as mesosulfuron and iodosulfuron in a variety of cereals. The main application of 8-quinolinoxy-acetate cloquintocet-mexyl is against clodinafop-propargyl in wheat. Dihydroisoxazole-carboxylate isoxadifen-ethyl can safen herbicides of various mode of action. First, it was applied in maize in combination with foramsulfuron but mixture with foramsul‐ furon and iodosulfuron-methyl is also in use. In rice, it can be used with fenoxafop-P-ethyl and ethoxysulfuron. The arylsulfonyl-benzamide, cyprosulfamide is the latest achievement in safener research. It protects maize against isoxaflutole pre-emergence and can also be used in maize with isoxaflutole plus thiencarbazone in pre-emergence and early post-emergence applications [10].

tries (Table 1) successfully applied to alleviate injury symptoms by various classes of herbicides

NA patented by Hoffmann [5] has been considered as the most versatile safener showing less botanical and chemical specificity than other safeners developed later. NA protected cereals as seed treatments against various herbicide chemistries [6]. NA was reported to be mildly phytotoxic to maize (chlorosis and growth inhibition) under some growing conditions. One problem in treating seeds with safeners prior to planting is that phytotoxicity can increase as the time the safener is exposed to the seed increases. With NA, the phytotoxicity to the crop increases with increased time the safener is in contact with the seed during storage. This problem has thus far prevented NA from being introduced to the commercial market [7].

The introduction of dichloroacetamide derivatives developed as safeners against thiocarba‐ mates and chloroacetanilides was a breakthough in the history of the safeners since these compounds can be applied to the soil in preplant incorporated (PPI) or preemergence (PRE) technology in prepackaged tank mixture with the herbicide. Generally, prepackaged herbi‐ cide-safener mixtures offer several advantages over seed safeners. First of all, the manufacturer controls all components of the formulation secondly, the farmers buy and use a single and reliable product which allows a wider selection of crop cultivars. Dichlormid exhibited a remarkable degree of chemical and botanical specificity in protection of maize against thiocarbamates such as EPTC, butylate, vernolate but the safener was less protective to maize against chloroacetanilides. In addition to dichlormid, a number of dichloroacetylated amine derivatives were marketed. Among them AD-67, a spiro-oxazolidine compound was com‐ mercialized to protect maize plants against acetochlor while benoxaxor can be used to safen *S*-metolachlor or racemic metolachlor in maize. Furilazole, in addition to providing protection against acetochlor, has a very good safening effect on sulfonylureas particularly halosulfuron. The dichloromethyl-1,3-dioxolane MG-191 the most active member of dichloroacetal and ketal derivatives, protects maize against thiocarbamate and chloroacetanilide injuries. MG-191, similarly to dichlormid, is more effective against thiocarbamates than chloroacetanilides.

The oxime ethers such as cyometrinil, oxabetrinil, and fluxofenim were marketed as seed treatment safeners to protect sorghum plants against chloroacetanilides, in particular, meto‐ lachlor. Flurazole, a 2,4-disubstituted 5-thiazolecarboxylate is also a seed safener allowing the safe use of alachlor in sorghum. The phenylpyrimidine safener fenclorim was introduced against pretilachlor in rice and can be used in tank mixture formulated together with the

The urea type dymron and the thiocarbamate dimepiperate are actually herbicidally active compounds that possess safening activity against pretilachlor [8] and bensulfuron [9] in rice.

Trends toward post-emergence herbicide treatments and the use of high-activity herbicide molecules have led to the development of safeners with post-emergence application in winter cereals. A new era in safener research began with the discovery of 1,2,4-triazolcarboxylates and fenchlorazole-ethyl was developed as a post-emergence safener against ACCase inhibitor fenoxaprop-ethyl in wheat in a tank mixture with the herbicide. Similarly, the dihydropyrazol dicarboxylate mefenpyr-diethyl was used against ACCase inhibitors including fenoxaprop-

in cereal crops.

590 Herbicides - Current Research and Case Studies in Use

chloroacetanilide herbicide.

Interestingly, no successful safeners have been developed for broad-leafed crops. Recently, the non-phytotoxic microbial inhibitor dietholate (*O*, *O*-diethyl-*O*-phenyl phosphorothioate) [11] used to inhibit soil microbes that degrade thiocarbamate herbicides was patented as a Table 1 safener for cotton plants against injuries by clomazone [12].

Despite large amount of information published on the activity, mode of action and uses of safeners during the 50-year history of these herbicide antagonists this overview will focus on several less addressed topics such as a) relationships between the molecular structure and the safening properties; b) basis for differential chemical selectivity; and c) safener effects on detoxifying enzymes in crop plants and weeds.



**Chemical class Name Structurea logP Herbicide Crop Appl.**

O H N

b Log P values unavailable were calculated by ALOGPS 2.1 program available online at www.vclab.org/articles/cite.html.

Structure-activity correlations are very important in the search for biological activity because they provide useful information about chemical substituents that are necessary for the required bioactivity. Published structure-activity correlation studies with safeners and analogous

Hoffmann's original patent for NA against EPTC in maize claimed only a few NA analogs such as alkyl esters, barium and tin salts as well as *N*,*N*'-diallyl naphthalene-1,8-dicarboxylic acid, *N*,*N*'-diallyloxamide, *N*,*N*'-dipropynyloxamide, *N*,*N*,*N*',*N*'-tetrapropynyloxamide and dipropynylmalonamide [5]. In addition to the original patent, the effects of other structural analogs of NA were tested against EPTC in maize as seed dressing [13]. The presence of the dicarboxylic anhydride group and at least one aromatic ring attached directly to the anhydride appeared to be essential for the protective activity of NA structural analogues. Derivatives such as acenaphthylene-1,2-dione, benzoisoquinoline-1,3-dione, 4-amino-NA, naphthalicdianhydride, phtalic anhydride as well as diphenic anhydride showed safening effects while chlorinated NA, 2-phenylglutaric anhydride and phenalene-1-one were toxic to maize.

Detailed structure-activity correlations were conducted mainly with various amide safeners that protect maize from thiocarbamate injury. Studies with several hundred of amides revealed that the most effective safeners were *N*,*N*-disubstituted acetamides [14] or substituted *N*acetyl-1,3-oxazolidines [15, 16]. Structure-activity studies with dichloroacetamides revealed that *N*,*N*-disubstituted derivatives were more effective than monosubstituted amides. A

3.83

ACCase inhibitors Sulfonylureas

Herbicide Safeners: Effective Tools to Improve Herbicide Selectivity

3.88b ACCase inhibitors Sulfonylureas

Cl N N O O

O O

Safeners used as racemic mixtures are indicated by *R*/*S* in their structures.

**Table 1.** Structure, logP and application of some important safeners.

**2. Structure–safening activity relationships**

NH <sup>S</sup> O O

<sup>O</sup> <sup>O</sup> R/S

> O O

Dihydropyrazole–

Dihydroisoxazole–

Arylsulfonyl–

a

dicarboxylate Mefenpyr–diethyl Cl

benzamide Cypro–sulfamide

compounds have been limited.

carboxylate Isoxadifen–ethyl <sup>O</sup> <sup>N</sup>

**method**

593

POST

Wheat, Rye, Triticale, Barley

http://dx.doi.org/10.5772/55168

2.09b Isoxaflutole Maize PRE, POST

Maize Rice POST


a Safeners used as racemic mixtures are indicated by *R*/*S* in their structures.

b Log P values unavailable were calculated by ALOGPS 2.1 program available online at www.vclab.org/articles/cite.html.

**Chemical class Name Structurea logP Herbicide Crop Appl.**

<sup>O</sup> 1.56

O 2.76

2.90

1.35b

2.70

O

O O

S <sup>N</sup> Cl F3C

Chloroacet– anilides (metolachlor)

Chloroacet– anilides (metolachlor)

Chloroacetanilides (metolachlor)

3.64b Alachlor Sorghum

Thiocarbamates Chloroacetanilides

Pyributicarb Pretilachlor Pyrazosulfuron– ethyl

<sup>N</sup> 4.02 Sulfonylureas Rice POST

5.03 Clodinafop– propargyl

4.52 Fenoxaprop– ethyl

4.17 Pretilachlor Rice PRE

CN

CN O

> CF3 O

O O

OO Cl Cl

> N N

H

S O

O

Cl

Cl Cl Cl

<sup>N</sup> <sup>N</sup> N

O C5H11

R/S

O O

O N H

Cl

Cl

Cyometrinil <sup>N</sup>

Oxabetrinil N

Fluxofenim N

Flurazole

592 Herbicides - Current Research and Case Studies in Use

MG–191

Urea Dymron <sup>N</sup>

Cloquintocet–

Fenchlorazole– ethyl

mexyl <sup>N</sup> <sup>O</sup>

Cl

Cl

Oxime ether

Thiazole carboxylic

Dichloromethyl-

Piperidine–1–

8–Quinolinoxy– carboxylic esters

1,2,4–Triazole– carboxylate

Phenyl–pyrimidine Fenclorim

carbothioate Dimepiperate

acid

ketal

**method**

Seed– treatment

Seedtreatment

Seed– treatment

Seed– treatment

Maize PRE

Rice PRE, POST

Cereals POST

Cereals POST

Sorghum

Sorghum

Sorghum

### **2. Structure–safening activity relationships**

Structure-activity correlations are very important in the search for biological activity because they provide useful information about chemical substituents that are necessary for the required bioactivity. Published structure-activity correlation studies with safeners and analogous compounds have been limited.

Hoffmann's original patent for NA against EPTC in maize claimed only a few NA analogs such as alkyl esters, barium and tin salts as well as *N*,*N*'-diallyl naphthalene-1,8-dicarboxylic acid, *N*,*N*'-diallyloxamide, *N*,*N*'-dipropynyloxamide, *N*,*N*,*N*',*N*'-tetrapropynyloxamide and dipropynylmalonamide [5]. In addition to the original patent, the effects of other structural analogs of NA were tested against EPTC in maize as seed dressing [13]. The presence of the dicarboxylic anhydride group and at least one aromatic ring attached directly to the anhydride appeared to be essential for the protective activity of NA structural analogues. Derivatives such as acenaphthylene-1,2-dione, benzoisoquinoline-1,3-dione, 4-amino-NA, naphthalicdianhydride, phtalic anhydride as well as diphenic anhydride showed safening effects while chlorinated NA, 2-phenylglutaric anhydride and phenalene-1-one were toxic to maize.

Detailed structure-activity correlations were conducted mainly with various amide safeners that protect maize from thiocarbamate injury. Studies with several hundred of amides revealed that the most effective safeners were *N*,*N*-disubstituted acetamides [14] or substituted *N*acetyl-1,3-oxazolidines [15, 16]. Structure-activity studies with dichloroacetamides revealed that *N*,*N*-disubstituted derivatives were more effective than monosubstituted amides. A variety of substituents on the nitrogen atom including alkyl, haloalkyl, alkenyl and heterocy‐ clic groups impart various degrees of protective activity. Nevertheless, mono- and trichloroa‐ cetamides exhibited less safening activity than dichloro analogues [17, 18]. Based on these SAR studies similarities between the chemical structure of the herbicide and its safener, the possible competitive antagonism between the thiocarbamate and the safener molecules for a common target site has been postulated [19]. Computer-aided molecular modeling (CAMM) studies supported this theory [20]. Superimposing of the structures of dichlormid and EPTC revealed that the two chlorine atoms of the safener do not superimpose over any functional group of the EPTC. If structure of EPTC sulfoxide, the very phytotoxic EPTC metabolite, and the dichlormid were superimposed, the two compounds were similar with functional groups in the same location on both molecules. Comparative three-dimensional quantitative structureactivity relationship studies using comparative molecular field analysis (CoMFA) also supported the competitive antagonism theory and predicted a structure of *N*-allyl-*N*-methox‐ yethoxymethyl dichloroacetamide as a potent highly effective safener [21].

derivatives among which 1,3-dithiolane derivative showed higher activity than the oxathio‐ lanes. Various 1,3-dioxolane-4-ones provided significant protection against the acetochlor. Benzo[1,3]-dioxoles were ineffective while benzo[1,3]dioxin-4-ones were protective in

Herbicide Safeners: Effective Tools to Improve Herbicide Selectivity

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595

Unfortunately, no publication has been reported for the other chemistry of safeners. However, no unifying structural motifs for compounds to be safeners can be predicted from these studies.

The importance of the chirality in the biological activity has long been recognized. Since biochemical processes in the cells take place in chiral environment and most enzymatic pathways are stereoselective, a high degree of enantiomeric and enantiotopic selectivity can be obtained when chiral or prochiral molecules are introduced into biological systems. About one fourth of the presently available pesticides are chiral, existing as two mirror images called enantiomers. These stereoisomers generally possess identical physico-chemical properties but widely different biological activities, such as toxicity, mutagenicity, and carciogenecity [27]. The active enantiomer of the chiral pesticide would have the desired effect on target species

Among the commercially available safeners, four such as benoxacor, furilazole, cloquintocetmexyl, and mefenpyr-dietyl are chiral compounds but used exclusively as racemic (*R*/*S*) mixtures in herbicidal compositions and no information accessible on the safening efficacy of the individual enantiomers. In one recent patent, the *R* enantiomer of furilazole is described

Nevertheless, only a few molecules have been reported as safeners in enantiomerically pure form. The optical isomers of 4-(dichloromethylene)-2-[*N*-(α-methylbenzyl)imino]-1,3-dithio‐ lane hydrochloride were synthesized and were tested against triallate in wheat [30]. The *R* enantiomer exhibited high safening activity and its activity exceeded that of the *S* and the racemic compound. The monoterpene *R*-carvone was found more effective than the *S* enantiomer to safen maize against acetochlor injury [31]. 2-Dichloromethyl-2-methyl- [1,3]oxathiolane 3-oxide, a structural analogue of the MG-191 safener, was prepared and the enantiomers were separated by chiral HPLC [32]. The more polar diastereomeric pair was as effective as MG-191 while the other exhibited only marginal protection against acetochlor. Inducibility of ZmGSTF1-2 from roots was more enhanced by the stereoisomers with higher safening efficacy while only one of these enantiomers was effective in shoots. The findings indicated the importance of the stereochemistry in the protective effectiveness. The safener (*S*)-3-dichloroacetyl-2,2-dimethyl-4-ethyl-1,3-oxazolidine was found to induce the GSH content and GST activity in root and shoot of maize seedlings but the effect of the *R* form was not reported in these experiments [33]. As a future prospect, the needs for broad application of the green technology in the sustainable agriculture will probably induce a shift in the use

safening maize. 5-Dichloromethyl-3-substituted-isoxazoles were also active safeners.

**3. Chiral safeners**

while the other may be inactive [28].

in a herbicidal mixture as a safener [29].

and development of enantiomerically pure safeners.

Structure-safening activity studies with oxime ether derivatives revealed that the safening activity is affected by the number of nucleophilic sites present in the molecule. An oxime ether with two nucleophilic sites was more effective than those with only one. In addition to cyometrinil, oxabetrinil and fluxofenim pyridin-2-aldoxime *O*-ethers such as benzyl and phenylethyl ethers were protective to grain sorghum in seed treatments against metolachlor. The oxime and aldehyde derivatives tested, in terms of decreasing safening effectiveness, were dimethyglyoxime > benzophenone oxime > pyridine-2-aldoxime > benzoin--oxime > methyl thioacetohydroxamate >pyridine-2-aldoxime methiodide > 5-nitro-furancarboxyaldehyde [22]. CAMM evaluations of the oxime ether analogues cyometrinil, oxabetrinil and fluxofenim revealed that as the effectiveness of the safener increases so does its molecular similarity to metolachlor [20].

Structure-safening activity relationships for thiazol-5-carboxylic acids against acetamide herbicides were described for 60 derivatives in the original patent [23]. Thiazolecarboxylates substituted by a trifluoromethyl in the 4-position are clearly superior to those substituted in the 4-position by methyl in reducing herbicidal injury to sorghum. Another preferred group of thiazolecarboxylates contained a halogen atom at position 2 preferably chlorine.

A structure-activity relationship study to safen maize against acetochlor was carried out with the herbicide safener MG-191 and its acetal and ketal analogues at preemergence application [24-26]. Open chain acetals formed from 1,1-dichloroacetaldehyde exhibited only marginal safening efficacy. Dialkyl ketals of 1,1-dichloroacetone showed increasing effectiveness up to 3 carbon length of the alkyl group with further increases in carbon atoms resulted in loss of activity. The 5-, 6- and 7-membered 1,3-dioxacycloalkanes prepared from dichloroacetalde‐ hyde had hardly detectable safening activity. However, introducing alkyl or aryl substitution at the 2-position of the 1,3-dioxacycloalkane ring remarkably increased the safening activity. Regarding ring size the highest activity observed was for 2-dichloromethyl-2-methyl-1,3 dioxepane. Replacing an oxygen in the 1,3-dioxolane ring for nitrogen resulted in oxazolidines with reduced safening activities but alkyl or aryl substitution on the nitrogen increased the safening activity of compounds. Replacement of oxygens by sulfur atoms leads to less active derivatives among which 1,3-dithiolane derivative showed higher activity than the oxathio‐ lanes. Various 1,3-dioxolane-4-ones provided significant protection against the acetochlor. Benzo[1,3]-dioxoles were ineffective while benzo[1,3]dioxin-4-ones were protective in safening maize. 5-Dichloromethyl-3-substituted-isoxazoles were also active safeners.

Unfortunately, no publication has been reported for the other chemistry of safeners. However, no unifying structural motifs for compounds to be safeners can be predicted from these studies.

### **3. Chiral safeners**

variety of substituents on the nitrogen atom including alkyl, haloalkyl, alkenyl and heterocy‐ clic groups impart various degrees of protective activity. Nevertheless, mono- and trichloroa‐ cetamides exhibited less safening activity than dichloro analogues [17, 18]. Based on these SAR studies similarities between the chemical structure of the herbicide and its safener, the possible competitive antagonism between the thiocarbamate and the safener molecules for a common target site has been postulated [19]. Computer-aided molecular modeling (CAMM) studies supported this theory [20]. Superimposing of the structures of dichlormid and EPTC revealed that the two chlorine atoms of the safener do not superimpose over any functional group of the EPTC. If structure of EPTC sulfoxide, the very phytotoxic EPTC metabolite, and the dichlormid were superimposed, the two compounds were similar with functional groups in the same location on both molecules. Comparative three-dimensional quantitative structureactivity relationship studies using comparative molecular field analysis (CoMFA) also supported the competitive antagonism theory and predicted a structure of *N*-allyl-*N*-methox‐

Structure-safening activity studies with oxime ether derivatives revealed that the safening activity is affected by the number of nucleophilic sites present in the molecule. An oxime ether with two nucleophilic sites was more effective than those with only one. In addition to cyometrinil, oxabetrinil and fluxofenim pyridin-2-aldoxime *O*-ethers such as benzyl and phenylethyl ethers were protective to grain sorghum in seed treatments against metolachlor. The oxime and aldehyde derivatives tested, in terms of decreasing safening effectiveness, were dimethyglyoxime > benzophenone oxime > pyridine-2-aldoxime > benzoin--oxime > methyl thioacetohydroxamate >pyridine-2-aldoxime methiodide > 5-nitro-furancarboxyaldehyde [22]. CAMM evaluations of the oxime ether analogues cyometrinil, oxabetrinil and fluxofenim revealed that as the effectiveness of the safener increases so does its molecular similarity to

Structure-safening activity relationships for thiazol-5-carboxylic acids against acetamide herbicides were described for 60 derivatives in the original patent [23]. Thiazolecarboxylates substituted by a trifluoromethyl in the 4-position are clearly superior to those substituted in the 4-position by methyl in reducing herbicidal injury to sorghum. Another preferred group

A structure-activity relationship study to safen maize against acetochlor was carried out with the herbicide safener MG-191 and its acetal and ketal analogues at preemergence application [24-26]. Open chain acetals formed from 1,1-dichloroacetaldehyde exhibited only marginal safening efficacy. Dialkyl ketals of 1,1-dichloroacetone showed increasing effectiveness up to 3 carbon length of the alkyl group with further increases in carbon atoms resulted in loss of activity. The 5-, 6- and 7-membered 1,3-dioxacycloalkanes prepared from dichloroacetalde‐ hyde had hardly detectable safening activity. However, introducing alkyl or aryl substitution at the 2-position of the 1,3-dioxacycloalkane ring remarkably increased the safening activity. Regarding ring size the highest activity observed was for 2-dichloromethyl-2-methyl-1,3 dioxepane. Replacing an oxygen in the 1,3-dioxolane ring for nitrogen resulted in oxazolidines with reduced safening activities but alkyl or aryl substitution on the nitrogen increased the safening activity of compounds. Replacement of oxygens by sulfur atoms leads to less active

of thiazolecarboxylates contained a halogen atom at position 2 preferably chlorine.

yethoxymethyl dichloroacetamide as a potent highly effective safener [21].

metolachlor [20].

594 Herbicides - Current Research and Case Studies in Use

The importance of the chirality in the biological activity has long been recognized. Since biochemical processes in the cells take place in chiral environment and most enzymatic pathways are stereoselective, a high degree of enantiomeric and enantiotopic selectivity can be obtained when chiral or prochiral molecules are introduced into biological systems. About one fourth of the presently available pesticides are chiral, existing as two mirror images called enantiomers. These stereoisomers generally possess identical physico-chemical properties but widely different biological activities, such as toxicity, mutagenicity, and carciogenecity [27]. The active enantiomer of the chiral pesticide would have the desired effect on target species while the other may be inactive [28].

Among the commercially available safeners, four such as benoxacor, furilazole, cloquintocetmexyl, and mefenpyr-dietyl are chiral compounds but used exclusively as racemic (*R*/*S*) mixtures in herbicidal compositions and no information accessible on the safening efficacy of the individual enantiomers. In one recent patent, the *R* enantiomer of furilazole is described in a herbicidal mixture as a safener [29].

Nevertheless, only a few molecules have been reported as safeners in enantiomerically pure form. The optical isomers of 4-(dichloromethylene)-2-[*N*-(α-methylbenzyl)imino]-1,3-dithio‐ lane hydrochloride were synthesized and were tested against triallate in wheat [30]. The *R* enantiomer exhibited high safening activity and its activity exceeded that of the *S* and the racemic compound. The monoterpene *R*-carvone was found more effective than the *S* enantiomer to safen maize against acetochlor injury [31]. 2-Dichloromethyl-2-methyl- [1,3]oxathiolane 3-oxide, a structural analogue of the MG-191 safener, was prepared and the enantiomers were separated by chiral HPLC [32]. The more polar diastereomeric pair was as effective as MG-191 while the other exhibited only marginal protection against acetochlor. Inducibility of ZmGSTF1-2 from roots was more enhanced by the stereoisomers with higher safening efficacy while only one of these enantiomers was effective in shoots. The findings indicated the importance of the stereochemistry in the protective effectiveness. The safener (*S*)-3-dichloroacetyl-2,2-dimethyl-4-ethyl-1,3-oxazolidine was found to induce the GSH content and GST activity in root and shoot of maize seedlings but the effect of the *R* form was not reported in these experiments [33]. As a future prospect, the needs for broad application of the green technology in the sustainable agriculture will probably induce a shift in the use and development of enantiomerically pure safeners.

### **4. Prosafeners and natural compounds with safening activity**

The term prosafeners refers to molecules with safening activity undergoing biotransformation to the actual safening agent prior to exhibiting their safening effect. Substituted *N*-phenylma‐ leamic acids and their progenitors *N*-phenylmaleimides and *N*-phenylisomaleimides exhibit‐ ed safening activity against alachlor in sorghum at preemergence application [34]. Simple hydrolytic ring-opening reaction of *N*-phenylmaleimides and *N*-phenylisomaleimides results in the *N*-phenylmaleamic acid derivatives with safening activity. Two thiazolidine derivative L-2-oxathiazolidine-4-carboxylic acid (OTC) [35] and thioproline (L-thiazolidine-4-carboxylic acid) [36] have been reported to safen sorghum against tridiphane injury. OTC is converted by 5-oxoprolinase to *S*-carboxy-L-cysteine which spontaneously decarboxylates to yield Lcysteine. The conversion of thioproline to cysteine takes place in two steps, first proline oxidase yields *N*-formyl-L-cysteine from which cysteine is forming by hydrolysis. Either source of cysteine elevates the glutathione level in plants and therefore enhance herbicide detoxication. toward various herbicides was reviewed [2]. Interestingly the majority of papers published report safener-enhanced herbicide uptake followed by no effect then reduced uptake results. According to a recent study mefenpyr-diethyl had no effect on the uptake of either mesosul‐ furon-methyl or iodosulfuron-methyl-sodium [10]. These results suggest that the influence of safeners on the herbicide uptake may not be a decisive factor in the protective action. However, the knowledge of absorbed amounts of safeners and herbicides by crops may help to determine the optimal herbicide/safener ratios applicable in the agricultural practice. In addition, determination of the site of safener and herbicide uptake can contribute to prepare the most selective herbicide-safener mixture. A suitable placement of soil-applied herbicides to roots or the emerging shoots is of great practical importance in achieving the most effective weed

Studies on how maize can differentiate in the absorbtion of herbicides and safeners were conducted with radiolabeled EPTC, acetochlor and MG-191 [42, 43]. Time-dependent uptake of root-applied [14C]EPTC reached a maximum after 6h and decreased up to 3 days (Figure 1). The first measurable shoot growth inhibition appeared just after 1-day-exposition to the herbicide and 38% shoot length inhibition was observed 3 DAT. In general, the MG-191 safener had no influence on the herbicide uptake except for 1 DAT when the safener enhanced the herbicide uptake by 1.5-fold as compared to that in the unsafened plants. Nevertheless, the safener conferred a complete protection to maize throughout the study. The highest amount

**3 6 24 48 72**

As a comparision, the amount of root-absorbed [14C]acetochlor was continuously increased up to 3 days (Figure 2). As a result of increasing uptake the first detectable shoot length inhibition occurred 6h after treatment. At 3 DAT 28% shoot and 52% root (data not shown) growth inhibition by the herbicide occurred. Addition of the MG-191 safener did not affect the acetochlor absorption by maize seedlings but completely antagonized the herbicide shoot

**50 μM EPTC\* 50 μM EPTC\* + 50 μM MG-191 50 μM EPTC 50 μM EPTC + 50 μM MG-191**

**Figure 1.** Influence of MG-191 safener on uptake and shoot length inhibition of root-applied [14C]EPTC.

**Time, hours**

**0**

**25**

**50**

**75**

**Shoot length, % of control** 

**100**

**125**

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control and the least injury to crop plants.

of herbicide uptake was 65 μg/g fresh weight.

**0**

**25**

m**g/g fwt % of control** 

**50**

**Uptake** m**g/ g fresh wt** **75**

**100**

Safening activities of natural cyclic hydroxamic acids (DIMBOA, DIBOA, and MBOA ) as well as synthetic analogues such as 1,4-benzoxazin-3-ones and 1,3-benzoxazolidin-2-ones were prepared and tested to safen maize against acetochlor and EPTC injuries [37]. Cyclic hydroxa‐ mic acids were supposed to act as safeners by catalyzing hydroxylation of herbicides contain‐ ing reactive chlorine in their structure and they are ineffective against herbicides not possessing leaving groups. While no safening activities of natural hydroxamic acids were detected, the synthetic analogues exhibited low to moderate activity.

Metabolismoftheherbicidesafener,fenclorimresulting,inasemi-naturalproductwithsafening activity has recently been described in *Arabidopsis thaliana* cell cultures [38]. The metabolism of fenclorim mediated by GSTs yielded *S*-(fenclorim)-glutathione conjugate that was sequential‐ ly catabolized to *S*-(fenclorim)-cysteine then to 4-chloro-6-(methylthio)-phenylpyrimidine (CMTP). Although the fenclorim conjugates tested showed little GST inducing activity in *Arabidopsis*,theformationofCMTPresultedinmetabolicreactivation,withtheproduct showing enhancing activity similar to that of parent safener. In addition, CMTP safened rice plants and induced rice GSTs. The formation of CMTP by metabolic bioactivation can contribute to the longevity of safener action since it was found stable 8 – 24 h after application.

Oxylipins constitute a family of oxygenated natural products which are formed from fatty acids. Safeners and reactive electrophilic oxylipins (RES oxylipins) have a common biological activity in that they both strongly induce the expression of defence genes and activate detoxification responses in plants [39, 40]. Surprisingly, the application of oxylipin A has been found to reduce the herbicidal injury [41].

### **5. Interaction of safeners and herbicides on the absorption and translocation**

Published results on how safeners affect the herbicide absorption are rather contradictory and, therefore, no general conlusion can be drawn. In an excellent summary the effect of 15 safeners toward various herbicides was reviewed [2]. Interestingly the majority of papers published report safener-enhanced herbicide uptake followed by no effect then reduced uptake results. According to a recent study mefenpyr-diethyl had no effect on the uptake of either mesosul‐ furon-methyl or iodosulfuron-methyl-sodium [10]. These results suggest that the influence of safeners on the herbicide uptake may not be a decisive factor in the protective action. However, the knowledge of absorbed amounts of safeners and herbicides by crops may help to determine the optimal herbicide/safener ratios applicable in the agricultural practice. In addition, determination of the site of safener and herbicide uptake can contribute to prepare the most selective herbicide-safener mixture. A suitable placement of soil-applied herbicides to roots or the emerging shoots is of great practical importance in achieving the most effective weed control and the least injury to crop plants.

**4. Prosafeners and natural compounds with safening activity**

596 Herbicides - Current Research and Case Studies in Use

detected, the synthetic analogues exhibited low to moderate activity.

longevity of safener action since it was found stable 8 – 24 h after application.

**5. Interaction of safeners and herbicides on the absorption and**

found to reduce the herbicidal injury [41].

**translocation**

The term prosafeners refers to molecules with safening activity undergoing biotransformation to the actual safening agent prior to exhibiting their safening effect. Substituted *N*-phenylma‐ leamic acids and their progenitors *N*-phenylmaleimides and *N*-phenylisomaleimides exhibit‐ ed safening activity against alachlor in sorghum at preemergence application [34]. Simple hydrolytic ring-opening reaction of *N*-phenylmaleimides and *N*-phenylisomaleimides results in the *N*-phenylmaleamic acid derivatives with safening activity. Two thiazolidine derivative L-2-oxathiazolidine-4-carboxylic acid (OTC) [35] and thioproline (L-thiazolidine-4-carboxylic acid) [36] have been reported to safen sorghum against tridiphane injury. OTC is converted by 5-oxoprolinase to *S*-carboxy-L-cysteine which spontaneously decarboxylates to yield Lcysteine. The conversion of thioproline to cysteine takes place in two steps, first proline oxidase yields *N*-formyl-L-cysteine from which cysteine is forming by hydrolysis. Either source of cysteine elevates the glutathione level in plants and therefore enhance herbicide detoxication. Safening activities of natural cyclic hydroxamic acids (DIMBOA, DIBOA, and MBOA ) as well as synthetic analogues such as 1,4-benzoxazin-3-ones and 1,3-benzoxazolidin-2-ones were prepared and tested to safen maize against acetochlor and EPTC injuries [37]. Cyclic hydroxa‐ mic acids were supposed to act as safeners by catalyzing hydroxylation of herbicides contain‐ ing reactive chlorine in their structure and they are ineffective against herbicides not possessing leaving groups. While no safening activities of natural hydroxamic acids were

Metabolismoftheherbicidesafener,fenclorimresulting,inasemi-naturalproductwithsafening activity has recently been described in *Arabidopsis thaliana* cell cultures [38]. The metabolism of fenclorim mediated by GSTs yielded *S*-(fenclorim)-glutathione conjugate that was sequential‐ ly catabolized to *S*-(fenclorim)-cysteine then to 4-chloro-6-(methylthio)-phenylpyrimidine (CMTP). Although the fenclorim conjugates tested showed little GST inducing activity in *Arabidopsis*,theformationofCMTPresultedinmetabolicreactivation,withtheproduct showing enhancing activity similar to that of parent safener. In addition, CMTP safened rice plants and induced rice GSTs. The formation of CMTP by metabolic bioactivation can contribute to the

Oxylipins constitute a family of oxygenated natural products which are formed from fatty acids. Safeners and reactive electrophilic oxylipins (RES oxylipins) have a common biological activity in that they both strongly induce the expression of defence genes and activate detoxification responses in plants [39, 40]. Surprisingly, the application of oxylipin A has been

Published results on how safeners affect the herbicide absorption are rather contradictory and, therefore, no general conlusion can be drawn. In an excellent summary the effect of 15 safeners Studies on how maize can differentiate in the absorbtion of herbicides and safeners were conducted with radiolabeled EPTC, acetochlor and MG-191 [42, 43]. Time-dependent uptake of root-applied [14C]EPTC reached a maximum after 6h and decreased up to 3 days (Figure 1). The first measurable shoot growth inhibition appeared just after 1-day-exposition to the herbicide and 38% shoot length inhibition was observed 3 DAT. In general, the MG-191 safener had no influence on the herbicide uptake except for 1 DAT when the safener enhanced the herbicide uptake by 1.5-fold as compared to that in the unsafened plants. Nevertheless, the safener conferred a complete protection to maize throughout the study. The highest amount of herbicide uptake was 65 μg/g fresh weight.

**Figure 1.** Influence of MG-191 safener on uptake and shoot length inhibition of root-applied [14C]EPTC.

As a comparision, the amount of root-absorbed [14C]acetochlor was continuously increased up to 3 days (Figure 2). As a result of increasing uptake the first detectable shoot length inhibition occurred 6h after treatment. At 3 DAT 28% shoot and 52% root (data not shown) growth inhibition by the herbicide occurred. Addition of the MG-191 safener did not affect the acetochlor absorption by maize seedlings but completely antagonized the herbicide shoot growth inhibition. The maize seedlings absorbed much higher amounts of acetochlor (377 μg/ g fresh weight).

to the roots of maize plants than the less water-soluble acetochlor (log P 4.14). The higher logP

**3 h 6h 1d 3d 6d**

**Figure 3.** Time-course uptake of root-applied [14C]MG-191 by 5-day-old maize seedlings and the influence of EPTC.

**Time**

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It is also difficult to put the results of safener affected translocation of absorbed herbicides in perspective. Reduction of translocation of herbicides such as acetochlor, methazachlor, and imidazolinones from roots of maize to the shoots following treatments with dichlormid, BAS 145,138 and NA is likely a consequence of the safener-enhanced herbicide metabolism to more polar and less mobile products [45-49]. On the other hand, no effect of MG-191 on EPTC and acetochlor translocation has been observed [42, 44]. It is interesting to note that safener MG-191 and the herbicide acetochlor exhibit different translocation patterns (Figure 4). While the majority of the absorbed radiolabel from [14C] acetochlor was found in the roots and coleoptiles of maize seedlings (Figure 4a), the root-applied [14C]MG-191 distributed evenly within the plants (Figure 4b) showing similar mobility and distribution as EPTC (data not shown). This may be further evidence for the higher protective efficacy of this safener against EPTC as compared to acetochlor. The similar translocation pattern of the herbicide and the safener may

**6. Action of safeners on the glutathione-mediated detoxification of**

Various chemistries of safeners were found to enhance the herbicide detoxification in the safened plants by elevating the activity of the mediating enzymes such as glutathione Stransferases (GSTs), cytochrome P450 mixed function oxidases (CYPs), glycosyltransferases (UGTs) and ATP-binding casette (ABC) transporter proteins as well as a cofactor endogenous glutathione (GSH) involved in detoxification of herbicides [2, 50-52]. The best studied group

of acetochlor also supports its higher uptake as compared to MG-191.

**0**

**2**

**4**

**Uptake mg/ g fresh wt**

**6**

**8**

**10**

**10 μM MG-191 50 μM EPTC + 50 μM MG-191 50 μM EPTC 50 μM EPTC + 50 μM MG-191**

be a prerequisite for the high level of safening activity.

**herbicides**

**Figure 2.** Influence of MG-191 safener on uptake and shoot length inhibition of root-applied [14C]acetochlor.

All previous efforts to elucidate modes of action of safeners focused on the fate of various herbicides as affected by the safener treatments while no studies were conducted on how uptake, translocation and metabolism of safeners were influenced by herbicides. For a better understanding of the herbicide-safener interaction, absorption of [14C]MG-191 by maize seedlings was studied as influenced by EPTC. Absorbed amount of the labeled safener following application to the roots of 5-day-old maize plants increased over the time and no influence of EPTC on uptake was observed (Figure 3). At a higher safener concentration (50 μM), plants absorbed higher amounts of radiolabel than at a lower concentration (10 μM) but plants contained low levels (3% and 1%) of the safener applied. The highest value for the safener content in the maize seedlings was less than 8 μg/g fresh weight.

These data clearly suggest that even this small amount of safener offer protection to maize. The absorbed herbicide/safener ratio (μg/μg) at 3 DAT accounted for 50 with acetochlor and 1.7 with EPTC at same concentrations of the herbicide. These results may partly explain why safening efficacy of MG-191 toward EPTC is higher than toward acetochlor under field conditions. Site of uptake can also affect the MG-191 effectiveness. In experiments using a charcoal barrier to separate shoot and root zones of maize, the influence of site of safener placement on acetochlor phytotoxicity was studied [44]. MG-191 was the most protective when both the safener and the herbicide were applied simultaneously to shoots and roots but also satisfactory protection was achieved when the safener was applied in the root zone and the herbicide to the emerging shoots. This also indicates the main site of uptake for acetochlor absorption is the coleoptile while the root-uptake is very significant in the safener performance. Under field conditions the more water-soluble MG-191 (log P, 1.35) can be more easily leached

growth inhibition. The maize seedlings absorbed much higher amounts of acetochlor (377 μg/

**50 μM Ac\* 50 μM Ac\* + 50 μM MG-191 50 μM Ac 50 μM Ac + 50 μM MG-191**

**3 6 24 48 72**

All previous efforts to elucidate modes of action of safeners focused on the fate of various herbicides as affected by the safener treatments while no studies were conducted on how uptake, translocation and metabolism of safeners were influenced by herbicides. For a better understanding of the herbicide-safener interaction, absorption of [14C]MG-191 by maize seedlings was studied as influenced by EPTC. Absorbed amount of the labeled safener following application to the roots of 5-day-old maize plants increased over the time and no influence of EPTC on uptake was observed (Figure 3). At a higher safener concentration (50 μM), plants absorbed higher amounts of radiolabel than at a lower concentration (10 μM) but plants contained low levels (3% and 1%) of the safener applied. The highest value for the

These data clearly suggest that even this small amount of safener offer protection to maize. The absorbed herbicide/safener ratio (μg/μg) at 3 DAT accounted for 50 with acetochlor and 1.7 with EPTC at same concentrations of the herbicide. These results may partly explain why safening efficacy of MG-191 toward EPTC is higher than toward acetochlor under field conditions. Site of uptake can also affect the MG-191 effectiveness. In experiments using a charcoal barrier to separate shoot and root zones of maize, the influence of site of safener placement on acetochlor phytotoxicity was studied [44]. MG-191 was the most protective when both the safener and the herbicide were applied simultaneously to shoots and roots but also satisfactory protection was achieved when the safener was applied in the root zone and the herbicide to the emerging shoots. This also indicates the main site of uptake for acetochlor absorption is the coleoptile while the root-uptake is very significant in the safener performance. Under field conditions the more water-soluble MG-191 (log P, 1.35) can be more easily leached

**Figure 2.** Influence of MG-191 safener on uptake and shoot length inhibition of root-applied [14C]acetochlor.

safener content in the maize seedlings was less than 8 μg/g fresh weight.

**Time, hours**

**0**

**25**

**50**

**75**

**Shoot length, % of control** 

**100**

**125**

g fresh weight).

**0**

**100**

**200**

**Uptake mg/ g fresh wt**

**300**

**400**

**500**

598 Herbicides - Current Research and Case Studies in Use

m**g/g fwt % of control** 

**Figure 3.** Time-course uptake of root-applied [14C]MG-191 by 5-day-old maize seedlings and the influence of EPTC.

to the roots of maize plants than the less water-soluble acetochlor (log P 4.14). The higher logP of acetochlor also supports its higher uptake as compared to MG-191.

It is also difficult to put the results of safener affected translocation of absorbed herbicides in perspective. Reduction of translocation of herbicides such as acetochlor, methazachlor, and imidazolinones from roots of maize to the shoots following treatments with dichlormid, BAS 145,138 and NA is likely a consequence of the safener-enhanced herbicide metabolism to more polar and less mobile products [45-49]. On the other hand, no effect of MG-191 on EPTC and acetochlor translocation has been observed [42, 44]. It is interesting to note that safener MG-191 and the herbicide acetochlor exhibit different translocation patterns (Figure 4). While the majority of the absorbed radiolabel from [14C] acetochlor was found in the roots and coleoptiles of maize seedlings (Figure 4a), the root-applied [14C]MG-191 distributed evenly within the plants (Figure 4b) showing similar mobility and distribution as EPTC (data not shown). This may be further evidence for the higher protective efficacy of this safener against EPTC as compared to acetochlor. The similar translocation pattern of the herbicide and the safener may be a prerequisite for the high level of safening activity.

### **6. Action of safeners on the glutathione-mediated detoxification of herbicides**

Various chemistries of safeners were found to enhance the herbicide detoxification in the safened plants by elevating the activity of the mediating enzymes such as glutathione Stransferases (GSTs), cytochrome P450 mixed function oxidases (CYPs), glycosyltransferases (UGTs) and ATP-binding casette (ABC) transporter proteins as well as a cofactor endogenous glutathione (GSH) involved in detoxification of herbicides [2, 50-52]. The best studied group

sion of *Zm*GSTU1 only in the roots. *Zm*GSTU1 has previously been shown to play a key role

Cl

NO-17 MG-191 contr. NO-17 MG-191 contr. NO-17 MG-191 contr.

contr. MG-191 NO-17 contr. MG-191 NO-17 contr. MG-191 NO-17

**Figure 5.** Western blots of crude GST extracts from maize roots and shoots (a) Analysis of GSTs from maize shoots using the anti-*Zm*GSTF1-2 serum.(b) Analysis of GSTs from maize roots using the anti-*Zm*GSTF1-2 serum.(c) Analysis of GSTs from maize shoots using the anti-*Zm*GSTU1-2 serum.(d) Analysis of GSTs from maize roots using the anti-

Analysis of isoenzyme profile of maize GSTs revealed that phi class of GSTs predominate, with ZmGSTF1 as the major subunit which is present constitutively and shows high specificity to 1-chloro-2,4-dinitrobenzene (CDNB) substrate [62]. A second phi type GST termed *Zm*GSTF2 accumulates following treatments with herbicide safeners. These subunits can dimerise together to form *Zm*GSTF1-1 and *Zm*GSTF2-2 homodimers as well as *Zm*GSTF1-2 heterodimer. In addition to these three phi GST isoenzymes a phi type GST *Zm*GSTF3 and three tau class GSTs *Zm*GSTU1, *Zm*GSTU2 and *Zm*GSTU3 are present in lower amounts [63, 64]. While the expression of *Zm*GSTF2 was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione, the *Zm*GSTU1 subunit was induced more selectively, only accumulating significantly in response to dichlormid treatment [63]. Although *Zm*GSTF2 has been consid‐

+ CH + CH + CH

**2 DAT**

**2 DAT**

**1 DAT**

contr. MG-191 NO-17 contr. MG-191 NO-17 + CH + CH + CH

NO-17 MG-191 contr

**1 DAT**

29 kDa

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27 kDa

in metabolism of nitrodiphenyl ether type herbicides [54].

**a**

**b**

**c**

**d**

+ CH + CH + CH

O

**1 DAT**

**2 DAT**

OO

Cl

contr. MG-191 NO-17

**1 DAT**

+ CH + CH + CH

NO-17 MG-191 contr. NO-17 MG-191 contr.

**2 DAT**

29 kDa

27 kDa

*Zm*GSTU1-2 serum.

**Figure 4.** Distribution of root- and shoot-applied [14C]acetochlor and root-applied [14C]MG-191 in maize seedlings.

of plant enzymes involved in herbicide metabolism is the GSTs that mediate the conjugation of the major cellular thiol tripeptide, GSH with herbicide substrates. GSTs are multifunctional enzymes, each composed of two subunits which catalyze conjugation of a broad range of electrophilic substrates with GSH [53]. Herbicides known to conjugate with GSH include thiocarbamates, chloro-*s*-triazines, triazinone sulfoxides, chloroacetanilides, diphenylethers, some sulfonylureas, aryloxyphenoxypropionates, thiazolidines, and sulfonamides [54, 55]. Plant GSTs comprise a large and diverse group, with 54 GST genes encoded by the *Arabidop‐ sis* genome, and have been classified on sequence similarity, genomic organization and functions into several distinct subclasses [56]. In plants, phi (F) and tau (U) classes are the most prominent GSTs involved in herbicide detoxification [57-59]. In addition to up-regulating GST expression, safeners also enhance the activity of enzymes involved in sulfate assimilation and GSH biosynthesis thereby elevating the level of GSH [50, 60].

Only two studies are available in the literature on how the safener structure affects the expression of GST isoforms. The herbicide safener MG-191 (2-dichloromethyl-2-methyl-1,3 dioxolane) and its less effective structural analogue dichloromethyl-dioxolanone (NO-17; 2 dichloromethyl-2,5-dimethyl-1,3-dioxolane-4-one) were reported to differentially enhance the expression of members of the GSTs in maize [61].

None of these safener molecules had influence on the expression of *Zm*GSTF1-2 (Figure 5a and b). However, MG-191 and, to a lesser extent NO-17 selectively enhanced the expression of tau class *Zm*GSTU1 in both root and shoot tissues after 1 day of treatment (Figure 5c and d). Addition of cycloheximide to the treatment solutions suppressed the enhancement of expres‐

sion of *Zm*GSTU1 only in the roots. *Zm*GSTU1 has previously been shown to play a key role in metabolism of nitrodiphenyl ether type herbicides [54].

of plant enzymes involved in herbicide metabolism is the GSTs that mediate the conjugation of the major cellular thiol tripeptide, GSH with herbicide substrates. GSTs are multifunctional enzymes, each composed of two subunits which catalyze conjugation of a broad range of electrophilic substrates with GSH [53]. Herbicides known to conjugate with GSH include thiocarbamates, chloro-*s*-triazines, triazinone sulfoxides, chloroacetanilides, diphenylethers, some sulfonylureas, aryloxyphenoxypropionates, thiazolidines, and sulfonamides [54, 55]. Plant GSTs comprise a large and diverse group, with 54 GST genes encoded by the *Arabidop‐ sis* genome, and have been classified on sequence similarity, genomic organization and functions into several distinct subclasses [56]. In plants, phi (F) and tau (U) classes are the most prominent GSTs involved in herbicide detoxification [57-59]. In addition to up-regulating GST expression, safeners also enhance the activity of enzymes involved in sulfate assimilation and

**Figure 4.** Distribution of root- and shoot-applied [14C]acetochlor and root-applied [14C]MG-191 in maize seedlings.

**Autoradiograph of [14C]MG-191-treated (50** m**M) maize seedling 3 DAT**

b)

Only two studies are available in the literature on how the safener structure affects the expression of GST isoforms. The herbicide safener MG-191 (2-dichloromethyl-2-methyl-1,3 dioxolane) and its less effective structural analogue dichloromethyl-dioxolanone (NO-17; 2 dichloromethyl-2,5-dimethyl-1,3-dioxolane-4-one) were reported to differentially enhance the

None of these safener molecules had influence on the expression of *Zm*GSTF1-2 (Figure 5a and b). However, MG-191 and, to a lesser extent NO-17 selectively enhanced the expression of tau class *Zm*GSTU1 in both root and shoot tissues after 1 day of treatment (Figure 5c and d). Addition of cycloheximide to the treatment solutions suppressed the enhancement of expres‐

GSH biosynthesis thereby elevating the level of GSH [50, 60].

expression of members of the GSTs in maize [61].

**Autoradiograph of [14C]acetochlortreated (2.5 kg/ha) maize seedling 11 DAT**

**Autoradiograph** 

a)

600 Herbicides - Current Research and Case Studies in Use

**Figure 5.** Western blots of crude GST extracts from maize roots and shoots (a) Analysis of GSTs from maize shoots using the anti-*Zm*GSTF1-2 serum.(b) Analysis of GSTs from maize roots using the anti-*Zm*GSTF1-2 serum.(c) Analysis of GSTs from maize shoots using the anti-*Zm*GSTU1-2 serum.(d) Analysis of GSTs from maize roots using the anti-*Zm*GSTU1-2 serum.

Analysis of isoenzyme profile of maize GSTs revealed that phi class of GSTs predominate, with ZmGSTF1 as the major subunit which is present constitutively and shows high specificity to 1-chloro-2,4-dinitrobenzene (CDNB) substrate [62]. A second phi type GST termed *Zm*GSTF2 accumulates following treatments with herbicide safeners. These subunits can dimerise together to form *Zm*GSTF1-1 and *Zm*GSTF2-2 homodimers as well as *Zm*GSTF1-2 heterodimer. In addition to these three phi GST isoenzymes a phi type GST *Zm*GSTF3 and three tau class GSTs *Zm*GSTU1, *Zm*GSTU2 and *Zm*GSTU3 are present in lower amounts [63, 64]. While the expression of *Zm*GSTF2 was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione, the *Zm*GSTU1 subunit was induced more selectively, only accumulating significantly in response to dichlormid treatment [63]. Although *Zm*GSTF2 has been consid‐ ered more active in detoxifying metolachlor and alachlor than *Zm*GSTF1 it is far less abundant [65]. The importance of *de novo* synthesis of the isoenzyme *Zm*GSTU1 in its safening action is difficult to explain. Nevertheless, these results indicate that dichloromethyl-dioxolane type MG-191 is a more specific inducer of maize GSTs than other compounds commonly used to safen thiocarbamate or chloroacetanilide herbicides in maize.

**CHXY**

 **1a-l 2a-k**

**Code R X Y R1 R2** Protectiona

**N**

**R2 <sup>C</sup>**

**GSHb GST(CDNB)c GST (Ac)d treated/control**

**R1**

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603

**O**

Herbicide Safeners: Effective Tools to Improve Herbicide Selectivity

**3a-d**

**XYCH**

**CHXY**

(%)

**O O**

**R1 R2**

2k Me Cl Cl -(CH2)6- 60 1.38 1.39 1.14 3a - Cl Cl allyl allyl 81 1.78 1.24 4.69 3b - H Cl H allyl 48 2.25 1.25 3.60 3c - H Cl allyl allyl 2 1.45 1.16 2.39 3d - H Br allyl allyl 22 0.98 0.90 2.98

based on shoot length; protection (%) = 100 x [(herbicide + safener)] / [control - herbicide]; shoot lengths 14 DAT:

**Table 2.** Safening activity and inducibility of shoot GSH content and GST activities by acetals, ketals and amides in

commonly used to safen thiocarbamate and chloroacetanilide herbicides in maize [61].

The exact mechanism of the safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated protec‐ tion of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. Our results indicate

In other, structure and GST isoform expressing ability studies with acetal and ketal analogues of MG-191 as well as mono-and dichloroacetamides (Table 2) demonstrated that the safener structure affects the specific expression of GSTs mediating the detoxication of acetochlor (Matola et al., 2003). Nevertheless, no correlation was found between the degree of induction of GSH and GSTs and the safening activity as related to the structure. A higher inducibility of these GST isoforms was observed in root tissues (Figure 6a and c). In shoots, when the heterodimer *Zm*GSTF1-2 was used the expression of the constitutive *Zm*GSTF1 and inducible *Zm*GSTF2 was enhanced only by **2f** (MG-191) and its analogue **2g** having a 6-membered ring (Figure 6b). These molecules and also **2h** were the most potent inducers of the expression of tau class *Zm*GSTU1 in shoot tissues (Figure 6c). *Zm*GSTU1 has previously been shown to play a key role in metabo‐ lism of nitrodiphenyl ether herbicides [54]. These results confirm previous findings that dichloromethyl-ketal safeners are more specific inducers *Zm*GSTU1-2 than other compounds

b GSH content relative to that of untreated control; GSHcontr.: 0.55±0.09 μmol/g fresh weight;

 GST(CDNB) activity as compared to that of untreated control; GSTcontr.: 3.87±0.33 nkat/mg protein; d GST(Ac) activity as compared to that of untreated control; GSTcontr.: 8.26±1.68 pkat/mg protein

**R**

**O O**

**R1 R2**

control, 27.9+5.3 cm, acetochlor, 3.1±0.3 cm;

**H**

a

c

maize

2h Me Cl Cl -CH2C(CH3)2CH2- 66 1.62 1.77 1.44 2i Me Cl Cl -(CH2)4- 70 1.71 1.48 0.92 2j Me Cl Cl -(CH2)5- 50 1.24 1.27 1.19

a based on shoot length; protection (%) = 100 x [(herbicide + safener)] / [control - herbicide]; shoot lengths 14 DAT: control, 27.9+5.3 cm, acetochlor, 3.1±0.3 cm;

b GSH content relative to that of untreated control; GSHcontr.: 0.55±0.09 μmol/g fresh weight;

ered more active in detoxifying metolachlor and alachlor than *Zm*GSTF1 it is far less abundant [65]. The importance of *de novo* synthesis of the isoenzyme *Zm*GSTU1 in its safening action is difficult to explain. Nevertheless, these results indicate that dichloromethyl-dioxolane type MG-191 is a more specific inducer of maize GSTs than other compounds commonly used to

**CHXY**

(%)

**O O**

**R1 R2**

Ac - - - - - - 1.11 1.48 3.74 1a - H Cl Et Et 24 1.49 0.69 2.03 1b - H Br Et Et 60 1.53 0.94 3.76 1c - Cl Cl Et Et 8 0.69 1.42 1.83 1d - Cl Cl Pr Pr 0 0.80 0.95 1.38 1e - Cl Cl Bu Bu -6 1.22 0.96 0.91 1f - Cl Cl i-Bu i-Bu -2 1.20 0.90 1.61 1g - Cl Cl -(CH2)2- 18 0.93 0.88 1.23 1h - Cl Cl -(CH2)3- 14 0.60 0.89 0.90 1i - Cl Cl -CH2C(CH3)2CH2- -3 0.91 0.88 1.33 1j - Cl Cl -(CH2)4- 11 0.95 1.03 1.33 1k - Cl Cl -(CH2)5- 0 0.98 1.24 0.58 1l - Cl Cl -(CH2)6- 3 0.82 1.32 0.83 2a Me Cl Cl Et Et 62 1.15 1.22 0.65 2b Me Cl Cl Pr Pr 63 0.98 1.18 0.85 2c Me Cl Cl Bu Bu 38 0.78 0.88 3.93 2d Me Cl Cl i-Bu i-Bu 14 0.85 1.07 4.72 2e Ph Cl Cl -(CH2)2- 41 2.00 1.94 2.23 2f Me Cl Cl -(CH2)2- 64 1.18 1.83 3.93 2g Me Cl Cl -(CH2)3- 68 1.31 1.49 1.96 2h Me Cl Cl -CH2C(CH3)2CH2- 66 1.62 1.77 1.44 2i Me Cl Cl -(CH2)4- 70 1.71 1.48 0.92 2j Me Cl Cl -(CH2)5- 50 1.24 1.27 1.19

**R**

**N**

**R2 <sup>C</sup>**

**GSHb GST(CDNB)c GST (Ac)d treated/control**

**R1**

**O**

**3a-d**

**XYCH**

safen thiocarbamate or chloroacetanilide herbicides in maize.

 **1a-l 2a-k**

**Code R X Y R1 R2** Protectiona

**CHXY**

**O O**

602 Herbicides - Current Research and Case Studies in Use

**R1 R2**

**H**

c GST(CDNB) activity as compared to that of untreated control; GSTcontr.: 3.87±0.33 nkat/mg protein;

d GST(Ac) activity as compared to that of untreated control; GSTcontr.: 8.26±1.68 pkat/mg protein

**Table 2.** Safening activity and inducibility of shoot GSH content and GST activities by acetals, ketals and amides in maize

In other, structure and GST isoform expressing ability studies with acetal and ketal analogues of MG-191 as well as mono-and dichloroacetamides (Table 2) demonstrated that the safener structure affects the specific expression of GSTs mediating the detoxication of acetochlor (Matola et al., 2003). Nevertheless, no correlation was found between the degree of induction of GSH and GSTs and the safening activity as related to the structure. A higher inducibility of these GST isoforms was observed in root tissues (Figure 6a and c). In shoots, when the heterodimer *Zm*GSTF1-2 was used the expression of the constitutive *Zm*GSTF1 and inducible *Zm*GSTF2 was enhanced only by **2f** (MG-191) and its analogue **2g** having a 6-membered ring (Figure 6b). These molecules and also **2h** were the most potent inducers of the expression of tau class *Zm*GSTU1 in shoot tissues (Figure 6c). *Zm*GSTU1 has previously been shown to play a key role in metabo‐ lism of nitrodiphenyl ether herbicides [54]. These results confirm previous findings that dichloromethyl-ketal safeners are more specific inducers *Zm*GSTU1-2 than other compounds commonly used to safen thiocarbamate and chloroacetanilide herbicides in maize [61].

The exact mechanism of the safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated protec‐ tion of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. Our results indicate that safener structure plays a decisive role in specific expression of GSTs mediating the detoxication of chloroacetamide herbicides. Since no correlation between the degree of induction of levels of GSH and GST isoforms and the safener activity was found, the mode of action of safeners is a more complex process than simply promoting the metabolism of herbicides.

moderate safening to *Bromus secalinus* (bromegrass) and flurazole was also moderately protective in *Setaria glauca* (yellow foxtail) at sublethal rate of EPTC. Safener-induced elevation of GSH contents and GST activities is widely considered as key element for increased tolerance to thiocarbamates and chloroacetanilides of safened plants [50]. Tolerance of plant species such as maize, soybean and several weeds to acetochlor has been correlated with their glutathione and homoglutathione content [70]. It was also apparent that a relationship exists between the relative GST activities toward alachlor and metolachlor in maize and various weed species ([71]. GST activities toward metolachlor were found to correlate well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds [72]. However, there was no correlation between total activity of cysteine biosynthesis from serine (CBS) and susceptibility to metolachlor of sorghum, maize, and various grassy weeds [73]. GST isozymes involved in herbicide metabolism is cell suspension culture of a grass weed S*etaria faberi* (giant

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foxtail) exhibited a similar level of complexity to those from maize cell cultures [74].

**7.1 Effect of safeners on weed glutathione (GSH) content and glutathione** *S***-transferase enzyme (GSTs) activities** Safeners such as MG-191, dichlormid, AD-67, BAS-145138, and flurazol were reported to reduce phytotoxicity of EPTC in grassy weeds [69]. MG-191, BAS-145138 and flurazole offered moderate safening to *Bromus secalinus* (bromegrass) and flurazole was also moderately protective in *Setaria glauca* (yellow foxtail) at sublethal rate of EPTC. Safener-induced elevation of GSH contents and GST activities is widely considered as key element for increased tolerance to thiocarbamates and chloroacetanilides of safened plants [50]. Tolerance of plant species such as maize, soybean and several weeds to acetochlor has been correlated with their glutathione and homoglutathione content [70]. It was also apparent that a relationship exists between the relative GST activities toward alachlor and metolachlor in maize and various weed species ([71]. GST activities toward metolachlor were found to correlate well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds [72]. However, there was no correlation between total activity of cysteine biosynthesis from serine (CBS) and susceptibility to metolachlor of sorghum, maize, and various grassy weeds [73]. GST isozymes involved in herbicide metabolism is cell suspension culture of a grass weed S*etaria faberi* (giant foxtail) exhibited a similar

protein thiols.

level of complexity to those from maize cell cultures [74].

**0**

**50**

**non-protein thiol,** 

m**g/g fresh weight**

**100**

**150**

**200**

remarkable increases (73% and 87%, respectively) in the levels of non-protein thiols.

Figure 7. Effect of treatments on non-protein thiol contents of mono- and dicot weed species.

*crus-galli* and *Amaranthus retroflexus* (AMARE, redroot pigweed) compared to less sensitive species.

**Figure 7.** Effect of treatments on non-protein thiol contents of mono- and dicot weed species.

Nevertheless, much less is known about GSH or other non-protein thiol contents and GST activities of different weed species following treatments by herbicides and safeners. In order to explain differential physiological and biochemical responses of monocot and dicot weeds to these herbicides, non-protein thiol levels and GST activities were studied in selected monoand dicot weeds species [75]. The most sensitive *Echinochloa crus-galli* (ECHCR, barnyard‐ grass) contained higher level of non-protein thiols than less sensitive dicot seedlings (Figure 5). Nevertheless, thiol contents in the most tolerant maize and in the least sensitive monoco‐ tyledonous *Bromus secalinus* (BROSE, cheatgrass) were comparable. In general, either herbicide or safener pretreatments did not alter thiol contents substantially. *Abuthilon theophrasti* (ABUTH, velvetleaf) was the only exception because 1 μM acetochlor and 10 μM AD-67 resulted in remarkable increases (73% and 87%, respectively) in the levels of non-

Nevertheless, much less is known about GSH or other non-protein thiol contents and GST activities of different weed species following treatments by herbicides and safeners. In order to explain differential physiological and biochemical responses of monocot and dicot weeds to these herbicides, non-protein thiol levels and GST activities were studied in selected mono- and dicot weeds species [75]. The most sensitive *Echinochloa crus-galli* (ECHCR, barnyardgrass) contained higher level of non-protein thiols than less sensitive dicot seedlings (Figure 7). Nevertheless, thiol contents in the most tolerant maize and in the least sensitive monocotyledonous *Bromus secalinus* (BROSE, cheatgrass) were comparable. In general, either herbicide or safener pretreatments did not alter thiol contents substantially. *Abuthilon theophrasti* (ABUTH, velvetleaf) was the only exception because 1 μM acetochlor and 10 μM AD-67 resulted in

**control EPTC Acetochlor AD-67 MG-191**

**ECHCR AVEFA BROSE AMARE ABUTH XANST Maize**

Glutathione S-transferase (GST) activities using CDNB substrate were not correlated with herbicide susceptibility of the selected weed species (Figure 8a). The GSTs extracted from monocot seedlings exhibited much higher activities than from dicot seedlings. GSTCDNB activity detected in *Avena fatua* (AVEFA, wild oats) exceeded that in maize. In general, elevation of GSTCDNB activities following pretreatments with both herbicides and safeners were more pronounced (2- to 10-fold of controls) in the highly sensitive *Echinochloa* 

**weed species**

**Figure 6.** Western blots of crude GST extracts from maize roots and shoots; (a) and (b) analysis of GSTs using the anti-*Zm*GSTF1-2 serum from maize roots and shoots; (c) and (d) analysis of GSTs using the anti-*Zm*GSTU1-2 serum from maize roots and shoots.

### **7. Effect of safeners on herbicide detoxification enyzmes in weeds**

Studies on the mechanism of action of safeners revealed that herbicide safeners improve crop tolerance to herbicides by regulating the expression of genes involved in herbicide metabolism [68]. It is widely accepted that safeners selectively protect crop plants against herbicide injury by stimulating the plant detoxifying mechanism at herbicide rates required for effective weed control. Nevertheless, only a few papers were published on the safener effect of GSTs and cytochrome P450 monooxygenases of various weed species. To a better understanding on why safeners do not provide protection to weeds it is essential to explore the safener action on detoxification enzymes of weeds.

#### **7.1. Effect of safeners on weed glutathione (GSH) content and glutathione** *S***-transferase enzyme (GSTs) activities**

Safeners such as MG-191, dichlormid, AD-67, BAS-145138, and flurazol were reported to reduce phytotoxicity of EPTC in grassy weeds [69]. MG-191, BAS-145138 and flurazole offered moderate safening to *Bromus secalinus* (bromegrass) and flurazole was also moderately protective in *Setaria glauca* (yellow foxtail) at sublethal rate of EPTC. Safener-induced elevation of GSH contents and GST activities is widely considered as key element for increased tolerance to thiocarbamates and chloroacetanilides of safened plants [50]. Tolerance of plant species such as maize, soybean and several weeds to acetochlor has been correlated with their glutathione and homoglutathione content [70]. It was also apparent that a relationship exists between the relative GST activities toward alachlor and metolachlor in maize and various weed species ([71]. GST activities toward metolachlor were found to correlate well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds [72]. However, there was no correlation between total activity of cysteine biosynthesis from serine (CBS) and susceptibility to metolachlor of sorghum, maize, and various grassy weeds [73]. GST isozymes involved in herbicide metabolism is cell suspension culture of a grass weed S*etaria faberi* (giant foxtail) exhibited a similar level of complexity to those from maize cell cultures [74]. **7.1 Effect of safeners on weed glutathione (GSH) content and glutathione** *S***-transferase enzyme (GSTs) activities**

that safener structure plays a decisive role in specific expression of GSTs mediating the detoxication of chloroacetamide herbicides. Since no correlation between the degree of induction of levels of GSH and GST isoforms and the safener activity was found, the mode of action of safeners is a more complex process than simply promoting the metabolism of

**Contr**. **2f 2g 2a 2b 2c 2d 2i 2k 2j 2h**

**Figure 6.** Western blots of crude GST extracts from maize roots and shoots; (a) and (b) analysis of GSTs using the anti-*Zm*GSTF1-2 serum from maize roots and shoots; (c) and (d) analysis of GSTs using the anti-*Zm*GSTU1-2 serum from

Studies on the mechanism of action of safeners revealed that herbicide safeners improve crop tolerance to herbicides by regulating the expression of genes involved in herbicide metabolism [68]. It is widely accepted that safeners selectively protect crop plants against herbicide injury by stimulating the plant detoxifying mechanism at herbicide rates required for effective weed control. Nevertheless, only a few papers were published on the safener effect of GSTs and cytochrome P450 monooxygenases of various weed species. To a better understanding on why safeners do not provide protection to weeds it is essential to explore the safener action on

**7.1. Effect of safeners on weed glutathione (GSH) content and glutathione** *S***-transferase**

Safeners such as MG-191, dichlormid, AD-67, BAS-145138, and flurazol were reported to reduce phytotoxicity of EPTC in grassy weeds [69]. MG-191, BAS-145138 and flurazole offered

**7. Effect of safeners on herbicide detoxification enyzmes in weeds**

**GSTF1-2 (roots)**

**GSTF1-2 (shoots)**

**GSTU1-2 (roots)**

**GSTU1-2 (shoots)**

herbicides.

**29 kDa**

604 Herbicides - Current Research and Case Studies in Use

**a**

**b**

**c**

**d**

**29 kDa**

**27 kDa**

**27 kDa**

maize roots and shoots.

detoxification enzymes of weeds.

**enzyme (GSTs) activities**

**MG-191**

Nevertheless, much less is known about GSH or other non-protein thiol contents and GST activities of different weed species following treatments by herbicides and safeners. In order to explain differential physiological and biochemical responses of monocot and dicot weeds to these herbicides, non-protein thiol levels and GST activities were studied in selected monoand dicot weeds species [75]. The most sensitive *Echinochloa crus-galli* (ECHCR, barnyard‐ grass) contained higher level of non-protein thiols than less sensitive dicot seedlings (Figure 5). Nevertheless, thiol contents in the most tolerant maize and in the least sensitive monoco‐ tyledonous *Bromus secalinus* (BROSE, cheatgrass) were comparable. In general, either herbicide or safener pretreatments did not alter thiol contents substantially. *Abuthilon theophrasti* (ABUTH, velvetleaf) was the only exception because 1 μM acetochlor and 10 μM AD-67 resulted in remarkable increases (73% and 87%, respectively) in the levels of nonprotein thiols. Safeners such as MG-191, dichlormid, AD-67, BAS-145138, and flurazol were reported to reduce phytotoxicity of EPTC in grassy weeds [69]. MG-191, BAS-145138 and flurazole offered moderate safening to *Bromus secalinus* (bromegrass) and flurazole was also moderately protective in *Setaria glauca* (yellow foxtail) at sublethal rate of EPTC. Safener-induced elevation of GSH contents and GST activities is widely considered as key element for increased tolerance to thiocarbamates and chloroacetanilides of safened plants [50]. Tolerance of plant species such as maize, soybean and several weeds to acetochlor has been correlated with their glutathione and homoglutathione content [70]. It was also apparent that a relationship exists between the relative GST activities toward alachlor and metolachlor in maize and various weed species ([71]. GST activities toward metolachlor were found to correlate well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds [72]. However, there was no correlation between total activity of cysteine biosynthesis from serine (CBS) and susceptibility to metolachlor of sorghum, maize, and various grassy weeds [73]. GST isozymes involved in herbicide metabolism is cell suspension culture of a grass weed S*etaria faberi* (giant foxtail) exhibited a similar level of complexity to those from maize cell cultures [74]. Nevertheless, much less is known about GSH or other non-protein thiol contents and GST activities of different weed species following treatments by herbicides and safeners. In order to explain differential physiological and biochemical responses of monocot and dicot weeds to these herbicides, non-protein thiol levels and GST activities were studied in selected mono- and dicot weeds species [75]. The most sensitive *Echinochloa crus-galli* (ECHCR, barnyardgrass) contained higher level of non-protein thiols than less sensitive dicot seedlings (Figure 7). Nevertheless, thiol contents in the most tolerant maize and in the least sensitive monocotyledonous *Bromus secalinus* (BROSE, cheatgrass) were comparable. In general, either herbicide or safener pretreatments did not alter thiol contents substantially. *Abuthilon theophrasti* (ABUTH, velvetleaf) was the only exception because 1 μM acetochlor and 10 μM AD-67 resulted in remarkable increases (73% and 87%, respectively) in the levels of non-protein thiols.

species (Figure 8a). The GSTs extracted from monocot seedlings exhibited much higher activities than from dicot seedlings. GSTCDNB activity detected in *Avena fatua* (AVEFA, wild oats) exceeded that in maize. In general, elevation of GSTCDNB activities following pretreatments with both herbicides and safeners were more pronounced (2- to 10-fold of controls) in the highly sensitive *Echinochloa* 

Glutathione S-transferase (GST) activities using CDNB substrate were not correlated with herbicide susceptibility of the selected weed **Figure 7.** Effect of treatments on non-protein thiol contents of mono- and dicot weed species.

*crus-galli* and *Amaranthus retroflexus* (AMARE, redroot pigweed) compared to less sensitive species.

Figure 7. Effect of treatments on non-protein thiol contents of mono- and dicot weed species.

Glutathione S-transferase (GST) activities using CDNB substrate were not correlated with herbicide susceptibility of the selected weed species (Figure 6a). The GSTs extracted from monocot seedlings exhibited much higher activities than from dicot seedlings. GSTCDNB activity detected in *Avena fatua* (AVEFA, wild oats) exceeded that in maize. In general, elevation of GSTCDNB activities following pretreatments with both herbicides and safeners were more pronounced (2- to 10-fold of controls) in the highly sensitive *Echinochloa crus-galli* and *Amaranthus retroflexus* (AMARE, redroot pigweed) compared to less sensitive species.

With [14C]acetochlor substrate, GSTacetochlor activities of both mono- and dicot seedlings were in the same range except for velvetleaf (ABUTH) (Figure 6b). Regardless of treatment, extractable GSTs from velvetleaf did not show specificity for acetochlor. Nevertheless, GSTacetochlor activities in all weed species were less expressed than in maize. No correlation was found between enzyme activity and acetochlor susceptibilities of these weed species. In monocot seedlings higher enzyme inductions (up to 2-fold increase) were observed as compared to those in dicots following safener treatment. Nevertheless, GSTacetochlor activity of the maize seedlings exceeded those of weed species which may indicate that the higher detoxication capability of crop plant is closely related to the herbicide tolerance. It is also noteworthy that both GSH and cysteine conjugates of chloroacetamides were found inhibitory to GSTs from maize, *Avena fatua*, and *Echinochloa crus-galli*suggesting that GSH conjugation in crops and weeds takes place in a

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Interestingly, *Arabidopsis* plant cultures were more responsive to induction by safeners than either maize or wheat [77]. Enhancement of GSTCDNB activity was greatest with fenclorim however treatment with flurazole, CMPI and benoxacor also offered significant increases. *O*-Glucosyltransferase and *N*-glucosyltransferase activities were also stimulated but to a lesser extents. Safeners mefenpyr diethyl and fenchlorazole-ethyl enhanced fenoxapropethyl tolerance of weed *Alopecurus myosuroides* (black-grass) [78]. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabo‐ lism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydrox‐ ymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respective‐ ly. In addition to enhanced glutathione metabolism safener treatment resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Safening of grass weeds was concluded as a mechanism associated with an inducible activation of antioxidant and secondary metabolism. The ability of safeners to induce GSTs of grassy weeds can be exploited in phytoremediating herbicide-contaminat‐ ed soils. In recent studies safener benoxacor was used to enhance GSTs of the perennial grass *Festuca arundinancea* to establish a basis for preventing environmental herbicide pollution [79]. Further studies revealed that in addition to benoxacor cloquintocet-ethyl, fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil were also able to enhance GST activity in *Festuca* [80]. These results indicate that herbicide diffusion following the runoff of surface waters can be prevented or significantly reduced by vegetating buffer strips with *Festuca* and by the combination of herbicide and a suitable safener. By this way, the application of safeners can be extended by using non crop-species in phytoremediating

**7.2. Interaction of safeners on weed cytochrome P450 monooxygenases**

The involvement of cytochrome P450 monooxygenases in herbicide detoxication and selec‐ tivity has been well demonstrated [81, 82]. The role of cytochrome P450 monooxygenases in enhanced metabolism of resistant weed species has also been documented [83, 84]. Neverthe‐

complex manner [76].

contaminated soils.

Figure 8. Effect of treatments on glutathione S-transferase activities of selected weed species. a) GSTCDNB activities; b) GSTacetochlor activities of untreated and treated 6-day-old etiolated seedlings. With [14C]acetochlor substrate, GSTacetochlor activities of both mono- and dicot seedlings were in the same range except for velvetleaf **Figure 8.** Effect of treatments on glutathione S-transferase activities of selected weed species. a) GSTCDNB activities; b) GSTacetochlor activities of untreated and treated 6-day-old etiolated seedlings.

(ABUTH) (Figure 8b). Regardless of treatment, extractable GSTs from velvetleaf did not show specificity for acetochlor. Nevertheless, GSTacetochlor activities in all weed species were less expressed than in maize. No correlation was found between enzyme activity and acetochlor susceptibilities of these weed species. In monocot seedlings higher enzyme inductions (up to 2-fold increase) were observed as compared to those in dicots following safener treatment. Nevertheless, GSTacetochlor activity of the maize seedlings exceeded those of weed species which may indicate that the higher detoxication capability of crop plant is closely related to the herbicide tolerance. It is also noteworthy that both GSH and cysteine conjugates of chloroacetamides were found inhibitory to GSTs from maize, *Avena fatua*, and

*Echinochloa crus-galli*suggesting that GSH conjugation in crops and weeds takes place in a complex manner [76].

With [14C]acetochlor substrate, GSTacetochlor activities of both mono- and dicot seedlings were in the same range except for velvetleaf (ABUTH) (Figure 6b). Regardless of treatment, extractable GSTs from velvetleaf did not show specificity for acetochlor. Nevertheless, GSTacetochlor activities in all weed species were less expressed than in maize. No correlation was found between enzyme activity and acetochlor susceptibilities of these weed species. In monocot seedlings higher enzyme inductions (up to 2-fold increase) were observed as compared to those in dicots following safener treatment. Nevertheless, GSTacetochlor activity of the maize seedlings exceeded those of weed species which may indicate that the higher detoxication capability of crop plant is closely related to the herbicide tolerance. It is also noteworthy that both GSH and cysteine conjugates of chloroacetamides were found inhibitory to GSTs from maize, *Avena fatua*, and *Echinochloa crus-galli*suggesting that GSH conjugation in crops and weeds takes place in a complex manner [76].

Glutathione S-transferase (GST) activities using CDNB substrate were not correlated with herbicide susceptibility of the selected weed species (Figure 6a). The GSTs extracted from monocot seedlings exhibited much higher activities than from dicot seedlings. GSTCDNB activity detected in *Avena fatua* (AVEFA, wild oats) exceeded that in maize. In general, elevation of GSTCDNB activities following pretreatments with both herbicides and safeners were more pronounced (2- to 10-fold of controls) in the highly sensitive *Echinochloa crus-galli* and

*Amaranthus retroflexus* (AMARE, redroot pigweed) compared to less sensitive species.

**ECHCR AVEFA BRO SE AMARE ABUTH XANST Maize**

**b) control Acetochlor AD-67 MG-191**

**ECHCR AVEFA BRO SE AMARE ABUTH XANST Maize**

Figure 8. Effect of treatments on glutathione S-transferase activities of selected weed species. a) GSTCDNB activities; b) GSTacetochlor

**Figure 8.** Effect of treatments on glutathione S-transferase activities of selected weed species. a) GSTCDNB activities; b)

With [14C]acetochlor substrate, GSTacetochlor activities of both mono- and dicot seedlings were in the same range except for velvetleaf (ABUTH) (Figure 8b). Regardless of treatment, extractable GSTs from velvetleaf did not show specificity for acetochlor. Nevertheless, GSTacetochlor activities in all weed species were less expressed than in maize. No correlation was found between enzyme activity and acetochlor susceptibilities of these weed species. In monocot seedlings higher enzyme inductions (up to 2-fold increase) were observed as compared to those in dicots following safener treatment. Nevertheless, GSTacetochlor activity of the maize seedlings exceeded those of weed species which may indicate that the higher detoxication capability of crop plant is closely related to the herbicide tolerance. It is also noteworthy that both GSH and cysteine conjugates of chloroacetamides were found inhibitory to GSTs from maize, *Avena fatua*, and

*Echinochloa crus-galli*suggesting that GSH conjugation in crops and weeds takes place in a complex manner [76].

**control EPTC Acetochlor AD-67 MG-191**

**weed species**

**weed species**

**0**

**1000**

**0**

GSTacetochlor activities of untreated and treated 6-day-old etiolated seedlings.

**1000**

activities of untreated and treated 6-day-old etiolated seedlings.

**2000**

**3000**

**GST(acetochlor)** 

**pmol/mg protein/min**

**4000**

**5000**

**6000**

**2000**

**3000**

**GST(CDNB) activity** 

**a)**

**nmol/mg protein/min**

**4000**

**5000**

606 Herbicides - Current Research and Case Studies in Use

Interestingly, *Arabidopsis* plant cultures were more responsive to induction by safeners than either maize or wheat [77]. Enhancement of GSTCDNB activity was greatest with fenclorim however treatment with flurazole, CMPI and benoxacor also offered significant increases. *O*-Glucosyltransferase and *N*-glucosyltransferase activities were also stimulated but to a lesser extents. Safeners mefenpyr diethyl and fenchlorazole-ethyl enhanced fenoxapropethyl tolerance of weed *Alopecurus myosuroides* (black-grass) [78]. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabo‐ lism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydrox‐ ymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respective‐ ly. In addition to enhanced glutathione metabolism safener treatment resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Safening of grass weeds was concluded as a mechanism associated with an inducible activation of antioxidant and secondary metabolism. The ability of safeners to induce GSTs of grassy weeds can be exploited in phytoremediating herbicide-contaminat‐ ed soils. In recent studies safener benoxacor was used to enhance GSTs of the perennial grass *Festuca arundinancea* to establish a basis for preventing environmental herbicide pollution [79]. Further studies revealed that in addition to benoxacor cloquintocet-ethyl, fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil were also able to enhance GST activity in *Festuca* [80]. These results indicate that herbicide diffusion following the runoff of surface waters can be prevented or significantly reduced by vegetating buffer strips with *Festuca* and by the combination of herbicide and a suitable safener. By this way, the application of safeners can be extended by using non crop-species in phytoremediating contaminated soils.

#### **7.2. Interaction of safeners on weed cytochrome P450 monooxygenases**

The involvement of cytochrome P450 monooxygenases in herbicide detoxication and selec‐ tivity has been well demonstrated [81, 82]. The role of cytochrome P450 monooxygenases in enhanced metabolism of resistant weed species has also been documented [83, 84]. Neverthe‐ less, only a few examples can be found in the literature as to cytochrome P450-dependent monooxygenase system in weed species [85].

furon decreased P450 levels by about 50% as compared to the untreated control. Interestingly, without herbicide pretreatment with NA had no influence on maize P450. The inhibitory effect of NA *in vitro* on maize P450 was reported by the formation of an enzyme-NA Type I complex [87]. Pretreatments with the combination of MG-191 and all herbicides yielded slight increases in the enzyme concentration. It is interesting to note that no binding of MG-191 to P450 was detected [88] which may indicate why MG-191 was not inhibitory to P450. The P450 inhibitor PBO simultaneously applied with bentazon and nicosulfuron substantially reduced P450

For further characterization of *in vivo* interaction of the combination of the herbicides with safeners and inhibitors microsomes isolated from etiolated maize seedlings were used (Figure 9). Treatment of maize seedlings with nicosulfuron resulted in 30% elevation in P450 level while no effect of EPTC was found. The combination of NA with either bentazon or nicosulfuron decreased P450 levels by about 50% as compared to the untreated control. Interestingly, without herbicide pretreatment with NA had no influence on maize P450. The inhibitory effect of NA *in vitro* on maize P450 was reported by the formation of an enzyme-NA Type I complex [87]. Pretreatments with the combination of MG-191 and all herbicides yielded slight increases in the enzyme concentration. It is interesting to note that no binding of MG-191 to P450 was detected [88] which may indicate why MG-191 was not inhibitory to P450. The P450 inhibitor PBO simultaneously applied with bentazon and nicosulfuron substantially reduced P450 levels while the ABT was less inhibitory.

levels while the ABT was less inhibitory.

**-**

**0 50 100**

of cofactors involved in the herbicide detoxication [50, 52, 89, 90].

NA 0.5%w/v; MG-191, 10 μM; ABT, 1 μM; PBO, 10 μM.

**8. Mechanism of safener action**

**150**

**% of control**

ABT, 1 μM; PBO, 10 μM.

**8. Mechanism of safener action**

**+NA**

to those in crops provide a basis for the botanical selectivity of safeners.

cofactors involved in the herbicide detoxication [50, 52, 89, 90].

**+MG-191**

**+ABT** 

**+PBO**

Figure 9. Interaction of herbicides with safeners and cytochrome P450 inhibitors on P450 enzymes extracted from 4-day-old etiolated maize seedlings. Treatments were as follows: bentazon, 10 μM; EPTC, 10 μM; nicosulfuron, 10 μM; NA 0.5%w/v; MG-191, 10 μM;

**Figure 9.** Interaction of herbicides with safeners and cytochrome P450 inhibitors on P450 enzymes extracted from 4 day-old etiolated maize seedlings. Treatments were as follows: bentazon, 10 μM; EPTC, 10 μM; nicosulfuron, 10 μM;

These results demonstrate that safeners can marginally protect weed species by stimulating the herbicide detoxifying enzymes but the lower level of these enzymes in weeds as compared to those in crops provide a basis for the botanical selectivity of safeners.

These results demonstrate that safeners can marginally protect weed species by stimulating the herbicide detoxifying enzymes but the lower level of these enzymes in weeds as compared

The mechanism by which safeners act is currently unknown despite the widespread agricultural use and the substantial experimental evidence accumulated on the biochemical basis of action. Safeners appear to induce a set of genes that encode enzymes and biosynthesis

The exact mechanism of safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated protection of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledonous crops. The reason(s) for this difference in safening is unknown. Treatment of *Arabidopsis* seedlings with various safeners resulted in enhanced GST activities and expression of GSHconjugate transporters such as *At*MRP1-4 [91]. Safeners also increased GSH content of *Arabidopsis* seedlings. However, treatment of *Arabidopsis* plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Immunoblot analysis confirmed that *At*GSTU19 was induced in response to several safeners. These results indicate that, although *Arabidopsis* may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds. Concerning the location of safener binding site(s) of plants few studies have been conducted. A high-affinity cytosolic-binding site for the dichloroacetamide safener (*R*,*S*)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidine was found in etiolated maize seedlings ([92]. The binding was highest in the coleoptiles and lowest in the leaves. A good correlation was shown between the safener effectiveness. Chloroacetanilide and thiocarbamate herbicides were effective inhibitors of safener binding at low concentrations. The inhibition by alachlor and EPTC was shown to be competitive. The safener binding protein (SafBP) was purified to homogeneity having a molecular

The mechanism by which safeners act is currently unknown despite the widespread agricul‐ tural use and the substantial experimental evidence accumulated on the biochemical basis of action. Safeners appear to induce a set of genes that encode enzymes and biosynthesis of

The exact mechanism of safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated protec‐ tion of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. Herbicide safeners

+

**Bentazon EPTC**

**Treatment**

**Nicosulfuron**

**Bentazon EPTC Nicosulfuron**

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Monocotyledonous (*Avena fatua*, *Bromus inermis*, *Echinochloa crus-galli*) and dicotyledonous (*Amaranthus retroflexus*, *Abuthilon threophrasti*, *Xantium strumarium*) weeds were used to study the interaction of safeners, herbicides metabolized by cytochrome P450 enzymes, and P450 inhibitors on herbicide phytotoxicity and P450 levels of weeds and maize [86]. The safener NA was slightly protective to all monocots at the reduced rate (50 g/ha) of nicosulfuron and also exhibited safening effects on dicots against all herbicides. MG-191 reduced growth inhibition of EPTC in *A. fatua* and *E. crus-galli*.


a 0.5 %w/v; b 1 μM; <sup>c</sup> 7-day-old etiolated weed seedlings; dND not detectable; e 4-day-old etiolated maize seedlings.

**Table 3.** Cytochrome P450 contents of mono- and dicot weeds and influence of treatment with the safener NA and P450 inhibitor ABT.

Weed microsomal cytochrome P450 enzymes were found less stable than those from maize. Carbon-monoxide difference spectra for *B. inermis* and *X. stumarium* could not be recorded probably due to dark colors of microsomal preparations and difficulties in resuspending the microsomes. Cytochrome P450 content in the microsomal membrane fraction of *A. fatua* was 2.4-fold greater than in *E. crus-galli* (Table 3). Among dicotyledonous plants, *A. theophrasti* contained 5.1-fold higher level of the enzyme as compared to that of *A. retroflexus*. However, the P450 level was higher in maize than in weeds.

It is difficult to evaluate changes in the enzyme contents of weed species pretreated with the safener NA or the P450 inhibitor ABT due to the high values of standard deviation of the data. Following treatments with NA, a stimulating tendency could be observed for weeds except *E. crus-galli*. With maize the NA treatment had no enhancing effect on the enzyme content. However, a significant increase (43%) was found when maize seedlings treated with ABT but the P450 inhibitor was uneffective on weed P450s.

For further characterization of *in vivo* interaction of the combination of the herbicides with safeners and inhibitors microsomes isolated from etiolated maize seedlings were used (Figure 7). Treatment of maize seedlings with nicosulfuron resulted in 30% elevation in P450 level while no effect of EPTC was found. The combination of NA with either bentazon or nicosul‐ furon decreased P450 levels by about 50% as compared to the untreated control. Interestingly, without herbicide pretreatment with NA had no influence on maize P450. The inhibitory effect of NA *in vitro* on maize P450 was reported by the formation of an enzyme-NA Type I complex [87]. Pretreatments with the combination of MG-191 and all herbicides yielded slight increases in the enzyme concentration. It is interesting to note that no binding of MG-191 to P450 was detected [88] which may indicate why MG-191 was not inhibitory to P450. The P450 inhibitor PBO simultaneously applied with bentazon and nicosulfuron substantially reduced P450 levels while the ABT was less inhibitory. For further characterization of *in vivo* interaction of the combination of the herbicides with safeners and inhibitors microsomes isolated from etiolated maize seedlings were used (Figure 9). Treatment of maize seedlings with nicosulfuron resulted in 30% elevation in P450 level while no effect of EPTC was found. The combination of NA with either bentazon or nicosulfuron decreased P450 levels by about 50% as compared to the untreated control. Interestingly, without herbicide pretreatment with NA had no influence on maize P450. The inhibitory effect of NA *in vitro* on maize P450 was reported by the formation of an enzyme-NA Type I complex [87]. Pretreatments with the combination of MG-191 and all herbicides yielded slight increases in the enzyme concentration. It is interesting to note that no

binding of MG-191 to P450 was detected [88] which may indicate why MG-191 was not inhibitory to P450. The P450 inhibitor PBO simultaneously applied with bentazon and nicosulfuron substantially reduced P450 levels while the ABT was less inhibitory.

Figure 9. Interaction of herbicides with safeners and cytochrome P450 inhibitors on P450 enzymes extracted from 4-day-old etiolated maize seedlings. Treatments were as follows: bentazon, 10 μM; EPTC, 10 μM; nicosulfuron, 10 μM; NA 0.5%w/v; MG-191, 10 μM; ABT, 1 μM; PBO, 10 μM. These results demonstrate that safeners can marginally protect weed species by stimulating the herbicide detoxifying enzymes but the **Figure 9.** Interaction of herbicides with safeners and cytochrome P450 inhibitors on P450 enzymes extracted from 4 day-old etiolated maize seedlings. Treatments were as follows: bentazon, 10 μM; EPTC, 10 μM; nicosulfuron, 10 μM; NA 0.5%w/v; MG-191, 10 μM; ABT, 1 μM; PBO, 10 μM.

lower level of these enzymes in weeds as compared to those in crops provide a basis for the botanical selectivity of safeners.

**8. Mechanism of safener action** The mechanism by which safeners act is currently unknown despite the widespread agricultural use and the substantial experimental evidence accumulated on the biochemical basis of action. Safeners appear to induce a set of genes that encode enzymes and biosynthesis These results demonstrate that safeners can marginally protect weed species by stimulating the herbicide detoxifying enzymes but the lower level of these enzymes in weeds as compared to those in crops provide a basis for the botanical selectivity of safeners.

The exact mechanism of safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated

Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledonous crops. The reason(s) for this difference in safening is unknown. Treatment of *Arabidopsis* seedlings with various safeners resulted in enhanced GST activities and expression of GSH-

#### protection of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. **8. Mechanism of safener action**

of cofactors involved in the herbicide detoxication [50, 52, 89, 90].

less, only a few examples can be found in the literature as to cytochrome P450-dependent

Monocotyledonous (*Avena fatua*, *Bromus inermis*, *Echinochloa crus-galli*) and dicotyledonous (*Amaranthus retroflexus*, *Abuthilon threophrasti*, *Xantium strumarium*) weeds were used to study the interaction of safeners, herbicides metabolized by cytochrome P450 enzymes, and P450 inhibitors on herbicide phytotoxicity and P450 levels of weeds and maize [86]. The safener NA was slightly protective to all monocots at the reduced rate (50 g/ha) of nicosulfuron and also exhibited safening effects on dicots against all herbicides. MG-191 reduced growth inhibition

Control NAa ABTb

4-day-old etiolated maize seedlings.

**Species Cytochrome P450, pmol/mg protein**

7-day-old etiolated weed seedlings; dND not detectable; e

the P450 level was higher in maize than in weeds.

the P450 inhibitor was uneffective on weed P450s.

**Table 3.** Cytochrome P450 contents of mono- and dicot weeds and influence of treatment with the safener NA and

Weed microsomal cytochrome P450 enzymes were found less stable than those from maize. Carbon-monoxide difference spectra for *B. inermis* and *X. stumarium* could not be recorded probably due to dark colors of microsomal preparations and difficulties in resuspending the microsomes. Cytochrome P450 content in the microsomal membrane fraction of *A. fatua* was 2.4-fold greater than in *E. crus-galli* (Table 3). Among dicotyledonous plants, *A. theophrasti* contained 5.1-fold higher level of the enzyme as compared to that of *A. retroflexus*. However,

It is difficult to evaluate changes in the enzyme contents of weed species pretreated with the safener NA or the P450 inhibitor ABT due to the high values of standard deviation of the data. Following treatments with NA, a stimulating tendency could be observed for weeds except *E. crus-galli*. With maize the NA treatment had no enhancing effect on the enzyme content. However, a significant increase (43%) was found when maize seedlings treated with ABT but

For further characterization of *in vivo* interaction of the combination of the herbicides with safeners and inhibitors microsomes isolated from etiolated maize seedlings were used (Figure 7). Treatment of maize seedlings with nicosulfuron resulted in 30% elevation in P450 level while no effect of EPTC was found. The combination of NA with either bentazon or nicosul‐

*A. fatua*<sup>c</sup> 41±11 49±12 36±17 *B. inermis* NDd ND ND *E. crus-galli* 17±8 14±9 ND *A. retroflexus* 10±4 21±8 ND *A. theophrasti* 51±24 89±32 54±27 *X. strumarium* ND ND ND Maizee 67±14 73±15 96±18

monooxygenase system in weed species [85].

608 Herbicides - Current Research and Case Studies in Use

of EPTC in *A. fatua* and *E. crus-galli*.

a

0.5 %w/v; b 1 μM; <sup>c</sup>

P450 inhibitor ABT.

conjugate transporters such as *At*MRP1-4 [91]. Safeners also increased GSH content of *Arabidopsis* seedlings. However, treatment of *Arabidopsis* plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Immunoblot analysis confirmed that *At*GSTU19 was induced in response to several safeners. These results indicate that, although *Arabidopsis* may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds. Concerning the location of safener binding site(s) of plants few studies have been conducted. A high-affinity cytosolic-binding site for the dichloroacetamide safener (*R*,*S*)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidine was found in etiolated maize seedlings ([92]. The The mechanism by which safeners act is currently unknown despite the widespread agricul‐ tural use and the substantial experimental evidence accumulated on the biochemical basis of action. Safeners appear to induce a set of genes that encode enzymes and biosynthesis of cofactors involved in the herbicide detoxication [50, 52, 89, 90].

binding was highest in the coleoptiles and lowest in the leaves. A good correlation was shown between the safener effectiveness.

Chloroacetanilide and thiocarbamate herbicides were effective inhibitors of safener binding at low concentrations. The inhibition by alachlor and EPTC was shown to be competitive. The safener binding protein (SafBP) was purified to homogeneity having a molecular The exact mechanism of safener-mediated enhancement of GST activity is not completely understood. GSTs are induced by a diverse range of chemicals and accompanied by the production of active oxygen species. Thus the connection between safener-mediated protec‐ tion of crops and oxidative stress tolerance has been suggested [66]. Many GSTs are effective not only in conjugating electrophilic substrates but also function as glutathione peroxidases. Safeners may induce GST expression by mimicking oxidative insult [67]. Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledonous crops. The reason(s) for this difference in safening is unknown. Treatment of *Arabidopsis* seedlings with various safeners resulted in enhanced GST activities and expression of GSH-conjugate transporters such as *At*MRP1-4 [91]. Safeners also increased GSH content of *Arabidopsis* seedlings. However, treatment of *Arabidopsis* plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Immunoblot analysis confirmed that *At*GSTU19 was induced in response to several safeners. These results indicate that, although *Arabidopsis* may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds.

xenobiotic detoxication whereas herbicide-regulated proteins belonged to several classes involved in general stress responses. Quantitative RT-PCR revealed that multidrug resistanceassociated protein (MRP) transcripts were highly induced by safeners and two MRP genes

Safener

TGA ? Nrf2

PPA1

Gene expression Gene expression Gene expression (e.g. GSTs and OPRs) (e.g. GSTs and P450s) (e.g. GSTs and P450s)

Detoxification of tolerance

**Figure 10.** Suggested safener-mediated signalling pathway for regulation of defense genes and activation of detoxifi‐ cation pathways in plants by Riechers et al. [52]. Dashed lines indicate possible but unproven signaling pathways while solid lines indicate known signaling pathways. ODPA: 12-oxo-phytodienoic acid; OPRs: ODPA-reductases; PPA1: A1 type phytoprostanes; JA: jasmonic acid; TGA: TGA transcription factor; Nrf2: nuclear factor (erythroid-derived 2)-like 2; Col1: coronative insensitive protein 1; JAZ: transcriptional repressor protein; TF: transcription factor/activator.

Safeners were suggested to trigger an unidentified, preexisting signaling pathway for detox‐ ification of endogenous toxins or xenobiotics [96]. According to a new hypothesis, safeners may be utilizing an oxidized lipid-mediated (oxylipins) or cyclopentenone-mediated signaling pathway which subsequently leads to the expression of GSTs and other proteins involved in detoxification and plant defense [52]. Some possible safener-mediated signaling pathways for the regulation of defense genes and activation of detoxification pathways have been suggested (Figure 8). Safeners may tap into a RES oxylipin-mediated signaling pathway and up-regulate TGA transcription factors, an Nrf2-Keap1-mediated as well as jasmonic acid-mediated signaling pathways. Safeners and oxylipins as reactive electrophilic species (RES oxylipins) have a common biological activity since both strongly induce the expression of defense genes

Safener metabolism Herbicide metabolism Plant defense responses

**Herbicide tolerance**

TF

Abiotic/biotic stress

JAZ

CoI1

JA

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were differently expressed.

ODPA

RES oxylipins

and activate detoxification responses in plants [39, 40].

Concerning the location of safener binding site(s) of plants few studies have been conducted. A high-affinity cytosolic-binding site for the dichloroacetamide safener (*R*,*S*)-3-dichloroace‐ tyl-2,2,5-trimethyl-1,3-oxazolidine was found in etiolated maize seedlings ([92]. The binding was highest in the coleoptiles and lowest in the leaves. A good correlation was shown between the safener effectiveness. Chloroacetanilide and thiocarbamate herbicides were effective inhibitors of safener binding at low concentrations. The inhibition by alachlor and EPTC was shown to be competitive. The safener binding protein (SafBP) was purified to homogeneity having a molecular mass of 39 kDa [93]. Based on the peptides obtained from proteolytic digests of SafBP a cDNA encoding SafBP was cloned and expressed in *E. coli*. The predicted primary structure of SafBP was related to a phenolic *O*-methyltransferase but SafBP did not catalyze O-methylation of catechol or caffeic acid. It was concluded that SafBP may not be the primary site of action of the dichloroacetamide safeners. Supporting the participation of *O*methyltransferases in the safener action, treatment of wheats (*Triticum aestivum* L.) with cloquintocet-mexyl resulted in an accelerated depletion of flavone *C*-glycosides and a selective shift in the metabolism of endogenous phenolics [94]. Changes in phenolic content were associated with an increase in *O*-methyltransferase and *C*-glucosyltransferase activity toward flavonoid substrates.

Proteomic methods were used to identify herbicide safener-induced proteins in the coleop‐ tile of *Triticum tauschii* [95]. The herbicide safener, fluxofenim, dramatically increased protein abundance in the molecular range in the molecular weight range of 24 to 30 kDa as well as a few higher molecular weight protein and overall 20 proteins were identified. Among the eighteen inducible proteins 15 were glutathione *S*-transferase subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from phi subclass, and one was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family with proteins that have roles in glycolysis and the Krebs cycle. One of the two constitutively expressed proteins showed the highest homology to the dehydroascorbate reductase subclass of GSTs while the other to an ascorbate peroxidase. Results indicated that the induced proteins were associated with herbicide detoxication and with general stress response. In another study with cloquintocet-mexyl safener and dimethenamid herbicide 29 safener-induced and 10 herbicide-regulated proteins were identified in *Triticum tauschii* seedlings [39]. Surprisingly, mutually exclusive sets of proteins were identified following herbicide or safener treatment suggesting a different signaling pathway for each chemical. Safener-responsive proteins were mostly involved in xenobiotic detoxication whereas herbicide-regulated proteins belonged to several classes involved in general stress responses. Quantitative RT-PCR revealed that multidrug resistanceassociated protein (MRP) transcripts were highly induced by safeners and two MRP genes were differently expressed.

increase herbicide tolerance in cereals but not in dicotyledonous crops. The reason(s) for this difference in safening is unknown. Treatment of *Arabidopsis* seedlings with various safeners resulted in enhanced GST activities and expression of GSH-conjugate transporters such as *At*MRP1-4 [91]. Safeners also increased GSH content of *Arabidopsis* seedlings. However, treatment of *Arabidopsis* plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Immunoblot analysis confirmed that *At*GSTU19 was induced in response to several safeners. These results indicate that, although *Arabidopsis* may not be protected from herbicide injury by safeners, at least one component of their detoxification

Concerning the location of safener binding site(s) of plants few studies have been conducted. A high-affinity cytosolic-binding site for the dichloroacetamide safener (*R*,*S*)-3-dichloroace‐ tyl-2,2,5-trimethyl-1,3-oxazolidine was found in etiolated maize seedlings ([92]. The binding was highest in the coleoptiles and lowest in the leaves. A good correlation was shown between the safener effectiveness. Chloroacetanilide and thiocarbamate herbicides were effective inhibitors of safener binding at low concentrations. The inhibition by alachlor and EPTC was shown to be competitive. The safener binding protein (SafBP) was purified to homogeneity having a molecular mass of 39 kDa [93]. Based on the peptides obtained from proteolytic digests of SafBP a cDNA encoding SafBP was cloned and expressed in *E. coli*. The predicted primary structure of SafBP was related to a phenolic *O*-methyltransferase but SafBP did not catalyze O-methylation of catechol or caffeic acid. It was concluded that SafBP may not be the primary site of action of the dichloroacetamide safeners. Supporting the participation of *O*methyltransferases in the safener action, treatment of wheats (*Triticum aestivum* L.) with cloquintocet-mexyl resulted in an accelerated depletion of flavone *C*-glycosides and a selective shift in the metabolism of endogenous phenolics [94]. Changes in phenolic content were associated with an increase in *O*-methyltransferase and *C*-glucosyltransferase activity toward

Proteomic methods were used to identify herbicide safener-induced proteins in the coleop‐ tile of *Triticum tauschii* [95]. The herbicide safener, fluxofenim, dramatically increased protein abundance in the molecular range in the molecular weight range of 24 to 30 kDa as well as a few higher molecular weight protein and overall 20 proteins were identified. Among the eighteen inducible proteins 15 were glutathione *S*-transferase subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from phi subclass, and one was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family with proteins that have roles in glycolysis and the Krebs cycle. One of the two constitutively expressed proteins showed the highest homology to the dehydroascorbate reductase subclass of GSTs while the other to an ascorbate peroxidase. Results indicated that the induced proteins were associated with herbicide detoxication and with general stress response. In another study with cloquintocet-mexyl safener and dimethenamid herbicide 29 safener-induced and 10 herbicide-regulated proteins were identified in *Triticum tauschii* seedlings [39]. Surprisingly, mutually exclusive sets of proteins were identified following herbicide or safener treatment suggesting a different signaling pathway for each chemical. Safener-responsive proteins were mostly involved in

systems is responsive to these compounds.

610 Herbicides - Current Research and Case Studies in Use

flavonoid substrates.

**Figure 10.** Suggested safener-mediated signalling pathway for regulation of defense genes and activation of detoxifi‐ cation pathways in plants by Riechers et al. [52]. Dashed lines indicate possible but unproven signaling pathways while solid lines indicate known signaling pathways. ODPA: 12-oxo-phytodienoic acid; OPRs: ODPA-reductases; PPA1: A1 type phytoprostanes; JA: jasmonic acid; TGA: TGA transcription factor; Nrf2: nuclear factor (erythroid-derived 2)-like 2; Col1: coronative insensitive protein 1; JAZ: transcriptional repressor protein; TF: transcription factor/activator.

Safeners were suggested to trigger an unidentified, preexisting signaling pathway for detox‐ ification of endogenous toxins or xenobiotics [96]. According to a new hypothesis, safeners may be utilizing an oxidized lipid-mediated (oxylipins) or cyclopentenone-mediated signaling pathway which subsequently leads to the expression of GSTs and other proteins involved in detoxification and plant defense [52]. Some possible safener-mediated signaling pathways for the regulation of defense genes and activation of detoxification pathways have been suggested (Figure 8). Safeners may tap into a RES oxylipin-mediated signaling pathway and up-regulate TGA transcription factors, an Nrf2-Keap1-mediated as well as jasmonic acid-mediated signaling pathways. Safeners and oxylipins as reactive electrophilic species (RES oxylipins) have a common biological activity since both strongly induce the expression of defense genes and activate detoxification responses in plants [39, 40].

### **9. Conclusions**

Fifty-year of herbicide safeners resesearch and use confirms that these molecules offered new ways to improve herbicide selectivity. Although this technology now competes with herbicidetolerant, genetically-modified or naturally-selelected crops, safeners still comprise an impor‐ tant part of the herbicide market in maize, cereals and rice [10]. Many of the commercial safeners are in off-patent status offering a chance for the generic manufacturers to enter the market together with off-patent herbicides. In contrast, recent herbicide mixture patents with new herbicides still allow their exclusive usage by the patent holder [10].

[3] Hoffmann O. L. (1978). Herbicide antidotes: From concept to practice. In: *Chemistry and action of herbicide antidotes*. Pallos F. M., Casida J. E. (Eds). pp 35-61, Academic

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[11] Tam A. C., Behki R. M., Khan S. U. (1988). Effect of dietholate (R-33865) on the degra‐ dation of thiocarbamate herbicide by an EPTC-degrading bacterium. *J. Agric. Food.*

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maize. *Pestic. Sci.*, 17, 25-32.

Although safeners do not improve herbicide tolerance in dicot plants, but the utilization of biotechnology tools may help in extending the safener response from monocot to dicots. It was found, however that *Arabidopsis* transgenic plants did not respond to safeners at whole-plant level despite the increase of the expression of tau class protein in the roots [91]. Additionally, knowledge of critical regulatory elements in the promoters or untranslated regions of genes encoding detoxification enzymes, or a comprehensive understanding how gene expression is up-regulated by safeners might lead to the precise manipulation of transgene expression of plants [52].

The use of safeners to enhance tolerance of plants to organic pollutants such as herbicides, heavy metals or oils in the environment (soil, water) could also be a promising application of these chemicals. Phytoremediation studies with soils contaminated with oils and heavy metals and safener-treated wheat seeds have recently been reported [97]. While untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants.

### **Author details**

#### Istvan Jablonkai

Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

### **References**


**9. Conclusions**

612 Herbicides - Current Research and Case Studies in Use

plants [52].

**Author details**

Istvan Jablonkai

**References**

of Sciences, Budapest, Hungary

growing before succumbing to the pollutants.

3-45, Academic Press, San Diego, USA.)

Fifty-year of herbicide safeners resesearch and use confirms that these molecules offered new ways to improve herbicide selectivity. Although this technology now competes with herbicidetolerant, genetically-modified or naturally-selelected crops, safeners still comprise an impor‐ tant part of the herbicide market in maize, cereals and rice [10]. Many of the commercial safeners are in off-patent status offering a chance for the generic manufacturers to enter the market together with off-patent herbicides. In contrast, recent herbicide mixture patents with

Although safeners do not improve herbicide tolerance in dicot plants, but the utilization of biotechnology tools may help in extending the safener response from monocot to dicots. It was found, however that *Arabidopsis* transgenic plants did not respond to safeners at whole-plant level despite the increase of the expression of tau class protein in the roots [91]. Additionally, knowledge of critical regulatory elements in the promoters or untranslated regions of genes encoding detoxification enzymes, or a comprehensive understanding how gene expression is up-regulated by safeners might lead to the precise manipulation of transgene expression of

The use of safeners to enhance tolerance of plants to organic pollutants such as herbicides, heavy metals or oils in the environment (soil, water) could also be a promising application of these chemicals. Phytoremediation studies with soils contaminated with oils and heavy metals and safener-treated wheat seeds have recently been reported [97]. While untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly

Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy

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**Chapter 24**

**Herbicides — A Double Edged Sword**

Weeds represent a global agronomic problem that threatens the productivity of cultivated crops. Weeds compete with cultivated crops for the available moisture, nutrients and light. Consequently, weeds significantly reduce either crop yield or quality. Control of weeds is essential to maintaining the production of economic crops. Weed control may be achieved either through manual eradication or herbicide application. Balanced usage of herbicides should be considered in controlling weeds. Low concentrations of herbicides may act as growth regulators for the main crop metabolism [1]. However, in some cases, herbicides may affect the main crop adversely by interfering with its essential biochemical processes such as

respiration, photosynthesis, protein metabolism, and hydrolytic enzyme activity [1].

has been studied extensively in many investigations such as in **El-Hadary** [1].

Herbicide interference with the morphology, physiology and biochemical pathways of treated plants varies according to the characteristic actions of the herbicide and depends upon the degree of tolerance or susceptibility of the crop plant species. Environmental factors and soil conditions affecting plant growth, as well as herbicide formulation, herbicide degradation and application method would significantly influence the effects of herbicides on treated plants. Once an herbicide reaches the site of action in the treated plants, the biochemical processes are affected. Herbicides differ in their site of action and may have more than one site of action. As the herbicide concentration increases in plant tissue, additional sites of action may become involved. The effect of herbicides on growth, productivity and different metabolic activities

Authors intended to give some examples for commercial herbicides that were applied in agronomic systems within the past fifty years. These examples include those herbicides which may now be internationally prohibited but are still used in the developing and under-

> © 2013 El-Hadary and Chung; 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 El-Hadary and Chung; 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.

Mona H. El-Hadary and Gyuhwa Chung

http://dx.doi.org/10.5772/55957

**1.1. A word from the authors**

**1. Introduction**

Additional information is available at the end of the chapter


## **Herbicides — A Double Edged Sword**

Mona H. El-Hadary and Gyuhwa Chung

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55957

### **1. Introduction**

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620 Herbicides - Current Research and Case Studies in Use

bot.2011.12.030.

Weeds represent a global agronomic problem that threatens the productivity of cultivated crops. Weeds compete with cultivated crops for the available moisture, nutrients and light. Consequently, weeds significantly reduce either crop yield or quality. Control of weeds is essential to maintaining the production of economic crops. Weed control may be achieved either through manual eradication or herbicide application. Balanced usage of herbicides should be considered in controlling weeds. Low concentrations of herbicides may act as growth regulators for the main crop metabolism [1]. However, in some cases, herbicides may affect the main crop adversely by interfering with its essential biochemical processes such as respiration, photosynthesis, protein metabolism, and hydrolytic enzyme activity [1].

Herbicide interference with the morphology, physiology and biochemical pathways of treated plants varies according to the characteristic actions of the herbicide and depends upon the degree of tolerance or susceptibility of the crop plant species. Environmental factors and soil conditions affecting plant growth, as well as herbicide formulation, herbicide degradation and application method would significantly influence the effects of herbicides on treated plants. Once an herbicide reaches the site of action in the treated plants, the biochemical processes are affected. Herbicides differ in their site of action and may have more than one site of action. As the herbicide concentration increases in plant tissue, additional sites of action may become involved. The effect of herbicides on growth, productivity and different metabolic activities has been studied extensively in many investigations such as in **El-Hadary** [1].

### **1.1. A word from the authors**

Authors intended to give some examples for commercial herbicides that were applied in agronomic systems within the past fifty years. These examples include those herbicides which may now be internationally prohibited but are still used in the developing and under-

developing countries due to their low price and the little information available about them. References have been included that cover a long era of research concerning herbicide appli‐ cation in order to include those prohibited herbicides. Also, references were included that focus on research that was conducted in under- and developing countries.

**3. Selectivity of herbicides**

**3.1. Biochemical differences**

**3.2. Morphological differences**

dicotyledon weeds.

**3.3. Chronological selectivity**

**3.4. Positional selectivity**

if those herbicides are applied too late (Figure 1) [1].

factors related to:

Selectivity of herbicides for eradicating weeds can be achieved through employing some

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 623

Based on the biochemical differences between weeds and crops, or even weeds between each other, selectivity can be achieved. There is a great diversity of types of weeds usually growing in one crop. When employing an herbicide based on biochemical differences, the crop plant would possess a defense mechanism that is usually absent in most of the competing weed species. Consequently, the herbicide would react with the biochemical metabolism of the

The selectivity which depends upon morphological differences is characteristic for postemergence herbicides. Dicotyledonous plants have leaves spread out and exposed meriste‐ matic tissue, so that the toxin is directed to the growing point situated at the center of a rosette. While upright leaves of monocotyledonous plants enable plants to form a sheath around the meristem that protects it from receiving the herbicidal spray (Figure 1) [1]. Therefore, such morphological differences can be recruited to work with monocotyledon crops against

Chronological selectivity utilizes the time period necessary for growing both weeds and crop plants. In other words, it depends upon the fact that some weeds are shallower rooted and grow more rapidly than the crop plants. In consequence, many of the potentially more competitive weeds that emerge before the crop can be sprayed by a foliage spray. The time of application of the herbicide is important for chronological selectivity to be successful. That means if the non-selective herbicides are applied too early, many of the germinating weed seedlings will escape and break through the soil surface; however, the crop may be damaged

Positional selectivity is based upon the localization of weeds on the soil surface related to the main plant crop position. If seeds, tubers, etc., of the crops are large compared with those of the weeds, they become sown or placed quite deeply in the soil compared with the more shallow competitive weed seeds. Consequently, positional selectivity can often be achieved by spraying the soil surface with soil acting herbicides. These herbicides are able to destroy weed seeds growing in the top few millimeters of the soil, whereas the large seeds of the crop are protected by the fact that they are sown deeper in the soil. Bacteria and other microorgan‐

weeds without any fatal interference on main crop metabolism.

#### **1.2. Herbicides**

This chapter will discuss different herbicide groups, classification, selectivity, interference with metabolic processes and hazardous action upon crop plants. Also, the relation to naturally occurring phenomena, such allelopathy and future prospects of genetic engineering in the production of plant herbicides themselves, will be mentioned.

### **2. Classification of herbicides (Broad lines)**

There are different broad lines upon which herbicides could be classified:

#### **2.1. Application timing**

Time of application of an herbicide is so critical for getting satisfying results. Herbicides application is achieved either pre-emergence or post-emergence of the weed seedlings. Preemergence involves herbicide application prior to seed germination while post-emergence means application after seed germination and active growth. Moreover, post-directed application refers to targeting the treatment to a particular portion of the plant once emerged and growing.

#### **2.2. Application method**

Herbicides may be applied either as a foliar spray or a soil treatment. The application method may take either the broadcast pattern through treatment of the entire area or the spot pattern through specified area treatment.

#### **2.3. Chemical groups**

The chemical group to which an herbicide belongs indicates its mode of action. A good classification and description for herbicides is provided by "Compendium of Pesticide Common Names" at the web site of *http*://www.alanwood.net/pesticides/class\_herbi‐ cides.html.

#### **2.4. Mode of action**

Herbicides poisonous action goes either by contact or systematically. Herbicides can be classified according to their mode of action into two categories; non-selective herbicides and selective herbicides. Non-selective herbicides are characterized by having a general poisonous effect to the plant cells while selective herbicides can recognize the plant which they affect and kill it by interference with its principle biochemical processes.

### **3. Selectivity of herbicides**

developing countries due to their low price and the little information available about them. References have been included that cover a long era of research concerning herbicide appli‐ cation in order to include those prohibited herbicides. Also, references were included that focus

This chapter will discuss different herbicide groups, classification, selectivity, interference with metabolic processes and hazardous action upon crop plants. Also, the relation to naturally occurring phenomena, such allelopathy and future prospects of genetic engineering in the

Time of application of an herbicide is so critical for getting satisfying results. Herbicides application is achieved either pre-emergence or post-emergence of the weed seedlings. Preemergence involves herbicide application prior to seed germination while post-emergence means application after seed germination and active growth. Moreover, post-directed application refers to targeting the treatment to a particular portion of the plant once emerged

Herbicides may be applied either as a foliar spray or a soil treatment. The application method may take either the broadcast pattern through treatment of the entire area or the spot pattern

The chemical group to which an herbicide belongs indicates its mode of action. A good classification and description for herbicides is provided by "Compendium of Pesticide Common Names" at the web site of *http*://www.alanwood.net/pesticides/class\_herbi‐

Herbicides poisonous action goes either by contact or systematically. Herbicides can be classified according to their mode of action into two categories; non-selective herbicides and selective herbicides. Non-selective herbicides are characterized by having a general poisonous effect to the plant cells while selective herbicides can recognize the plant which they affect and

kill it by interference with its principle biochemical processes.

on research that was conducted in under- and developing countries.

production of plant herbicides themselves, will be mentioned.

There are different broad lines upon which herbicides could be classified:

**2. Classification of herbicides (Broad lines)**

**1.2. Herbicides**

622 Herbicides - Current Research and Case Studies in Use

**2.1. Application timing**

**2.2. Application method**

**2.3. Chemical groups**

**2.4. Mode of action**

cides.html.

through specified area treatment.

and growing.

Selectivity of herbicides for eradicating weeds can be achieved through employing some factors related to:

### **3.1. Biochemical differences**

Based on the biochemical differences between weeds and crops, or even weeds between each other, selectivity can be achieved. There is a great diversity of types of weeds usually growing in one crop. When employing an herbicide based on biochemical differences, the crop plant would possess a defense mechanism that is usually absent in most of the competing weed species. Consequently, the herbicide would react with the biochemical metabolism of the weeds without any fatal interference on main crop metabolism.

#### **3.2. Morphological differences**

The selectivity which depends upon morphological differences is characteristic for postemergence herbicides. Dicotyledonous plants have leaves spread out and exposed meriste‐ matic tissue, so that the toxin is directed to the growing point situated at the center of a rosette. While upright leaves of monocotyledonous plants enable plants to form a sheath around the meristem that protects it from receiving the herbicidal spray (Figure 1) [1]. Therefore, such morphological differences can be recruited to work with monocotyledon crops against dicotyledon weeds.

### **3.3. Chronological selectivity**

Chronological selectivity utilizes the time period necessary for growing both weeds and crop plants. In other words, it depends upon the fact that some weeds are shallower rooted and grow more rapidly than the crop plants. In consequence, many of the potentially more competitive weeds that emerge before the crop can be sprayed by a foliage spray. The time of application of the herbicide is important for chronological selectivity to be successful. That means if the non-selective herbicides are applied too early, many of the germinating weed seedlings will escape and break through the soil surface; however, the crop may be damaged if those herbicides are applied too late (Figure 1) [1].

#### **3.4. Positional selectivity**

Positional selectivity is based upon the localization of weeds on the soil surface related to the main plant crop position. If seeds, tubers, etc., of the crops are large compared with those of the weeds, they become sown or placed quite deeply in the soil compared with the more shallow competitive weed seeds. Consequently, positional selectivity can often be achieved by spraying the soil surface with soil acting herbicides. These herbicides are able to destroy weed seeds growing in the top few millimeters of the soil, whereas the large seeds of the crop are protected by the fact that they are sown deeper in the soil. Bacteria and other microorgan‐ isms attack and inactivate most herbicides when used at economic concentrations so the potential hazard to the crop is reduced (Figure 1) [1].

#### **3.5. Placement selectivity**

Placement selectivity can be achieved for non-selective substances when it is possible to direct a foliar spray in such a way that it makes contact only with the leaves of weeds and not the crop [2].

#### **3.6. Genetic engineering**

If the mode of action of an herbicide is known and the target proves to be a protein, genetic engineering may well allow the crop gene coding for that protein to be isolated. It is then possible to alter that crop gene so that it is less affected by the herbicide [2].This will be discussed in detail at the end of the chapter.

## **4. Herbicide interference with physiological and biochemical processes and plant response**

Mode of action of herbicides can lead to various physiological and biochemical effects on both growth and development of the emerging seedlings as well as the established plants. These physiological and biochemical effects are followed by various types of visual injury symptoms on susceptible plants. The incidental damage extent depends on the selectivity of the herbicide as well as the applied concentration. The herbicide application is always recommended at a certain dose termed as recommended dose (R), above which, a great damage to the crop plant may be obtained. Overdoses threaten not only the crop plant but also the environment and human health. Some herbicides in lower than recommended doses may act as growth regulators for crop plants [1,2].

Even recommended doses may have undesired effects upon the crop. The undesired effects might occur in the form of chlorosis, defoliation, necrosis, morphological aberrations, growth stimulation, cupping of leaves, marginal leaf burn, delayed emergence, germination failure, etc. These injury symptoms may appear on any part of the plant.

The various physiological and biochemical processes affected by herbicides are grouped under five broad categories including: respiration, mitochondrial activities, photosynthesis, protein synthesis, nucleic acid metabolism, and hydrolytic enzyme activities. Most herbicides can affect at least one or all of these processes. The following discusses their effect on various biochemical processes.

the mitochondrial activities by uncoupling the reaction responsible for ATP synthesis or interfering with electron transport and energy transfer. Uncouplers act on the membranes of the mitochondria in which phosphorylation takes place. Electrons leak through the membranes so that the charges that they normally separate are lost. As a result, energy is not accumulated

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 625

**Figure 1.** Factors Exploitable to Achieve Selectivity of Herbicides [1] as adapted from [2].

for ATP synthesis [3].

#### **4.1. Respiration and mitochondrial activities**

Cellular respiration that takes place in mitochondria involves the synthesis of ATP and the transport of electrons and protons from respiratory substances to oxygen. Herbicides affect

**Figure 1.** Factors Exploitable to Achieve Selectivity of Herbicides [1] as adapted from [2].

isms attack and inactivate most herbicides when used at economic concentrations so the

Placement selectivity can be achieved for non-selective substances when it is possible to direct a foliar spray in such a way that it makes contact only with the leaves of weeds and not the

If the mode of action of an herbicide is known and the target proves to be a protein, genetic engineering may well allow the crop gene coding for that protein to be isolated. It is then possible to alter that crop gene so that it is less affected by the herbicide [2].This will be

**4. Herbicide interference with physiological and biochemical processes**

Mode of action of herbicides can lead to various physiological and biochemical effects on both growth and development of the emerging seedlings as well as the established plants. These physiological and biochemical effects are followed by various types of visual injury symptoms on susceptible plants. The incidental damage extent depends on the selectivity of the herbicide as well as the applied concentration. The herbicide application is always recommended at a certain dose termed as recommended dose (R), above which, a great damage to the crop plant may be obtained. Overdoses threaten not only the crop plant but also the environment and human health. Some herbicides in lower than recommended doses may act as growth

Even recommended doses may have undesired effects upon the crop. The undesired effects might occur in the form of chlorosis, defoliation, necrosis, morphological aberrations, growth stimulation, cupping of leaves, marginal leaf burn, delayed emergence, germination failure,

The various physiological and biochemical processes affected by herbicides are grouped under five broad categories including: respiration, mitochondrial activities, photosynthesis, protein synthesis, nucleic acid metabolism, and hydrolytic enzyme activities. Most herbicides can affect at least one or all of these processes. The following discusses their effect on various

Cellular respiration that takes place in mitochondria involves the synthesis of ATP and the transport of electrons and protons from respiratory substances to oxygen. Herbicides affect

etc. These injury symptoms may appear on any part of the plant.

potential hazard to the crop is reduced (Figure 1) [1].

discussed in detail at the end of the chapter.

**3.5. Placement selectivity**

624 Herbicides - Current Research and Case Studies in Use

**3.6. Genetic engineering**

**and plant response**

regulators for crop plants [1,2].

biochemical processes.

**4.1. Respiration and mitochondrial activities**

crop [2].

the mitochondrial activities by uncoupling the reaction responsible for ATP synthesis or interfering with electron transport and energy transfer. Uncouplers act on the membranes of the mitochondria in which phosphorylation takes place. Electrons leak through the membranes so that the charges that they normally separate are lost. As a result, energy is not accumulated for ATP synthesis [3].

#### **4.2. Photosynthesis**

Pigment content and photosynthetic activity are affected by herbicidal applications. The mode of action of herbicides on the photosynthesis process depends on the chemical group to which the herbicide belongs [3]. Herbicides affect chloroplast organization and pigment formation especially chlorophyll which is the principle absorbing pigment. Chlorophyll bleaching is a potent inhibitor for photosynthetic electron transport and CO2 fixation.

synthesis as reported in barley [9-12]. Also, metalachlor inhibited protein synthesis in barley [13]. RNA and protein synthesis in tomato were found to be inhibited by propanil [14].

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 627

Benzoic and phenylacetic herbicides had variable effects on protein. For example, chloramben had no effect on RNA and protein synthesis on susceptible species [15]. On the other hand, it was suggested that foliar-applications of dicamba increased RNA and protein levels in

Carbamate herbicide groups include a large number of herbicides such as asulam, barban, chlorpropham, propham, desmedipham and phenmedipham. [17]. Barban was found to inhibit protein synthesis and the degree of inhibition was related to the susceptibility of the plant species. For example, barban increased nucleotide content of wild oat shoots associated with disruption of RNA and protein synthesis. Chlorproham and propham inhibited amino acid incorporation into protein and induced a reduction in protein synthesis [18]. DNA, RNA and protein synthesis are also inhibited at high concentrations (10-3 M) of propham [19].

Fluridone, paraquat, perfluidone and propanil treatments were found to reduce soluble protein levels in soybean [20]. Paraquat and diquat readily act on proteins, modifying their structure and function (e.g.lysozome) since they interact with dibasic and dicarboxylic amino

Oxadiazon at high doses inhibited protein synthesis in soybean while RNA and DNA synthesis were less sensitive to oxadiazon [22]. Combination of 2,4-D and glufosinate had an additive effect on protein synthesis in both sorghum and soybean [22]. On the other hand, sethoxydim, R- 25788 [N, N dichloroacetamide] or R- 28725 at low doses did not inhibit protein or RNA synthesis in cells of both sorghum and soybean but sethoxydim significantly inhibited DNA synthesis while R-25788 stimulated it [23]. Thus, the combined effects of sethoxydim and the two Safeners (R- 25788 and R- 28725) on protein and RNA synthesis were additive while on

The application of haloxyfop to *Zea mays* and soybean cell suspension, increased 14-C labeled free amino acids level and incorporation of 14C leucine as a precursor revealed that haloxyfop

Napropamide reduced DNA synthesis, RNA root cells of *Pea* and protein [25]. The inhibitory effect of napropamide on the mitotic cycle resulted from an inhibition in the synthesis of cell cycle specific protein. In contrast, 0.5 R, 1R and 1.5 R of metribuzin stimulated total and protein-N accumulation in soybean. Consequently, protein content was increased while RNA and DNA levels decreased [26]. Protein content of soybean yield was reported to be increased by application of 100 ppm GA3 (gibbrellic acid) and 2g/L Librel separately or together [27].

Metoxuron had a remarkable inhibition on the total protein biosynthesis, while bromoxynil accelerated the biosynthesis of low molecular proteins (water-soluble proteins) and inhibited the biosynthesis of high molecular proteins (sodium hydroxid soluble proteins) in wheat (*Triticum aestivum*, var. Sakha 69) [28]. Bromoxynil at low doses (0.4 and 0.8 kg / fed) enhanced protein content and RNA synthesis in wheat plants after 30 to 60 days from foliar spraying [29].

susceptible plants by removal of histone from the DNA template [16]

acids like ornithine and glutamic acid [21].

DNA synthesis they were antagonistic.

did not inhibit protein synthesis [24].

Herbicides affect photosynthetic activity via different ways including photosynthetic pig‐ ments. The primary site of action is located at photosystem II (PSII) since they cause blocking of the Hill reaction. The oxygen evolution step is inhibited by interfering with the reducing side rather than the oxidizing side of PSII [4]. The inhibition of electron transfer through PSII causes a block in the whole transport chain as the inhibition of the noncyclic photophosphor‐ ylation and ATP synthesis. Consequently, the production of NADP is blocked and the function of the protective carotenoid system is prevented [5]. Urea herbicides inhibit both noncyclic and cyclic electron transport by forming a complex with oxidized form of an unknown component located in the electron transfer pathway close to PSII. This component also takes part in cyclic electron transport.

The photosystem I (PSI) also could be reduced by some herbicides but it requires much higher concentrations of the herbicide than that required for the inhibition of PSII. Since PSII precedes PSI and the former is blocked completely at concentrations which do not affect PSI.

In a study conducted by **El-Hadary** [1], it was observed that photosynthetic activity measured in wheat chloroplasts (variety Giza 163) was greatly reduced throughout the growth by using Brominal as an example for bromphenol herbicides but lower concentrations (1/4R, 1/2R and R) increased the activity. Pigment content represented as chlorophyll, a/b ratio and carotenoids showed a similar results [1]. In the same study, sulfonyl-urea herbicides such as Granstar were examined. It was observed that low Granstar concentrations stimulated the photolytic activity of chloroplasts while high concentrations reduced it. However, Granstar reduced a/b ratios throughout the growth stages, except a slight increase at the fruiting stage with 1/2R. Carote‐ noids were decreased only with high Granstar concentrations [1].

#### **4.3. Protein and nucleic acid metabolism**

Protein synthesis takes place mainly in three stages involving initiation, elongation and termination of the polypeptide chain. Blocking any one of these stages by the herbicide will cause inhibition of protein and nucleic acid synthesis. The herbicides that inhibit photosyn‐ thesis and ATP formation can lead to inhibition of protein synthesis as a secondary effect. The damage that is caused by an herbicide is governed by its chemical group. There are numerous studies that investigate effects of the herbicidal chemical groups upon protein and nucleic acid metabolism [2].

For instance, sulfonyl-urea herbicides block the biosynthesis of the branched chain amino acids in higher plants [6,7]. Aliphatic herbicides like Dalapon cause degradation of protein to ammonium compounds as detected in *Setaria lutescens* and sugar beets [8]. While acetamide herbicides such as propachlor, alachlor and prynaclor inhibited the protein content and RNA synthesis as reported in barley [9-12]. Also, metalachlor inhibited protein synthesis in barley [13]. RNA and protein synthesis in tomato were found to be inhibited by propanil [14].

**4.2. Photosynthesis**

626 Herbicides - Current Research and Case Studies in Use

part in cyclic electron transport.

Pigment content and photosynthetic activity are affected by herbicidal applications. The mode of action of herbicides on the photosynthesis process depends on the chemical group to which the herbicide belongs [3]. Herbicides affect chloroplast organization and pigment formation especially chlorophyll which is the principle absorbing pigment. Chlorophyll bleaching is a

Herbicides affect photosynthetic activity via different ways including photosynthetic pig‐ ments. The primary site of action is located at photosystem II (PSII) since they cause blocking of the Hill reaction. The oxygen evolution step is inhibited by interfering with the reducing side rather than the oxidizing side of PSII [4]. The inhibition of electron transfer through PSII causes a block in the whole transport chain as the inhibition of the noncyclic photophosphor‐ ylation and ATP synthesis. Consequently, the production of NADP is blocked and the function of the protective carotenoid system is prevented [5]. Urea herbicides inhibit both noncyclic and cyclic electron transport by forming a complex with oxidized form of an unknown component located in the electron transfer pathway close to PSII. This component also takes

The photosystem I (PSI) also could be reduced by some herbicides but it requires much higher concentrations of the herbicide than that required for the inhibition of PSII. Since PSII precedes

In a study conducted by **El-Hadary** [1], it was observed that photosynthetic activity measured in wheat chloroplasts (variety Giza 163) was greatly reduced throughout the growth by using Brominal as an example for bromphenol herbicides but lower concentrations (1/4R, 1/2R and R) increased the activity. Pigment content represented as chlorophyll, a/b ratio and carotenoids showed a similar results [1]. In the same study, sulfonyl-urea herbicides such as Granstar were examined. It was observed that low Granstar concentrations stimulated the photolytic activity of chloroplasts while high concentrations reduced it. However, Granstar reduced a/b ratios throughout the growth stages, except a slight increase at the fruiting stage with 1/2R. Carote‐

Protein synthesis takes place mainly in three stages involving initiation, elongation and termination of the polypeptide chain. Blocking any one of these stages by the herbicide will cause inhibition of protein and nucleic acid synthesis. The herbicides that inhibit photosyn‐ thesis and ATP formation can lead to inhibition of protein synthesis as a secondary effect. The damage that is caused by an herbicide is governed by its chemical group. There are numerous studies that investigate effects of the herbicidal chemical groups upon protein and nucleic acid

For instance, sulfonyl-urea herbicides block the biosynthesis of the branched chain amino acids in higher plants [6,7]. Aliphatic herbicides like Dalapon cause degradation of protein to ammonium compounds as detected in *Setaria lutescens* and sugar beets [8]. While acetamide herbicides such as propachlor, alachlor and prynaclor inhibited the protein content and RNA

PSI and the former is blocked completely at concentrations which do not affect PSI.

noids were decreased only with high Granstar concentrations [1].

**4.3. Protein and nucleic acid metabolism**

metabolism [2].

potent inhibitor for photosynthetic electron transport and CO2 fixation.

Benzoic and phenylacetic herbicides had variable effects on protein. For example, chloramben had no effect on RNA and protein synthesis on susceptible species [15]. On the other hand, it was suggested that foliar-applications of dicamba increased RNA and protein levels in susceptible plants by removal of histone from the DNA template [16]

Carbamate herbicide groups include a large number of herbicides such as asulam, barban, chlorpropham, propham, desmedipham and phenmedipham. [17]. Barban was found to inhibit protein synthesis and the degree of inhibition was related to the susceptibility of the plant species. For example, barban increased nucleotide content of wild oat shoots associated with disruption of RNA and protein synthesis. Chlorproham and propham inhibited amino acid incorporation into protein and induced a reduction in protein synthesis [18]. DNA, RNA and protein synthesis are also inhibited at high concentrations (10-3 M) of propham [19].

Fluridone, paraquat, perfluidone and propanil treatments were found to reduce soluble protein levels in soybean [20]. Paraquat and diquat readily act on proteins, modifying their structure and function (e.g.lysozome) since they interact with dibasic and dicarboxylic amino acids like ornithine and glutamic acid [21].

Oxadiazon at high doses inhibited protein synthesis in soybean while RNA and DNA synthesis were less sensitive to oxadiazon [22]. Combination of 2,4-D and glufosinate had an additive effect on protein synthesis in both sorghum and soybean [22]. On the other hand, sethoxydim, R- 25788 [N, N dichloroacetamide] or R- 28725 at low doses did not inhibit protein or RNA synthesis in cells of both sorghum and soybean but sethoxydim significantly inhibited DNA synthesis while R-25788 stimulated it [23]. Thus, the combined effects of sethoxydim and the two Safeners (R- 25788 and R- 28725) on protein and RNA synthesis were additive while on DNA synthesis they were antagonistic.

The application of haloxyfop to *Zea mays* and soybean cell suspension, increased 14-C labeled free amino acids level and incorporation of 14C leucine as a precursor revealed that haloxyfop did not inhibit protein synthesis [24].

Napropamide reduced DNA synthesis, RNA root cells of *Pea* and protein [25]. The inhibitory effect of napropamide on the mitotic cycle resulted from an inhibition in the synthesis of cell cycle specific protein. In contrast, 0.5 R, 1R and 1.5 R of metribuzin stimulated total and protein-N accumulation in soybean. Consequently, protein content was increased while RNA and DNA levels decreased [26]. Protein content of soybean yield was reported to be increased by application of 100 ppm GA3 (gibbrellic acid) and 2g/L Librel separately or together [27].

Metoxuron had a remarkable inhibition on the total protein biosynthesis, while bromoxynil accelerated the biosynthesis of low molecular proteins (water-soluble proteins) and inhibited the biosynthesis of high molecular proteins (sodium hydroxid soluble proteins) in wheat (*Triticum aestivum*, var. Sakha 69) [28]. Bromoxynil at low doses (0.4 and 0.8 kg / fed) enhanced protein content and RNA synthesis in wheat plants after 30 to 60 days from foliar spraying [29]. Nitrogen in wheat grains, consequently protein, was found to be increased by treating wheat plants with Brominal at the 2-leaf stage [30]. Different bromoxynil levels increased the protein percentages in wheat grains [31]. The foliar spray with bromoxynil increased significantly the protein content in wheat grains [32]. Application of bromoxynil at the full recommended rate significantly increased grain nitrogen and proteins in both wheat and barley. The increase was evaluated by multiplying grain nitrogen by 5.7 as a factor in both wheat and barley [33]. Protein content in wheat vegetation (Giza 163) was significantly increased at the vegetative stage and flowering stage while decreased at the fruiting stage as a response to either low or high Brominal treatments [1]. In contrast, the protein content of wheat root was reduced. Also, protein profiling of grains is greatly altered with an induction for 19kDa and 25kDa but an inhibition for 66kDa, 100kDa and 110kDa was obtained [1].

Dalapon, which is an aliphatic herbicide, did not affect the activity of hydrolytic enzymes like protease, α- amylase and dipeptidase in barley seeds [38]. Acetamides such as alachlor, propachlor and prynachlor which all were applied at pre-emergence caused an inhibition for

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 629

It was reported that propaclor inhibited the gibberellic acid (GA3) induced production of αamylase in barley seeds [40]. Similarly, alachlor, propachlor and prynachlor were found to inhibit α-amylase as well as protease synthesis in barley seeds [41, 42]. It was suggested that these herbicides may act as repressors for gene action preventing the normal expression of the hormonal effect of GA3 through the synthesis of DNA-dependent RNA. This was confirmed when higher levels of GA3 overcame alachlor inhibition by removing the repressor effect [42]. In addition, the effect of these acetamide herbicides on α- amylase and protease was suggested to be secondary and these herbicides possibly act on the biosynthetic reactions (like protein

Chloroamben and dicamba, which belong to the benzoic and phenylacetic acid herbicide groups, were found to inhibit GA3-induced α-amylase synthesis and the development of amylase activity in barley seeds [40, 43]. This agrees with effect of trifluarlin, as an example for dinitroanilines, which was found to inhibit the *de novo* synthesis of hydrolytic enzymes such as protease [44] and dipeptidase in squash cotyledons [45], phytase in barley seedlings,

Nitriles such as bromoxynil and ioxynil also inhibited proteolytic and amylolytic enzyme activities [46, 45]. Also, thiocarbamate herbicides were found to inhibit GA3- induced αamylase synthesis in susceptible weeds [17]. Acifluorfon was found to stimulate the activity of chalcone synthase, phenylalanine ammonia lyase and isoflavone 7-0- glucosy transferase

The increase of galactonolactone oxidase was reported in common beans as a result of acifluorfen application; this enzyme is responsible for lipid peroxidation. Acifluorfen was found to increase the activity of galactonactone reductase, which prevented further oxidation of lipids [48]. Other herbicides, alachlor and glyphosate, were observed to inhibit 5- enolpyr‐ uvyl shikimate-3-phosphate (EPSP) synthase enzyme. This enzyme is responsible for the synthesis of all cinnamate derivatives (intermediates in flavonoids biosynthesis pathway)

Sulfonylureas herbicides act by inhibiting acetolactate synthase enzymes, thereby blocking the biosynthesis of the branched chain amino acids in higher plants [7]. According to **Gronwald** [50], carbomothioate herbicides inhibited one or more acyl- CoA elongase enzymes which catalyze the condensation of malonyl CoA with fatty acid acyl-CoA substrates to form a very

The effects of triazine, urea and nitroaniline herbicides on amylase and acid proteolytic activities of wheat grain cultivars, Salwa, Grana and Liwilla were studied by **Wybieralshi and Wybieralska** [51]. The studied herbicides were found to inhibit amylase activity in Salwa and Liwilla, but increased it in Grana. Acid proteolytic activity in Liwilla and Salwa was reduced especially by Igran 80 (terbutryn) and Dicuran 60 (Chlorotoluron), while the activity in Grana

squash cotyledons and maize embryos [39], and α-amylase in barley seeds [40].

which are responsible for the accumulation of isoflavonoids in soybean leaves [47].

seed germination in barley by reducing the synthesis of α-amylase enzyme [39].

synthesis) required for the synthesis of these hydrolytic enzymes.

leading to reduced flavonoid synthesis in higher plants [49].

long chain fatty acid, used in the synthesis of surface lipids.

The action of urea herbicides on protein and nucleic acid metabolism has been reported by many researchers. Although fluometron can cause an increase in the low molecular weight fraction of DNA, RNA and protein synthesis [34], diuron and monuron inhibited the same parameters as reported [35]. However, the monomethylated derivative of isouron [ N-[5-(1,1 dimethy ethyl-3-iso) (azol]-N-methylurea] suppressed the protein synthesis in soybean[36].

Sulfonylurea herbicides were found to inhibit branched chain amino acids valine, leucine and isoleucine (e.g. Granstar; DPX- L 5300; tribenuron) [6, 7]. Aflon (urea herbicide), when sprayed at 1/2 R and R doses on *Phaseolus vulgaris,* induced a DNA increase in both shoot and root while RNA content was increased in shoot only [37]. Moreover, RNA content of roots was mostly decreased in response to R and 2R aflon treatments but increased as a result of the 1/2 R application [37]. Protein content of the wheat shoot system was increased with all Granstar concentrations at the vegetative stage and with low concentrations (1/2R and R) at both flowering and fruiting stages. In contrast, protein levels were decreased with 5/2R at the flowering stage and with 3/2R and 2R- and 5/2R at the fruiting stage [1]. Granstar treatments reduced the contents of root proteins at the vegetative stage and flowering stages but increased it at the fruiting stage. Protein profiling of grain proteins exerted an induction for 19kDa and 25kDa and complete suppression for 66kDa, 100kDa and 110KDa [1].

#### **4.4. Hydrolytic enzyme activities**

Enzymes of plants were affected greatly by herbicide treatments and their effect differs according to the chemical group to which the herbicide belongs. The following examples represent some effects of herbicides on the enzyme activities of some plant species.

One of the major metabolic processes that take place during seed germination is the production of hydrolytic enzymes such as α-, β-amylases that degrade stored carbohydrates into simple sugars. The production of hydrolytic enzymes requires the synthesis and presence of proteins, polyribosomes and nucleic acids. Thus, an effect of the herbicide on protein formation as mentioned above, would affect the synthesis of the hydrolytic enzymes [1, 3]. **El-Hadary** [1] reported that use of either Brominal or Granstar at different levels below and above the recommended rate induced stimulation for amylolytic enzyme activity (α and *β*-amylase); however, an incidence of a slight reduction in *β*-amylase activity was observed with 2R and higher doses of Granstar [1].

Dalapon, which is an aliphatic herbicide, did not affect the activity of hydrolytic enzymes like protease, α- amylase and dipeptidase in barley seeds [38]. Acetamides such as alachlor, propachlor and prynachlor which all were applied at pre-emergence caused an inhibition for seed germination in barley by reducing the synthesis of α-amylase enzyme [39].

Nitrogen in wheat grains, consequently protein, was found to be increased by treating wheat plants with Brominal at the 2-leaf stage [30]. Different bromoxynil levels increased the protein percentages in wheat grains [31]. The foliar spray with bromoxynil increased significantly the protein content in wheat grains [32]. Application of bromoxynil at the full recommended rate significantly increased grain nitrogen and proteins in both wheat and barley. The increase was evaluated by multiplying grain nitrogen by 5.7 as a factor in both wheat and barley [33]. Protein content in wheat vegetation (Giza 163) was significantly increased at the vegetative stage and flowering stage while decreased at the fruiting stage as a response to either low or high Brominal treatments [1]. In contrast, the protein content of wheat root was reduced. Also, protein profiling of grains is greatly altered with an induction for 19kDa and 25kDa but an

The action of urea herbicides on protein and nucleic acid metabolism has been reported by many researchers. Although fluometron can cause an increase in the low molecular weight fraction of DNA, RNA and protein synthesis [34], diuron and monuron inhibited the same parameters as reported [35]. However, the monomethylated derivative of isouron [ N-[5-(1,1 dimethy ethyl-3-iso) (azol]-N-methylurea] suppressed the protein synthesis in soybean[36]. Sulfonylurea herbicides were found to inhibit branched chain amino acids valine, leucine and isoleucine (e.g. Granstar; DPX- L 5300; tribenuron) [6, 7]. Aflon (urea herbicide), when sprayed at 1/2 R and R doses on *Phaseolus vulgaris,* induced a DNA increase in both shoot and root while RNA content was increased in shoot only [37]. Moreover, RNA content of roots was mostly decreased in response to R and 2R aflon treatments but increased as a result of the 1/2 R application [37]. Protein content of the wheat shoot system was increased with all Granstar concentrations at the vegetative stage and with low concentrations (1/2R and R) at both flowering and fruiting stages. In contrast, protein levels were decreased with 5/2R at the flowering stage and with 3/2R and 2R- and 5/2R at the fruiting stage [1]. Granstar treatments reduced the contents of root proteins at the vegetative stage and flowering stages but increased it at the fruiting stage. Protein profiling of grain proteins exerted an induction for 19kDa and

Enzymes of plants were affected greatly by herbicide treatments and their effect differs according to the chemical group to which the herbicide belongs. The following examples

One of the major metabolic processes that take place during seed germination is the production of hydrolytic enzymes such as α-, β-amylases that degrade stored carbohydrates into simple sugars. The production of hydrolytic enzymes requires the synthesis and presence of proteins, polyribosomes and nucleic acids. Thus, an effect of the herbicide on protein formation as mentioned above, would affect the synthesis of the hydrolytic enzymes [1, 3]. **El-Hadary** [1] reported that use of either Brominal or Granstar at different levels below and above the recommended rate induced stimulation for amylolytic enzyme activity (α and *β*-amylase); however, an incidence of a slight reduction in *β*-amylase activity was observed with 2R and

represent some effects of herbicides on the enzyme activities of some plant species.

inhibition for 66kDa, 100kDa and 110kDa was obtained [1].

628 Herbicides - Current Research and Case Studies in Use

25kDa and complete suppression for 66kDa, 100kDa and 110KDa [1].

**4.4. Hydrolytic enzyme activities**

higher doses of Granstar [1].

It was reported that propaclor inhibited the gibberellic acid (GA3) induced production of αamylase in barley seeds [40]. Similarly, alachlor, propachlor and prynachlor were found to inhibit α-amylase as well as protease synthesis in barley seeds [41, 42]. It was suggested that these herbicides may act as repressors for gene action preventing the normal expression of the hormonal effect of GA3 through the synthesis of DNA-dependent RNA. This was confirmed when higher levels of GA3 overcame alachlor inhibition by removing the repressor effect [42]. In addition, the effect of these acetamide herbicides on α- amylase and protease was suggested to be secondary and these herbicides possibly act on the biosynthetic reactions (like protein synthesis) required for the synthesis of these hydrolytic enzymes.

Chloroamben and dicamba, which belong to the benzoic and phenylacetic acid herbicide groups, were found to inhibit GA3-induced α-amylase synthesis and the development of amylase activity in barley seeds [40, 43]. This agrees with effect of trifluarlin, as an example for dinitroanilines, which was found to inhibit the *de novo* synthesis of hydrolytic enzymes such as protease [44] and dipeptidase in squash cotyledons [45], phytase in barley seedlings, squash cotyledons and maize embryos [39], and α-amylase in barley seeds [40].

Nitriles such as bromoxynil and ioxynil also inhibited proteolytic and amylolytic enzyme activities [46, 45]. Also, thiocarbamate herbicides were found to inhibit GA3- induced αamylase synthesis in susceptible weeds [17]. Acifluorfon was found to stimulate the activity of chalcone synthase, phenylalanine ammonia lyase and isoflavone 7-0- glucosy transferase which are responsible for the accumulation of isoflavonoids in soybean leaves [47].

The increase of galactonolactone oxidase was reported in common beans as a result of acifluorfen application; this enzyme is responsible for lipid peroxidation. Acifluorfen was found to increase the activity of galactonactone reductase, which prevented further oxidation of lipids [48]. Other herbicides, alachlor and glyphosate, were observed to inhibit 5- enolpyr‐ uvyl shikimate-3-phosphate (EPSP) synthase enzyme. This enzyme is responsible for the synthesis of all cinnamate derivatives (intermediates in flavonoids biosynthesis pathway) leading to reduced flavonoid synthesis in higher plants [49].

Sulfonylureas herbicides act by inhibiting acetolactate synthase enzymes, thereby blocking the biosynthesis of the branched chain amino acids in higher plants [7]. According to **Gronwald** [50], carbomothioate herbicides inhibited one or more acyl- CoA elongase enzymes which catalyze the condensation of malonyl CoA with fatty acid acyl-CoA substrates to form a very long chain fatty acid, used in the synthesis of surface lipids.

The effects of triazine, urea and nitroaniline herbicides on amylase and acid proteolytic activities of wheat grain cultivars, Salwa, Grana and Liwilla were studied by **Wybieralshi and Wybieralska** [51]. The studied herbicides were found to inhibit amylase activity in Salwa and Liwilla, but increased it in Grana. Acid proteolytic activity in Liwilla and Salwa was reduced especially by Igran 80 (terbutryn) and Dicuran 60 (Chlorotoluron), while the activity in Grana was not affected. In contrast, amylase, dehydrogenase, cellulase and xylanase activities were increased by application of the herbicides Pyramin (chloridazon), Ro-neet (cycloate) and Venzar (lenacil) when applied on the soil with 5% (w/w) addition of wheat straw [52]. Other studies suggested that application of SAN 9789 (norflurazon) as a metabolic inhibitor to *Sinapis alba* seedlings destroyed the chloroplasts but had no effect on α-amylase activity. This is due to the fact that α-amylase is a cytosolic enzyme [53].

Lipid peroxidation and galactonlactone oxidase increased in response to the treatment of *Phaseolus vulgaris* leaves with acifluorfen [48] and the activity of glutathione reductase also increased to prevent further oxidation. Gronowald studies on herbicides concluded that the carbothioates group impaired the synthesis of surface lipids (waxes, cutin, and subrin) by inhibiting acyl- CoA elongases while chloroacetamide herbicides inhibited *de novo* fatty acid biosynthesis. Similarly, pyridazinones herbicides decreased the degree of unsaturation of plastidic galactolipids while aryloxyphenoxy pypropionic acid and cyclohexanedione herbicides inhibited *de novo* fatty acid synthesis. The target site for all these classes is the

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 631

The total lipid content as well as *gluco-*and *phospho-*lipid content of maize seedlings markedly decreased by application of perfluidone while in sunflower cotyledons total lipids were not affected but glycolipids increased at the expense of phospholipids [62]. Also, a decrease in lipid synthesis in soybean by Isouron was reported [36] but an increase in seed oil of soybean was

Carbohydrate content is one of the most affected parameters in response to herbicide appli‐ cation**. Yakout** [28] demonstrated that treating wheat (*Triticum aestivum* var. Sakha 69) with metoxuron showed a slight reduction in the available carbohydrates with relatively no change in sucrose content while bromoxynil showed an increase in different carbohydrate levels. Also, the total reducing substances (may include sugars, phenolic substances, ascorbic acid, organic

Inhibition of the accumulated reducing sugars, sucrose and polysaccharides, in soybean leaves was observed in response to 1R and 1.5R metribuzin application and, consequently, seed carbohydrate content decreased with increasing metribuzin concentration [26]. Terbytryn herbicide was found to decrease starch content and increase sugar content in pre-emergence and post emergence applications [63]. On the other hand, bromoxynil was reported to significantly increase soluble and total carbohydrates at low doses while a higher dose (1.2 kg/ Fed) inhibited their synthetic rate in wheat plants [29]. Similarly, the results of **El-Hadary** [1] found that *mono-, di- and poly-*saccharides and, consequently, total carbohydrates were increased with low doses but decreased with high doses of either Brominal or Granstar [1]. The incidental increase with low concentrations was attributed to that some herbicides act as

Urea herbicides including afalon-S at low doses of 1/2R and R increased the soluble and insoluble sugar contents of shoots at different stages of growth and development of *Phaseo‐ lus vulgaris* while a reverse situation was obtained in the case of a 2R application. The root tissue treated with various concentrations suffered from an obvious decrease in the content of

The content of reducing sugars and sucrose of *Ricinus communis* cultivar Balada and maize cultivar Giza 2 seedlings and adult plants supplemented with low concentrations (0.5-2.5μg/g) of metribuzin either alone or in combination with NaCl at 50μg/g were increased significantly

the different carbohydrate fraction relative to those of the control [37].

enzyme acetyl-CoA carboxylase [50].

**4.6. Carbohydrate content**

growth regulators in low doses.

obtained by 0.5R or 1R metribuzin application [26].

acid, etc.) were increased for both treatments [28].

The levels of leaf β-amylase and starch debranching enzyme in pea seedlings were found to slightly decrease in response to norflurazon-treatment [54]. However, inhibitors of chloro‐ plastic functions, i.e.; diuron (DCMU), atrazine, tentoxin, paclobutrazol and San 9785 (4 chloro-5-( dimethylamino)-2-phenyl-3 (2H)- pyridazinone) caused either no or only slight increases in α-amylase activity. In contrast were the inhibitors of plastidic protein synthesis lincomycin and chloramphenicol that cause an increase in α-amylase activity in pea seedlings. It is concluded that there was an inverse relationship between α-amylase activity and chloro‐ phyll concentration in pea petals and stems [55]. Similarly an inhibition of α-amylase induction in barley seeds was reported [56]. Also, Li found that juglone decreased the content of total soluble protein and α-amylase activity induced by gibberellin by 74% and 78% in the aleuron cells of barley. It was concluded that juglone may be a metabolic inhibitor which prevents many (if not all) physiological and biochemical processes involving SH-groups in compounds such as amino acids, peptides and enzymes [57].

The activities of α-and β-amylases of castor bean and maize Giza 2 seedlings and adult plants supplemented with low concentration (0.5-2.5 μg/g) of metribuzin either alone or in combi‐ nation with NaCl at 50 μg/g were increased significantly [58] but higher metribuzin concen‐ tration (5-10μg) had an opposite response. Application of 1.5-4.5kg/ha thiobencarb and butachlor six days after transplanting of 30-day-old rice seedlings affected the enzyme activities of the seedlings whether they were grown alone or with the competitive barnyard grass [59]. Moreover, both herbicides reduced α-amylase activity by increasing the concen‐ tration but a sharp increase in α-amylase activity was noted at 96h post-treatment in both species. In addition, protease (proteinase) activity was maximized after post-treatment at both 48h.and 24h in rice and grass, respectively.

Butachor (1000-3000 g/ha) and oxyfluorfen (100-300g/ha) effect on α-amylase activity and chlorophyll content in 46 rice cultivars was dependent on the degree of tolerance of each cultivar [60]. It was concluded that rice cultivars ADT-37, ASD-16 and ASD-18 were highly tolerant to butachor, whereas ADT-36, ADT-38 and PY-3 were highly susceptible. Howev‐ er, tolerance to oxyfluorfen was high in ASD-18 and AS-18696, while IR-50 was highly susceptible [60].

#### **4.5. Lipid synthesis and oxidation**

Substituted ureas, uracils, triazine, benzonitriles and bipyridyls markedly accelerated the photo-oxidations (lipids- per-oxidation) but peroxidation was completely prevented by NADH or NADPH [5]. Lipid peroxidation in higher plants (Duranta and Cassia) was induced by oxyfluorfen [61] but the peroxidative cell damage is controlled by antioxidative systems such as vitamins "C" and "E".

Lipid peroxidation and galactonlactone oxidase increased in response to the treatment of *Phaseolus vulgaris* leaves with acifluorfen [48] and the activity of glutathione reductase also increased to prevent further oxidation. Gronowald studies on herbicides concluded that the carbothioates group impaired the synthesis of surface lipids (waxes, cutin, and subrin) by inhibiting acyl- CoA elongases while chloroacetamide herbicides inhibited *de novo* fatty acid biosynthesis. Similarly, pyridazinones herbicides decreased the degree of unsaturation of plastidic galactolipids while aryloxyphenoxy pypropionic acid and cyclohexanedione herbicides inhibited *de novo* fatty acid synthesis. The target site for all these classes is the enzyme acetyl-CoA carboxylase [50].

The total lipid content as well as *gluco-*and *phospho-*lipid content of maize seedlings markedly decreased by application of perfluidone while in sunflower cotyledons total lipids were not affected but glycolipids increased at the expense of phospholipids [62]. Also, a decrease in lipid synthesis in soybean by Isouron was reported [36] but an increase in seed oil of soybean was obtained by 0.5R or 1R metribuzin application [26].

#### **4.6. Carbohydrate content**

was not affected. In contrast, amylase, dehydrogenase, cellulase and xylanase activities were increased by application of the herbicides Pyramin (chloridazon), Ro-neet (cycloate) and Venzar (lenacil) when applied on the soil with 5% (w/w) addition of wheat straw [52]. Other studies suggested that application of SAN 9789 (norflurazon) as a metabolic inhibitor to *Sinapis alba* seedlings destroyed the chloroplasts but had no effect on α-amylase activity. This is due

The levels of leaf β-amylase and starch debranching enzyme in pea seedlings were found to slightly decrease in response to norflurazon-treatment [54]. However, inhibitors of chloro‐ plastic functions, i.e.; diuron (DCMU), atrazine, tentoxin, paclobutrazol and San 9785 (4 chloro-5-( dimethylamino)-2-phenyl-3 (2H)- pyridazinone) caused either no or only slight increases in α-amylase activity. In contrast were the inhibitors of plastidic protein synthesis lincomycin and chloramphenicol that cause an increase in α-amylase activity in pea seedlings. It is concluded that there was an inverse relationship between α-amylase activity and chloro‐ phyll concentration in pea petals and stems [55]. Similarly an inhibition of α-amylase induction in barley seeds was reported [56]. Also, Li found that juglone decreased the content of total soluble protein and α-amylase activity induced by gibberellin by 74% and 78% in the aleuron cells of barley. It was concluded that juglone may be a metabolic inhibitor which prevents many (if not all) physiological and biochemical processes involving SH-groups in compounds

The activities of α-and β-amylases of castor bean and maize Giza 2 seedlings and adult plants supplemented with low concentration (0.5-2.5 μg/g) of metribuzin either alone or in combi‐ nation with NaCl at 50 μg/g were increased significantly [58] but higher metribuzin concen‐ tration (5-10μg) had an opposite response. Application of 1.5-4.5kg/ha thiobencarb and butachlor six days after transplanting of 30-day-old rice seedlings affected the enzyme activities of the seedlings whether they were grown alone or with the competitive barnyard grass [59]. Moreover, both herbicides reduced α-amylase activity by increasing the concen‐ tration but a sharp increase in α-amylase activity was noted at 96h post-treatment in both species. In addition, protease (proteinase) activity was maximized after post-treatment at both

Butachor (1000-3000 g/ha) and oxyfluorfen (100-300g/ha) effect on α-amylase activity and chlorophyll content in 46 rice cultivars was dependent on the degree of tolerance of each cultivar [60]. It was concluded that rice cultivars ADT-37, ASD-16 and ASD-18 were highly tolerant to butachor, whereas ADT-36, ADT-38 and PY-3 were highly susceptible. Howev‐ er, tolerance to oxyfluorfen was high in ASD-18 and AS-18696, while IR-50 was highly

Substituted ureas, uracils, triazine, benzonitriles and bipyridyls markedly accelerated the photo-oxidations (lipids- per-oxidation) but peroxidation was completely prevented by NADH or NADPH [5]. Lipid peroxidation in higher plants (Duranta and Cassia) was induced by oxyfluorfen [61] but the peroxidative cell damage is controlled by antioxidative systems

to the fact that α-amylase is a cytosolic enzyme [53].

630 Herbicides - Current Research and Case Studies in Use

such as amino acids, peptides and enzymes [57].

48h.and 24h in rice and grass, respectively.

**4.5. Lipid synthesis and oxidation**

such as vitamins "C" and "E".

susceptible [60].

Carbohydrate content is one of the most affected parameters in response to herbicide appli‐ cation**. Yakout** [28] demonstrated that treating wheat (*Triticum aestivum* var. Sakha 69) with metoxuron showed a slight reduction in the available carbohydrates with relatively no change in sucrose content while bromoxynil showed an increase in different carbohydrate levels. Also, the total reducing substances (may include sugars, phenolic substances, ascorbic acid, organic acid, etc.) were increased for both treatments [28].

Inhibition of the accumulated reducing sugars, sucrose and polysaccharides, in soybean leaves was observed in response to 1R and 1.5R metribuzin application and, consequently, seed carbohydrate content decreased with increasing metribuzin concentration [26]. Terbytryn herbicide was found to decrease starch content and increase sugar content in pre-emergence and post emergence applications [63]. On the other hand, bromoxynil was reported to significantly increase soluble and total carbohydrates at low doses while a higher dose (1.2 kg/ Fed) inhibited their synthetic rate in wheat plants [29]. Similarly, the results of **El-Hadary** [1] found that *mono-, di- and poly-*saccharides and, consequently, total carbohydrates were increased with low doses but decreased with high doses of either Brominal or Granstar [1]. The incidental increase with low concentrations was attributed to that some herbicides act as growth regulators in low doses.

Urea herbicides including afalon-S at low doses of 1/2R and R increased the soluble and insoluble sugar contents of shoots at different stages of growth and development of *Phaseo‐ lus vulgaris* while a reverse situation was obtained in the case of a 2R application. The root tissue treated with various concentrations suffered from an obvious decrease in the content of the different carbohydrate fraction relative to those of the control [37].

The content of reducing sugars and sucrose of *Ricinus communis* cultivar Balada and maize cultivar Giza 2 seedlings and adult plants supplemented with low concentrations (0.5-2.5μg/g) of metribuzin either alone or in combination with NaCl at 50μg/g were increased significantly but decreased in response to higher concentrations (5-10μg). On the other hand, polysacchar‐ ide content of *R. communis* and maize seedlings as well as adult plants were significantly decreased in response to low concentrations of metribuzin and increased significantly at higher concentrations either alone or in combination with NaCl. Total carbohydrate content detected in *R. communis* treated with metribuzin were greater than those detected in presence of herbicide and NaCl combination [58].

Herbicidal effects may be varied when they are applied in combination. For example, a marked increase was observed in the grain yield, ears/plant and number of ears in barley by using a combination of bromoxynil, ioxnil and mercoprop [72]. An increase of about 20% was recorded in grain wheat yield when oxitril 4, which is a combination of oxitril and bromoxynil, was used at 130g/liter and applied at rates of 1.5,4 and 5 liters/ha [73]. In winter wheat a marked increase in yield was mentioned in response to half rate applications of various commercial herbicides (active ingredients bromoxynil, ioxynil, mocoprop, cyanazine, fluroxypyr, metasulfuron-

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 633

Urea herbicide such as Granstar (metasulfuron- methyl 75% water dispersible granules) was found to suppress the growth rate of wheat and barley by about 20% while weeds were completely destroyed[75]. Its application with a dose of 20-40 g/ha in 200-500 liter/ha prior to planting resulted in 50% suppression [76]. The author in a previous work applied Granstar at a dose of 0.5R, 1R, 1.5R and 2R on wheat at 40-days old and reported an increase in grains no./ spike [1]. However, a great decrease in monosaccharides, disaccharides, polysaccharides and, consequently, total carbohydrate levels was obtained in wheat grains with both low and high Granstar concentrations [1]. Also, chlorsulfuron was mentioned to reduce both the third leaf growth rate and shoot dry weight of wheat seedlings but not the root dry weight [76].

The urea herbicide metoxuron was reported to decrease wheat grain yield (var. Sakha 69) [28]. It was found that 100-seed weight of soybean was decreased by using metribuzin at rates of 0.5R, 1R and 1.5R [26]. Wheat yield was markedly increased by using tribenuron at a rate of 0-125g [77]. However, sulfonylurea herbicides, Chisel [Chlorosulfuron+thifensulfuron methyl] and Granstar, significantly increased the productive tillering in some wheat varieties [78]. Application of trifluralin alone in the spring followed by some post herbicides resulted in a reduction in vegetative growth, shoot dry weight and wheat grain yield [79]. An applica‐ tion of 0.126 mM perfluidon herbicide was reported not only to decrease both fresh and dry

**5. Hazardous action of herbicides in the agricultural environment and**

Although the benefits gained from herbicides usage in weeds control, herbisides have undesired effects on man health and environment. Their residues remain in the soil for many years, affecting crops, water canals, grazing animals and human health and even the pollution

Herbicides and pesticides have been suspected by the "National Cancer Research Institute" as a probable cause of certain cancers especially cancers of the brain, prostate, stomach and lip, as well as leukemia, skin melanomas and Hodgkin's lymphoma [80]. They also cause repro‐ ductive problems as well as infertility and nervous system diseases. The National Academy of Sciences reported that infants and children, because of their developing physiology, are more susceptible to the negative effects of herbicides and pesticides in comparison to adults. Herbicides may cause human poisoning since they affect humans through three mechanisms

methyl, and clopyralid) [74].

**human health**

of air.

weight but also shoot length of maize seedlings [62].

Thiobencarb and butaclor herbicides when applied at 1.5-4.5 kg/ha after transplanting 30 days old rice seedlings and barnyard grass grown alone or with rice were found to have no effect either on total carbohydrate or starch and reducing sugars in rice and grass [59].

#### **4.7. Plant growth response and yield**

Plant growth and yield are greatly affected by herbicidal applications depending on the age, tolerance, dose and the active chemical group of the herbicide. The author in a previous work pointed that Brominal application on wheat induced an increase in the number of grains per spike with 1/4 R. 1/2R and R while higher doses caused a significant reduction [1]. Also, grain yield showed a detectable reduction in monosaccharides, disaccharides, polysaccharides and, consequently, total carbohydrate levels with all Brominal concentrations [1].

The percentage of germination and seedling growth of barley was decreased greatly by applications of bromoxynil [64]. But the same herbicide in different concentrations encouraged wheat growth [31]. Also, growth parameters such as plant height, weight and leaf area of wheat plants at 75 days after sowing were increased significantly by foliar application of bromoxynil at rate of 1.0 L/Fed [32, 65]. Moreover, a good seedling establishment of wheat was obtained by combinations of bromoxynil and fenoxaprop [66]. Low metribuzin concentrations (0.5-2.5μg/g) either alone or in combination with NaCl (50 μg/g) caused an increase in different growth parameters such as leaf area, length of shoot and root, water content and dry matter accumulation in both *Ricinus communis* cultivars, and maize cultivars Giza 2 throughout the different growth stages [58]. In contrast, the higher metribuzin concentration (5-10μg) affected the same parameters oppositely [58].

Productivity of the plant is affected in terms of 100 grains weight in response to herbicides treatment. The yield of wheat grains (var.Sakha 69) increased by bromoxynil application [28]. A dose of 1.5 kg/ha of bromoxynil brought an increase in weight of 100 grains [30,67]. The highest yield was obtained when one liter/fed bromoxynil was applied at the third-leaf stage [68]. The number of wheat grains/ear and grain yield were increased at a low dose (0.8kg/ fed.) of bromoxynil [29,69] while a higher dose of the same herbicide (1.2 kg/ fed) reduced the yield of wheat varieties; i.e. Sakha 69, Giza 157 and Giza 160 [29]. On the other hand, it was noticed that higher doses of bromoxynil resulted in a marked increase in both yield and grains/ear when crops were poorly developed at the time of spraying [70]. However, the application of 2.5, 3.0 liter bromoxynil /ha at the third-leaf and flowering stages on wheat significantly decreased the grain yield [71] as well as the number of spikes per plant, main spike length, weight of 100 grains and straw per plant [32].

Herbicidal effects may be varied when they are applied in combination. For example, a marked increase was observed in the grain yield, ears/plant and number of ears in barley by using a combination of bromoxynil, ioxnil and mercoprop [72]. An increase of about 20% was recorded in grain wheat yield when oxitril 4, which is a combination of oxitril and bromoxynil, was used at 130g/liter and applied at rates of 1.5,4 and 5 liters/ha [73]. In winter wheat a marked increase in yield was mentioned in response to half rate applications of various commercial herbicides (active ingredients bromoxynil, ioxynil, mocoprop, cyanazine, fluroxypyr, metasulfuronmethyl, and clopyralid) [74].

but decreased in response to higher concentrations (5-10μg). On the other hand, polysacchar‐ ide content of *R. communis* and maize seedlings as well as adult plants were significantly decreased in response to low concentrations of metribuzin and increased significantly at higher concentrations either alone or in combination with NaCl. Total carbohydrate content detected in *R. communis* treated with metribuzin were greater than those detected in presence of

Thiobencarb and butaclor herbicides when applied at 1.5-4.5 kg/ha after transplanting 30 days old rice seedlings and barnyard grass grown alone or with rice were found to have no effect

Plant growth and yield are greatly affected by herbicidal applications depending on the age, tolerance, dose and the active chemical group of the herbicide. The author in a previous work pointed that Brominal application on wheat induced an increase in the number of grains per spike with 1/4 R. 1/2R and R while higher doses caused a significant reduction [1]. Also, grain yield showed a detectable reduction in monosaccharides, disaccharides, polysaccharides and,

The percentage of germination and seedling growth of barley was decreased greatly by applications of bromoxynil [64]. But the same herbicide in different concentrations encouraged wheat growth [31]. Also, growth parameters such as plant height, weight and leaf area of wheat plants at 75 days after sowing were increased significantly by foliar application of bromoxynil at rate of 1.0 L/Fed [32, 65]. Moreover, a good seedling establishment of wheat was obtained by combinations of bromoxynil and fenoxaprop [66]. Low metribuzin concentrations (0.5-2.5μg/g) either alone or in combination with NaCl (50 μg/g) caused an increase in different growth parameters such as leaf area, length of shoot and root, water content and dry matter accumulation in both *Ricinus communis* cultivars, and maize cultivars Giza 2 throughout the different growth stages [58]. In contrast, the higher metribuzin concentration (5-10μg) affected

Productivity of the plant is affected in terms of 100 grains weight in response to herbicides treatment. The yield of wheat grains (var.Sakha 69) increased by bromoxynil application [28]. A dose of 1.5 kg/ha of bromoxynil brought an increase in weight of 100 grains [30,67]. The highest yield was obtained when one liter/fed bromoxynil was applied at the third-leaf stage [68]. The number of wheat grains/ear and grain yield were increased at a low dose (0.8kg/ fed.) of bromoxynil [29,69] while a higher dose of the same herbicide (1.2 kg/ fed) reduced the yield of wheat varieties; i.e. Sakha 69, Giza 157 and Giza 160 [29]. On the other hand, it was noticed that higher doses of bromoxynil resulted in a marked increase in both yield and grains/ear when crops were poorly developed at the time of spraying [70]. However, the application of 2.5, 3.0 liter bromoxynil /ha at the third-leaf and flowering stages on wheat significantly decreased the grain yield [71] as well as the number of spikes per plant, main spike length,

either on total carbohydrate or starch and reducing sugars in rice and grass [59].

consequently, total carbohydrate levels with all Brominal concentrations [1].

herbicide and NaCl combination [58].

632 Herbicides - Current Research and Case Studies in Use

**4.7. Plant growth response and yield**

the same parameters oppositely [58].

weight of 100 grains and straw per plant [32].

Urea herbicide such as Granstar (metasulfuron- methyl 75% water dispersible granules) was found to suppress the growth rate of wheat and barley by about 20% while weeds were completely destroyed[75]. Its application with a dose of 20-40 g/ha in 200-500 liter/ha prior to planting resulted in 50% suppression [76]. The author in a previous work applied Granstar at a dose of 0.5R, 1R, 1.5R and 2R on wheat at 40-days old and reported an increase in grains no./ spike [1]. However, a great decrease in monosaccharides, disaccharides, polysaccharides and, consequently, total carbohydrate levels was obtained in wheat grains with both low and high Granstar concentrations [1]. Also, chlorsulfuron was mentioned to reduce both the third leaf growth rate and shoot dry weight of wheat seedlings but not the root dry weight [76].

The urea herbicide metoxuron was reported to decrease wheat grain yield (var. Sakha 69) [28]. It was found that 100-seed weight of soybean was decreased by using metribuzin at rates of 0.5R, 1R and 1.5R [26]. Wheat yield was markedly increased by using tribenuron at a rate of 0-125g [77]. However, sulfonylurea herbicides, Chisel [Chlorosulfuron+thifensulfuron methyl] and Granstar, significantly increased the productive tillering in some wheat varieties [78]. Application of trifluralin alone in the spring followed by some post herbicides resulted in a reduction in vegetative growth, shoot dry weight and wheat grain yield [79]. An applica‐ tion of 0.126 mM perfluidon herbicide was reported not only to decrease both fresh and dry weight but also shoot length of maize seedlings [62].

## **5. Hazardous action of herbicides in the agricultural environment and human health**

Although the benefits gained from herbicides usage in weeds control, herbisides have undesired effects on man health and environment. Their residues remain in the soil for many years, affecting crops, water canals, grazing animals and human health and even the pollution of air.

Herbicides and pesticides have been suspected by the "National Cancer Research Institute" as a probable cause of certain cancers especially cancers of the brain, prostate, stomach and lip, as well as leukemia, skin melanomas and Hodgkin's lymphoma [80]. They also cause repro‐ ductive problems as well as infertility and nervous system diseases. The National Academy of Sciences reported that infants and children, because of their developing physiology, are more susceptible to the negative effects of herbicides and pesticides in comparison to adults. Herbicides may cause human poisoning since they affect humans through three mechanisms of entry: ingestion, inhalation and dermal absorption. In under-developed countries, the least expensive pesticides are utilized due the inability of farmers to purchase more expensive, safer products. As a byproduct of pesticide use, farmers and their families are affected daily with health problems directly resulting from pesticide exposure [81]. Herbicide toxicity and risks are not only limited by their direct use but can also present risks indirectly. Indirect risks are represented by herbicidal traces that remain in the edible plants themselves as well as the residues in the soil that may remain for a number of years before it can be degraded. Moreover, the leakage of these herbicides and their residues in water canals, vaporization and sublimation in air may be poisonous to the surrounding living organisms.

through volatilization, leaching, root exudation, and decomposition of plant residues. Rainfall causes the leaching of allelopathic substances from leaves which fall to the ground during period of stress, leading to inhibition of growth and germination of crop plants [84, 85].

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 635

According to the different structures and properties of allelochemicals, they can be classified into the following categories: water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, and ketones; simple unsaturated lactones; long-chain fatty acids and polyacety‐ lenes; quinines (benzoquinone, anthraquinone and complex quinines); phenolics; cinnamic acid and its derivatives; coumarins; flavonoids; tannins; steroids and terpenoids (sesquiter‐ pene lactones, diterpenes, and triterpenoids) [86]. The biosynthetic pathways of the major

The allelochemical interference implies their interference with each other as well the interfer‐ ence with other surrounding plants. Several chemicals can be released together and may exert toxicities in an additive or synergistic manner. Allelopathic interferences often result from the mixing action of several different compounds. Allelopathic plant extracts can effectively control weeds since mixtures of allelopathic water extracts are more effective than the application of single-plant extract. Combined application of allelopathic extracts and reduced herbicide dose (up to half the standard dose) give as much weed control as the standard herbicide dose in several field crops. Lower doses of herbicides may help to reduce the development of herbicide resistance in weed ecotypes [88]. Allelopathy thus offers an attrac‐ tive environmentally friendly alternative to pesticides in agricultural pest management [88].

Response of the receiver plants to allelochemicals is not only concentration dependent but also controlled by the biochemical pathway in the receiver plant. Generally, low concentrations of allelochemicals are stimulatory while it is inhibitory with higher concentrations [89]. Allelo‐ chemical concentrations in the producer plant may also vary over time and in the plant tissue produced. Foliar and leaf litter leachates of Eucalyptus species, for example, are more toxic than bark leachates to some food crops. Typically, allelochemical concentration in field

Receiver plant response to antagonistic allelochemicals is detected as certain signs on growth and development of the plants that are exposed to allelochemicals. The effect includes the inhibition or retardation of germination rate; seeds darkness and swelling; root or radicle reduction, curling of the root axis, lack of root hairs; increased number of seminal roots, swelling or necrosis of root tips; shoot or coleoptile extension; discolouration, reduced dry weight accumulation; and lowered reproductive capacity. These morphological effects may be secondary for primary events due to interference with different biochemical pathways of

Biological activity of allelochemicals could be increased by some modifications so the end product could be more active, selective, or persistent. This is attributed to the potential

situations is below the required inhibitory level that can affect sensitive plants.

**6.4. Allelochemical classification and biosynthesis**

allelopathic substances are shown in Figure 2 [87].

the receiver plant [90].

**6.5. Allelochemical interference and biological activity**

### **6. Natural herbicides**

Allelopathy phenomenon serves the agricultural community so much. The following section discusses the related concepts to allelopathy and recruiting it as natural herbicides for weed management to be an alternative or to minimize conventional herbicide use.

#### **6.1. Allelopathy term**

Allelopathy is a natural biological phenomenon of interference among organisms in such a way that an organism produces one or more biochemicals that influence the growth, survival, and reproduction of other organisms. Allelopathy is the favorable or adverse effect of one plant on another due to direct or indirect release of chemicals from live or dead plants (including microorganisms).

#### **6.2. Allelochemical term**

Allelochemicals, or allelochemics, are a subset of low molecular weight secondary metabolites such as alkaloids, phenolics, flavonoids, terpenoids, and glucosinolates which are produced during growth and development but are not used by the allelopathic plant [82]. Allelochemi‐ cals may have beneficial (positive allelopathy) or detrimental (negative allelopathy) effects on the target organisms. Allelochemicals with negative allelopathic effects contribute in plant defense against herbivory. Also, allelochemicals could be recruited in weed management as alternatives to herbicides.

Allelochemicals are listed as six classes [83] that possess actual or potential phytotoxicity. The classes are namely alkaloids, benzoxazinones, cinnamic acid derivatives, cyanogenic com‐ pounds, ethylene and other seed germination stimulants, and flavonoids which have been isolated from over 30 families of terrestrial and aquatic plants. Like synthetic herbicides, there is no common mode of action or physiological target site for all allelochemicals.

#### **6.3. Allelochemical occurrence**

Allelochemics are present in different parts of the plant; leaves, flowers, fruits, stems, bark, roots, rhizomes, seeds and pollen. They may be released from plants into the environment through volatilization, leaching, root exudation, and decomposition of plant residues. Rainfall causes the leaching of allelopathic substances from leaves which fall to the ground during period of stress, leading to inhibition of growth and germination of crop plants [84, 85].

#### **6.4. Allelochemical classification and biosynthesis**

of entry: ingestion, inhalation and dermal absorption. In under-developed countries, the least expensive pesticides are utilized due the inability of farmers to purchase more expensive, safer products. As a byproduct of pesticide use, farmers and their families are affected daily with health problems directly resulting from pesticide exposure [81]. Herbicide toxicity and risks are not only limited by their direct use but can also present risks indirectly. Indirect risks are represented by herbicidal traces that remain in the edible plants themselves as well as the residues in the soil that may remain for a number of years before it can be degraded. Moreover, the leakage of these herbicides and their residues in water canals, vaporization and sublimation

Allelopathy phenomenon serves the agricultural community so much. The following section discusses the related concepts to allelopathy and recruiting it as natural herbicides for weed

Allelopathy is a natural biological phenomenon of interference among organisms in such a way that an organism produces one or more biochemicals that influence the growth, survival, and reproduction of other organisms. Allelopathy is the favorable or adverse effect of one plant on another due to direct or indirect release of chemicals from live or dead plants (including

Allelochemicals, or allelochemics, are a subset of low molecular weight secondary metabolites such as alkaloids, phenolics, flavonoids, terpenoids, and glucosinolates which are produced during growth and development but are not used by the allelopathic plant [82]. Allelochemi‐ cals may have beneficial (positive allelopathy) or detrimental (negative allelopathy) effects on the target organisms. Allelochemicals with negative allelopathic effects contribute in plant defense against herbivory. Also, allelochemicals could be recruited in weed management as

Allelochemicals are listed as six classes [83] that possess actual or potential phytotoxicity. The classes are namely alkaloids, benzoxazinones, cinnamic acid derivatives, cyanogenic com‐ pounds, ethylene and other seed germination stimulants, and flavonoids which have been isolated from over 30 families of terrestrial and aquatic plants. Like synthetic herbicides, there

Allelochemics are present in different parts of the plant; leaves, flowers, fruits, stems, bark, roots, rhizomes, seeds and pollen. They may be released from plants into the environment

is no common mode of action or physiological target site for all allelochemicals.

management to be an alternative or to minimize conventional herbicide use.

in air may be poisonous to the surrounding living organisms.

**6. Natural herbicides**

634 Herbicides - Current Research and Case Studies in Use

**6.1. Allelopathy term**

microorganisms).

**6.2. Allelochemical term**

alternatives to herbicides.

**6.3. Allelochemical occurrence**

According to the different structures and properties of allelochemicals, they can be classified into the following categories: water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes, and ketones; simple unsaturated lactones; long-chain fatty acids and polyacety‐ lenes; quinines (benzoquinone, anthraquinone and complex quinines); phenolics; cinnamic acid and its derivatives; coumarins; flavonoids; tannins; steroids and terpenoids (sesquiter‐ pene lactones, diterpenes, and triterpenoids) [86]. The biosynthetic pathways of the major allelopathic substances are shown in Figure 2 [87].

#### **6.5. Allelochemical interference and biological activity**

The allelochemical interference implies their interference with each other as well the interfer‐ ence with other surrounding plants. Several chemicals can be released together and may exert toxicities in an additive or synergistic manner. Allelopathic interferences often result from the mixing action of several different compounds. Allelopathic plant extracts can effectively control weeds since mixtures of allelopathic water extracts are more effective than the application of single-plant extract. Combined application of allelopathic extracts and reduced herbicide dose (up to half the standard dose) give as much weed control as the standard herbicide dose in several field crops. Lower doses of herbicides may help to reduce the development of herbicide resistance in weed ecotypes [88]. Allelopathy thus offers an attrac‐ tive environmentally friendly alternative to pesticides in agricultural pest management [88].

Response of the receiver plants to allelochemicals is not only concentration dependent but also controlled by the biochemical pathway in the receiver plant. Generally, low concentrations of allelochemicals are stimulatory while it is inhibitory with higher concentrations [89]. Allelo‐ chemical concentrations in the producer plant may also vary over time and in the plant tissue produced. Foliar and leaf litter leachates of Eucalyptus species, for example, are more toxic than bark leachates to some food crops. Typically, allelochemical concentration in field situations is below the required inhibitory level that can affect sensitive plants.

Receiver plant response to antagonistic allelochemicals is detected as certain signs on growth and development of the plants that are exposed to allelochemicals. The effect includes the inhibition or retardation of germination rate; seeds darkness and swelling; root or radicle reduction, curling of the root axis, lack of root hairs; increased number of seminal roots, swelling or necrosis of root tips; shoot or coleoptile extension; discolouration, reduced dry weight accumulation; and lowered reproductive capacity. These morphological effects may be secondary for primary events due to interference with different biochemical pathways of the receiver plant [90].

Biological activity of allelochemicals could be increased by some modifications so the end product could be more active, selective, or persistent. This is attributed to the potential

natural herbicide abilities is the black walnut tree whose leaf extraction is often used in

**Crops Scientific name Allelochemicals** Rice Oryza sativa L. Phenolic acids Wheat Triticum aestivumL. Hydroxamic acids

Cucumber Cucumis sativus L. Benzoic and Cinnamic acids

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 637

Clovers and *Trifolium spp.* Isoflavonoids and Phenolics

Oats Avena sativa L Phenolic acids and Scopoletin

Sudangrass Phenolic acids and Dhurrin

Black mustard Brassica nigra L. Allyl isothiocyanate

Cereals - Hydroxamic acids

Buck wheat Fagopyrium esculentum L. Fatty acids

Sweet clover *Melilotus spp.* Phenolics

Sorghum Sorghum bicolor L. Sorgoleone

Other natural pre-emergent herbicides are used to control weed growth such the natural herbicide corn gluten meal. Corn gluten meal was originally developed as a medium for growing fungus, but its inhibitory effect upon the germination of weeds and grasses was detected. A cover crop of rye could work as a natural herbicide between soybean crops [94].

Herbicidal effects have been identified and quantified for more than twenty allelochemicals in *Vulpia* residues. Those present in large quantities possessed low biological activities, while those present in small quantities possessed strong inhibitory activities. Interference between different allelochemicals controls the overall phytotoxicity of *Vulpia* residues which varies according to the individual chemical structure and occurred quantity. This interference provides a pattern for suggested artificial combinations of these allelochemicals prepared in aqueous solution. Biological tests for different combinations of *Vulpia* extracts demonstrated the existence of strong synergistic effects among the identified allelochemics. Moreover, exploration of the composition of a cluster of allelochemicals, which are simple in structure, possess various biological activities and few barriers to synthesis and production; this provides an alternative option for developing new herbicides from individual plant allelochemicals [94].

Selective activity of tree allelochemicals on crops and other plants has also been reported. For example, *Leucaena leucocephala*, the miracle tree promoted for revegetation, soil and water conservation and animal improvements in India, also contains a toxic, non-protein amino acid in leaves and foliage that inhibits the growth of other trees but not its own seedlings. *Leucae‐ na* species have also been shown to reduce the yield of wheat but increase the yield of rice. Leachates of the chaste tree or box elder can retard the growth of pangolagrass but stimulate growth of bluestem, another pasture grass. Examples that are shown in Table 2 represent some

allelopathic plants and their impact as reported in published research [95].

commercially-produced natural herbicides [94].

**Table 1.** Allelochemicals of Some Important Crops

**Figure 2.** The Biosynthetic Pathways of the Major Allelopathic Substances [87]

phytotoxicity of alkaloids, benzoxazinones, cinnamic acid derivatives, cyanogenic com‐ pounds, ethylene and other seed germination stimulants, and flavonoids that always represent the secondary products of allelopathic plants. Biodegradable natural plant products rarely contain halogenated atoms and possess structural diversity and complexity, constituting one such class of chemicals and these can act directly as herbicides or may provide lead structures for herbicidal discovery [91]. Selection of allelopathic plants is a good and commonly used approach for identification of plants with biologically active natural products [91].

Different crops such as beet (*Beta vulgaris* L.), lupin (*Lupinus lutens* L.), maize (*Zea mays* L.), wheat (*Triticum aestivum* L.), oats (*Avena sativa* L.) and barley (*Hordeum vulgare* L.) are known to have an allelopathic effect on other crops (Rice, 1984b). For instance, some wheat cultivars were found to significantly inhibit both germination and radicle growth of annual ryegrass. The allelopathic potential of wheat cultivars was positively correlated with their allelochemical (total phenolics) content [92]. However, different allelopathic compounds of some crops important in weed management are presented in Table 1 [93].

#### **6.6. Allelopathic plants impact**

There are some examples of plants that act as natural herbicides, such as black walnut, sunflowers, sagebrush and spotted knapweed. An herbicidal chemical called catechin was extracted from the roots of spotted knapweed and can be synthesized on a larger scale and applied to a number of other invasive plants due to selectivity. Another popular species with


**Table 1.** Allelochemicals of Some Important Crops

phytotoxicity of alkaloids, benzoxazinones, cinnamic acid derivatives, cyanogenic com‐ pounds, ethylene and other seed germination stimulants, and flavonoids that always represent the secondary products of allelopathic plants. Biodegradable natural plant products rarely contain halogenated atoms and possess structural diversity and complexity, constituting one such class of chemicals and these can act directly as herbicides or may provide lead structures for herbicidal discovery [91]. Selection of allelopathic plants is a good and commonly used

Different crops such as beet (*Beta vulgaris* L.), lupin (*Lupinus lutens* L.), maize (*Zea mays* L.), wheat (*Triticum aestivum* L.), oats (*Avena sativa* L.) and barley (*Hordeum vulgare* L.) are known to have an allelopathic effect on other crops (Rice, 1984b). For instance, some wheat cultivars were found to significantly inhibit both germination and radicle growth of annual ryegrass. The allelopathic potential of wheat cultivars was positively correlated with their allelochemical (total phenolics) content [92]. However, different allelopathic compounds of some crops

There are some examples of plants that act as natural herbicides, such as black walnut, sunflowers, sagebrush and spotted knapweed. An herbicidal chemical called catechin was extracted from the roots of spotted knapweed and can be synthesized on a larger scale and applied to a number of other invasive plants due to selectivity. Another popular species with

approach for identification of plants with biologically active natural products [91].

important in weed management are presented in Table 1 [93].

**Figure 2.** The Biosynthetic Pathways of the Major Allelopathic Substances [87]

636 Herbicides - Current Research and Case Studies in Use

**6.6. Allelopathic plants impact**

natural herbicide abilities is the black walnut tree whose leaf extraction is often used in commercially-produced natural herbicides [94].

Other natural pre-emergent herbicides are used to control weed growth such the natural herbicide corn gluten meal. Corn gluten meal was originally developed as a medium for growing fungus, but its inhibitory effect upon the germination of weeds and grasses was detected. A cover crop of rye could work as a natural herbicide between soybean crops [94].

Herbicidal effects have been identified and quantified for more than twenty allelochemicals in *Vulpia* residues. Those present in large quantities possessed low biological activities, while those present in small quantities possessed strong inhibitory activities. Interference between different allelochemicals controls the overall phytotoxicity of *Vulpia* residues which varies according to the individual chemical structure and occurred quantity. This interference provides a pattern for suggested artificial combinations of these allelochemicals prepared in aqueous solution. Biological tests for different combinations of *Vulpia* extracts demonstrated the existence of strong synergistic effects among the identified allelochemics. Moreover, exploration of the composition of a cluster of allelochemicals, which are simple in structure, possess various biological activities and few barriers to synthesis and production; this provides an alternative option for developing new herbicides from individual plant allelochemicals [94].

Selective activity of tree allelochemicals on crops and other plants has also been reported. For example, *Leucaena leucocephala*, the miracle tree promoted for revegetation, soil and water conservation and animal improvements in India, also contains a toxic, non-protein amino acid in leaves and foliage that inhibits the growth of other trees but not its own seedlings. *Leucae‐ na* species have also been shown to reduce the yield of wheat but increase the yield of rice. Leachates of the chaste tree or box elder can retard the growth of pangolagrass but stimulate growth of bluestem, another pasture grass. Examples that are shown in Table 2 represent some allelopathic plants and their impact as reported in published research [95].

#### **6.7. Allelochemical modes of action**

Allelochemical action goes mainly through affecting photosynthesis, respiration cell division, enzymes function and activity, endogenou*s* hormones and protein synthesis. This suggests allelochemical action on the molecular level and gene expression [86]. Some phenolics such as ferulic acid and cinnamic acid can inhibit protein synthesis or amino acid transport and the subsequent growth of treated plants. This is attributed to the ability of all phenolics to reduce integrity of DNA and RNA [86]. A series of physiological and biochemical changes in plants induced by phenolic compounds are shown in Figure 3 [87].


**Figure 3.** Mechanism of Allelochemicals [87].

**6.8. Strategies of allelopathic plants application as natural herbicides**

The strategy of allelochemical application is based on their antagonistic or synergistic action. Antagonistic properties of allelopathic plants are utilized in companion cropping system. Growing a companion plant which is selectively allelopathic against certain weeds and does not interfere appreciably with crop growth can greatly reduce weed establishment [96].

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 639

The interaction of weeds with crops may be positive; for instance, controlled densities of wild mustard (*Brassica campestris* L.) were interplanted with broccoli (*Brassica oleracea* var. Premium crop), crop yield increased by as much as 50% compared with broccoli planted alone [97].

Allelochemicals may be utilized as stimulators to weed seed germination before sowing the main crops, so that the germinated weeds could be eradicated easily. *Striga asiatica* is a good example for this case since it grows as a parasite to cereal grains in the southeastern United States. *Striga* normally germinates in response to compounds released from its host plants [98]. A germination stimulant, a p-benzoquinone compound from a natural host (sorghum) for *Striga* was identified. This stimulatory compound is used to induce germination of *Striga* and eradicate it before cropping its host. Ethylene was found to be a very effective germination stimulant. Also, ethylene stimulates *Striga* to germinate in the absence of a host [99] since its

**Table 2.** Examples of Allelopathy from Published Research.

**Figure 3.** Mechanism of Allelochemicals [87].

**6.7. Allelochemical modes of action**

638 Herbicides - Current Research and Case Studies in Use






**Table 2.** Examples of Allelopathy from Published Research.

alley cropping system

alley cropping system

citrus tristeza virus

red cedar

citrus

induced by phenolic compounds are shown in Figure 3 [87].

Allelochemical action goes mainly through affecting photosynthesis, respiration cell division, enzymes function and activity, endogenou*s* hormones and protein synthesis. This suggests allelochemical action on the molecular level and gene expression [86]. Some phenolics such as ferulic acid and cinnamic acid can inhibit protein synthesis or amino acid transport and the subsequent growth of treated plants. This is attributed to the ability of all phenolics to reduce integrity of DNA and RNA [86]. A series of physiological and biochemical changes in plants

**Allelopathic Plant Impact**

from trees

yield of maize and rice

and lambsquarters


species - Mango - Dried mango leaf powder completely inhibited sprouting of




crops that follow

purple nutsedge tubers.






grass but stimulated the growth of bluestem, another grass

reported to possess non-selecitve post-emergence herbicial

activity similar to glyphosate and paraquat

or when crop residues are retained as mulch.

#### **6.8. Strategies of allelopathic plants application as natural herbicides**

The strategy of allelochemical application is based on their antagonistic or synergistic action. Antagonistic properties of allelopathic plants are utilized in companion cropping system. Growing a companion plant which is selectively allelopathic against certain weeds and does not interfere appreciably with crop growth can greatly reduce weed establishment [96].

The interaction of weeds with crops may be positive; for instance, controlled densities of wild mustard (*Brassica campestris* L.) were interplanted with broccoli (*Brassica oleracea* var. Premium crop), crop yield increased by as much as 50% compared with broccoli planted alone [97].

Allelochemicals may be utilized as stimulators to weed seed germination before sowing the main crops, so that the germinated weeds could be eradicated easily. *Striga asiatica* is a good example for this case since it grows as a parasite to cereal grains in the southeastern United States. *Striga* normally germinates in response to compounds released from its host plants [98]. A germination stimulant, a p-benzoquinone compound from a natural host (sorghum) for *Striga* was identified. This stimulatory compound is used to induce germination of *Striga* and eradicate it before cropping its host. Ethylene was found to be a very effective germination stimulant. Also, ethylene stimulates *Striga* to germinate in the absence of a host [99] since its use as a gas at about 1.5kg/ha has been used effectively via a soil injection to trigger "suicidal" germination of *Striga* and to deplete the numbers of dormant seeds in soil [100].

unrelated plants into commercial crop cultivars through conventional plant breeding methods or other genetic recombination strategies. There are two methods for creating herbicidal plant crops that have been suggested; regulation of gene expression related to alleochemicales biosynthesis; or insertion of genes to produce allelochemicals that are not found in the crop [88].

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 641

The allelopathic phenomenon as mentioned before refers to the ability of some plant species to suppress other species by releasing allelochemicals, which are not toxic to the originating plant but toxic to surrounding vegetation. Breeding allelopathic cultivars by molecular approaches are more complicated than developing an herbicide-resistant crop. Genetic engineering of allelochemicals bases on their overexpression as valuable secondary metabo‐ lites in plants [111]. Most secondary metabolites being used as allelochemicals are products of a multi-gene system might which have to be developed and transformed into the specific crop

Gene insertion targets the change of the recent biochemical pathways into another one which is able to produce new allelochemicals through the insertion of transgenes. Although there is great difficulty to satisfy this approach, it represents the promising molecular approaches available for application in the near future. Various reviews in this trend and reference book

Regulation of gene expression by a biologist first requires accurate identification of the target allelochemical(s), to determine enzymes and the genes encoding them. Accordingly, a specific promoter can be inserted into crop plants to enhance allelochemical production. Allelochem‐ icals are conditionally expressed by biotic and abiotic factors since some metabolites having allelopathic potential might be newly synthesized or highly elevated in rice plants by UV irradiation [114]. For instance, there is a differential response to UV or other environmental stresses among rice cultivars. The phenylpropanoid pathway intermediates of several allelopathic rice cultivars have the highest content of *p*-coumaric acid. The latter is a key reaction in the biosynthesis of a large number of phenolic compounds in higher plants. Phenolic compounds are derived from cinnamic acid by the catalysis of 4-hydroxylase (CA4H) enzyme. The activity of CA4H was measured to determine its response to UV irradiation in rice leaves of different varieties. *Kouketsumochi* showed induction for CA4H activity by UV after 24 h of UV irradiation for 20 min while the rice cultivar AUS 196 showed no response. The increase in CA4H enzyme activity as a required enzyme in conversion of cinammic acid into *p*-coumaric acid suggested a role for CA4H gene in the elevation of the allelopathic

Responsiveness to environmental stresses and plant-plant interaction may be conferred by a specific promoter. A promoter which its induction is responsive to an elicitor can be used to regulate genes that are responsible for coding allelochemicals. The expression of phytoalexins and pathogenesis related genes in plants were reported in response to UV treatment and other

on molecular biology of weed control [112, 113] were conducted.

**7.2. Regulation of gene expression related to allelochemicals**

**7.1. Gene insertion**

to produce allelochemicals [112, 113].

function in rice plants [114].

#### **6.9. Limitation of using allelopathic plants as herbicides**

Recruiting allelopathy in weed management is limited by both the allelopathic plant itself and the environment. Production, release and phytotoxicity of allelochemicals are altered by biotic and abiotic soil factors [101, 102] such as plant age, temperature, light and soil conditions, microflora, nutritional status, and herbicide treatments. Toxicity of allelochemicals may be either cleared or increased after releasing into the soil by action of microbes [103] since the toxicity is influenced by soil texture. For instance, amounts of water-soluble phenolics in *P. lanceolata* leaf leachate amended soil varied depending on the soil textural classes if it is clay, sandy-loam, sand, or silty-loam [104]. Some allelopathic agents are active only under hot and dry climates as they work in the vapor phase such as monoterpenes because the high vapor density of the essential oils may penetrate into soil, affecting adversely the under growing plants [105].

High costs for synthesizing many allelochemicals stands as a limiting factor for utilizing allelochemicals. Also, the hazardous action of allelochemicals on human beings limits their use. They may be toxic [91] carcinogenic [106] or even cause thyroid, liver and kidney diseases in monogastric animals [107].

Allelopathic potentiality of some plants is influenced either by the availability or deficiency of nutrient. The deficiency of nutrients favors the production of secondary metabolites. For example in aerobic P-deficient soil, rice roots excrete organic anions, particularly citrate, to solubilize and enhance phosphorus uptake [108]. Some allelochemicals affect the growth of the plant itself, i.e., autotoxic effect as some derivatives of benzoic and cinnamic acids from the root exudates of cucumber since it inhibits root antioxidant enzymes and leaf photosyn‐ thesis, transpiration and stomatal conductance in cucumber [109].

Natural herbicides sound attractive as alternatives for herbicides but their application is still surrounded with much concern since they affect humans and environmental equilibrium. The agricultural community cannot discard the use of synthetic herbicides completely at the present time but their use can be reduced up to a certain extent by utilizing allelopathic potentiality as an alternative weed management strategy for crop production.

### **7. Future prospects for rationalization of herbicide usage by molecular biology**

Rationalization of herbicidal use targets mainly the production of plants which are herbicidal themselves by recruiting allelopathic characters. Allelopathy is considered a genetically influenced factor [91]. Allelopathic characteristics are more likely to evolve in competitive populations such as in wild types [110]. Therefore, it is possible to enhance weed suppressive potential of crop cultivars or to transfer allelopathic characteristics from wild types or unrelated plants into commercial crop cultivars through conventional plant breeding methods or other genetic recombination strategies. There are two methods for creating herbicidal plant crops that have been suggested; regulation of gene expression related to alleochemicales biosynthesis; or insertion of genes to produce allelochemicals that are not found in the crop [88].

### **7.1. Gene insertion**

use as a gas at about 1.5kg/ha has been used effectively via a soil injection to trigger "suicidal"

Recruiting allelopathy in weed management is limited by both the allelopathic plant itself and the environment. Production, release and phytotoxicity of allelochemicals are altered by biotic and abiotic soil factors [101, 102] such as plant age, temperature, light and soil conditions, microflora, nutritional status, and herbicide treatments. Toxicity of allelochemicals may be either cleared or increased after releasing into the soil by action of microbes [103] since the toxicity is influenced by soil texture. For instance, amounts of water-soluble phenolics in *P. lanceolata* leaf leachate amended soil varied depending on the soil textural classes if it is clay, sandy-loam, sand, or silty-loam [104]. Some allelopathic agents are active only under hot and dry climates as they work in the vapor phase such as monoterpenes because the high vapor density of the essential oils may penetrate into soil, affecting adversely the under growing

High costs for synthesizing many allelochemicals stands as a limiting factor for utilizing allelochemicals. Also, the hazardous action of allelochemicals on human beings limits their use. They may be toxic [91] carcinogenic [106] or even cause thyroid, liver and kidney diseases

Allelopathic potentiality of some plants is influenced either by the availability or deficiency of nutrient. The deficiency of nutrients favors the production of secondary metabolites. For example in aerobic P-deficient soil, rice roots excrete organic anions, particularly citrate, to solubilize and enhance phosphorus uptake [108]. Some allelochemicals affect the growth of the plant itself, i.e., autotoxic effect as some derivatives of benzoic and cinnamic acids from the root exudates of cucumber since it inhibits root antioxidant enzymes and leaf photosyn‐

Natural herbicides sound attractive as alternatives for herbicides but their application is still surrounded with much concern since they affect humans and environmental equilibrium. The agricultural community cannot discard the use of synthetic herbicides completely at the present time but their use can be reduced up to a certain extent by utilizing allelopathic

**7. Future prospects for rationalization of herbicide usage by molecular**

Rationalization of herbicidal use targets mainly the production of plants which are herbicidal themselves by recruiting allelopathic characters. Allelopathy is considered a genetically influenced factor [91]. Allelopathic characteristics are more likely to evolve in competitive populations such as in wild types [110]. Therefore, it is possible to enhance weed suppressive potential of crop cultivars or to transfer allelopathic characteristics from wild types or

thesis, transpiration and stomatal conductance in cucumber [109].

potentiality as an alternative weed management strategy for crop production.

germination of *Striga* and to deplete the numbers of dormant seeds in soil [100].

**6.9. Limitation of using allelopathic plants as herbicides**

640 Herbicides - Current Research and Case Studies in Use

plants [105].

**biology**

in monogastric animals [107].

The allelopathic phenomenon as mentioned before refers to the ability of some plant species to suppress other species by releasing allelochemicals, which are not toxic to the originating plant but toxic to surrounding vegetation. Breeding allelopathic cultivars by molecular approaches are more complicated than developing an herbicide-resistant crop. Genetic engineering of allelochemicals bases on their overexpression as valuable secondary metabo‐ lites in plants [111]. Most secondary metabolites being used as allelochemicals are products of a multi-gene system might which have to be developed and transformed into the specific crop to produce allelochemicals [112, 113].

Gene insertion targets the change of the recent biochemical pathways into another one which is able to produce new allelochemicals through the insertion of transgenes. Although there is great difficulty to satisfy this approach, it represents the promising molecular approaches available for application in the near future. Various reviews in this trend and reference book on molecular biology of weed control [112, 113] were conducted.

#### **7.2. Regulation of gene expression related to allelochemicals**

Regulation of gene expression by a biologist first requires accurate identification of the target allelochemical(s), to determine enzymes and the genes encoding them. Accordingly, a specific promoter can be inserted into crop plants to enhance allelochemical production. Allelochem‐ icals are conditionally expressed by biotic and abiotic factors since some metabolites having allelopathic potential might be newly synthesized or highly elevated in rice plants by UV irradiation [114]. For instance, there is a differential response to UV or other environmental stresses among rice cultivars. The phenylpropanoid pathway intermediates of several allelopathic rice cultivars have the highest content of *p*-coumaric acid. The latter is a key reaction in the biosynthesis of a large number of phenolic compounds in higher plants. Phenolic compounds are derived from cinnamic acid by the catalysis of 4-hydroxylase (CA4H) enzyme. The activity of CA4H was measured to determine its response to UV irradiation in rice leaves of different varieties. *Kouketsumochi* showed induction for CA4H activity by UV after 24 h of UV irradiation for 20 min while the rice cultivar AUS 196 showed no response. The increase in CA4H enzyme activity as a required enzyme in conversion of cinammic acid into *p*-coumaric acid suggested a role for CA4H gene in the elevation of the allelopathic function in rice plants [114].

Responsiveness to environmental stresses and plant-plant interaction may be conferred by a specific promoter. A promoter which its induction is responsive to an elicitor can be used to regulate genes that are responsible for coding allelochemicals. The expression of phytoalexins and pathogenesis related genes in plants were reported in response to UV treatment and other plant defense inducers [115, 116]. UV was found to stimulate phytoalexine production in pepper. The effective motifs response to UV light was determined in tobacco by examining the expression of GUS activity of plants transformed with the constructs of various CASC (*Capsicum annuum* sesqiterpene cyclase) promoters fused into GUS gene [115]. This was followed by UV irradiation of the transgenic plants to assure the induction of the CASC promoters through examining GUS activity of the transgenic plants. The levels of GUS activity for transgenic plants with pBI121-KF1 and pBI121-KF6 were significantly elevated by UVirradiation and had a two-to-threefold increase approximately over the untreated-transgenic plants. In contrast, GUS expression in the transgenic plants with pBI121-CaMV 35S was not changed by UV, and in the other constructs had only a very small increase [117]. The CASC promoters of both KF-1 and KF-6 were suggested to contain cis-acting elements capable of conferring quantitative expression patterns that were exclusively associated with UV irradia‐ tion. The regulation of genes associated with allelopathy could be achieved by developing a specific promoter responsive to plant-weed competition or environmental stresses. The CASC promoters of KF-1 and KF-6 obtained may be specific to UV. Thus, this promoter can be used for the overexpression of specific promoters constructed to allelochemical-producing genes [116]. To regulate the CA4H gene in the phenylpropanoid pathway, specific promoters, the CASC-KF1 and KF6, were fused to CA4H gene. The gene constructs were introduced into the binary plant expression vector pIG121-HMR with reverse primer harbouring *Bam*HI site and forward primer harbouring *Hin*dIII site as illustrated in Figure 4 [118].

**8. Conclusion**

difficult to transfer genes into crops.

Mona H. El-Hadary1,2\* and Gyuhwa Chung3

Institute (GEBRI) Minufiya University, Egypt

\*Address all correspondence to: drmona3000@yahoo.com

**Author details**

**References**

Egypt; (1988).

10.1002/anie.199202422.

Herbicides are widely used in agricultural communities on a large scale for eradicating weeds. Herbicides function by affecting different biochemical processes in weeds. Herbicides in low doses act as growth regulators for the main crop but high doses may cause crop damage. However, uncontrolled herbicide use can cause hazardous effects not only upon the main crop but also human health and the surrounding environment [80, 81]. Moreover, heavy doses of herbicides create the problem of herbicide resistance development in weeds. There is an urgent need to identify natural alternatives that can meet the demands of agrosystems without affecting the surrounding environment. Hence, the idea of recruiting the allelopathic phe‐ nomenon of some plants in inhibiting the growth of weed vegetation has been investigated. Allelopathy cannot cancel the use of herbicides completely but can minimize it. Allelopathic plant use has limitations in the application because of the potential toxicity. Thus, molecular biology can aid the agricultural community by engineering crops to be herbicides themselves through gene insertion and regulation depending on well-defined allelopathic genes or promoters, respectively. Even with well-characterized allelopathic genes, it might be very

Herbicides — A Double Edged Sword http://dx.doi.org/10.5772/55957 643

1 Department of Molecular Biology, Genetic Engineering and Biotechnology Research

[1] El-Hadary, M. H. Effect of Brominal and Granstar Herbicides on Growth and Some Metabolic Activities in Wheat. MSc. thesis. Faculty of Science Tanta University

[2] Hassall, K. A, Editors- Ebert, E, & Kayser, H. Staub T- Book Review: The Biochemis‐ try and Uses of Pesticides. Structure, Metabolism, Mode of Action and Uses in Crop Protection. (2nd Edition). VCH Verlagsgesellschaft mb: Germany; (2003). DOI:

2 Department of Botany, Faculty of Science, Damanhour University, Egypt

3 Department of Biotechnology, Chonnam National University, Korea

**Figure 4.** The Gene Cassette with Specific Promoters Responsive to UV Irradiation in pIG121-HmR [117].

### **8. Conclusion**

plant defense inducers [115, 116]. UV was found to stimulate phytoalexine production in pepper. The effective motifs response to UV light was determined in tobacco by examining the expression of GUS activity of plants transformed with the constructs of various CASC (*Capsicum annuum* sesqiterpene cyclase) promoters fused into GUS gene [115]. This was followed by UV irradiation of the transgenic plants to assure the induction of the CASC promoters through examining GUS activity of the transgenic plants. The levels of GUS activity for transgenic plants with pBI121-KF1 and pBI121-KF6 were significantly elevated by UVirradiation and had a two-to-threefold increase approximately over the untreated-transgenic plants. In contrast, GUS expression in the transgenic plants with pBI121-CaMV 35S was not changed by UV, and in the other constructs had only a very small increase [117]. The CASC promoters of both KF-1 and KF-6 were suggested to contain cis-acting elements capable of conferring quantitative expression patterns that were exclusively associated with UV irradia‐ tion. The regulation of genes associated with allelopathy could be achieved by developing a specific promoter responsive to plant-weed competition or environmental stresses. The CASC promoters of KF-1 and KF-6 obtained may be specific to UV. Thus, this promoter can be used for the overexpression of specific promoters constructed to allelochemical-producing genes [116]. To regulate the CA4H gene in the phenylpropanoid pathway, specific promoters, the CASC-KF1 and KF6, were fused to CA4H gene. The gene constructs were introduced into the binary plant expression vector pIG121-HMR with reverse primer harbouring *Bam*HI site and

642 Herbicides - Current Research and Case Studies in Use

forward primer harbouring *Hin*dIII site as illustrated in Figure 4 [118].

**Figure 4.** The Gene Cassette with Specific Promoters Responsive to UV Irradiation in pIG121-HmR [117].

Herbicides are widely used in agricultural communities on a large scale for eradicating weeds. Herbicides function by affecting different biochemical processes in weeds. Herbicides in low doses act as growth regulators for the main crop but high doses may cause crop damage. However, uncontrolled herbicide use can cause hazardous effects not only upon the main crop but also human health and the surrounding environment [80, 81]. Moreover, heavy doses of herbicides create the problem of herbicide resistance development in weeds. There is an urgent need to identify natural alternatives that can meet the demands of agrosystems without affecting the surrounding environment. Hence, the idea of recruiting the allelopathic phe‐ nomenon of some plants in inhibiting the growth of weed vegetation has been investigated. Allelopathy cannot cancel the use of herbicides completely but can minimize it. Allelopathic plant use has limitations in the application because of the potential toxicity. Thus, molecular biology can aid the agricultural community by engineering crops to be herbicides themselves through gene insertion and regulation depending on well-defined allelopathic genes or promoters, respectively. Even with well-characterized allelopathic genes, it might be very difficult to transfer genes into crops.

### **Author details**

Mona H. El-Hadary1,2\* and Gyuhwa Chung3

\*Address all correspondence to: drmona3000@yahoo.com

1 Department of Molecular Biology, Genetic Engineering and Biotechnology Research Institute (GEBRI) Minufiya University, Egypt

2 Department of Botany, Faculty of Science, Damanhour University, Egypt

3 Department of Biotechnology, Chonnam National University, Korea

### **References**


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## *Edited by Andrew J. Price and Jessica A. Kelton*

Herbicide use is a common component of many weed management strategies in both agricultural and non-crop settings. However, herbicide use practices and recommendations are continuously updated and revised to provide control of everchanging weed compositions and to preserve efficacy of current weed control options. Herbicides - Current Research and Case Studies in Use provides information about current trends in herbicide use and weed control in different land and aquatic settings as well as case studies in particular weed control situations.

Herbicides - Current Research and Case Studies in Use

Herbicides

Current Research and Case Studies in Use

*Edited by Andrew J. Price and Jessica A. Kelton*