Novel Approaches to Plant Disease

**3**

**Chapter 1**

**Abstract**

**1. Introduction**

Downy Mildew of Basil: A New

Destructive Disease Worldwide

Oomycete pseudofungus (*Peronospora belbahrii*) is a causal of devastating basil downy mildew disease because once infected basil plants are no longer marketable. The host range is limited to basil and hyssop. Coleus was previously considered as host as well, but pathogen causing downy mildew on coleus has been shown genetically different and specified as *P. belbahrii sensu lato*. Therefore, *P. belbahrii* is described as a complex species, likely defined by plant host. The *P. belbahrii* is air-borne and seed-borne pathogen and it does not need a vector for dispersal. The disease was firstly reported from Africa where it is assumed to have originated on sweet basil and 70 years later it was reported from Europe. Currently, basil downy mildew is of pandemic occurrence and the pathogen is present in almost all areas around the world where basil is cultivated. Since the pathogen is transmitted by the

*Snježana Topolovec-Pintarić and Katarina Martinko*

seed, there is a high risk of the pathogen spread by the seed trade.

is assumed to have originated on sweet basil [1, 2].

**Keywords:** *Agastache*, downy mildew, *Lamiaceae*, *Ocimum*, *Peronospora*

Downy mildew caused by *Peronospora belbahrii* Thines is one of the most destructive diseases of sweet basil (*Ocimum basilicum*) of the family *Lamiaceae* Lindl. (alternatively *Labiaceae* Dulac) which except field farming is also grown as a specialty crop in greenhouses. Downy mildew of basil was first reported in 1932 from Uganda, Africa as *Peronospora* spp. and again in 1937 as *P. lamii* from where it

First report from Europe was in 2001 from Switzerland where it was observed in greenhouses [3]. After that, the disease was detected in Italy in 2003 on sweet basil in several greenhouses located in Liguria region (Northern Italy). In 2004, it was found in France on some basil crops near Saint Tropez (Southern France) [4]. In the same year, it was found in Belgium, but there are no data about the first detection. After those first European reports, the pathogen was rapidly spread through Europe. In summer 2009, it was detected in United Kingdom in *Agastache* (hyssop) plants (*Agastache mexicana* and *Agastache* sp.) at Wisley gardens (Surrey) and on the summer of 2010 in protected basil plants in south-east England [5, 6]. In 2010, a significant incidence of downy mildew was reported in Hungary at two plant stands at Budapest-Soroksár and Tordasal though a similar disease had been observed in 2003 in a greenhouse at Albertirsa [7]. It was reported from Czech Republic in 2012 as well as from Cyprus [8]. In 2014, it was found again in United Kingdom but in several plants of coleus (*Solenostemon scutellarioides* cv. 'Chocolate Mint') but in 2016 has been shown that the pathogen causing coleus downy mildew is *P. belbahrii* 

#### **Chapter 1**

## Downy Mildew of Basil: A New Destructive Disease Worldwide

*Snježana Topolovec-Pintarić and Katarina Martinko*

#### **Abstract**

Oomycete pseudofungus (*Peronospora belbahrii*) is a causal of devastating basil downy mildew disease because once infected basil plants are no longer marketable. The host range is limited to basil and hyssop. Coleus was previously considered as host as well, but pathogen causing downy mildew on coleus has been shown genetically different and specified as *P. belbahrii sensu lato*. Therefore, *P. belbahrii* is described as a complex species, likely defined by plant host. The *P. belbahrii* is air-borne and seed-borne pathogen and it does not need a vector for dispersal. The disease was firstly reported from Africa where it is assumed to have originated on sweet basil and 70 years later it was reported from Europe. Currently, basil downy mildew is of pandemic occurrence and the pathogen is present in almost all areas around the world where basil is cultivated. Since the pathogen is transmitted by the seed, there is a high risk of the pathogen spread by the seed trade.

**Keywords:** *Agastache*, downy mildew, *Lamiaceae*, *Ocimum*, *Peronospora*

#### **1. Introduction**

Downy mildew caused by *Peronospora belbahrii* Thines is one of the most destructive diseases of sweet basil (*Ocimum basilicum*) of the family *Lamiaceae* Lindl. (alternatively *Labiaceae* Dulac) which except field farming is also grown as a specialty crop in greenhouses. Downy mildew of basil was first reported in 1932 from Uganda, Africa as *Peronospora* spp. and again in 1937 as *P. lamii* from where it is assumed to have originated on sweet basil [1, 2].

First report from Europe was in 2001 from Switzerland where it was observed in greenhouses [3]. After that, the disease was detected in Italy in 2003 on sweet basil in several greenhouses located in Liguria region (Northern Italy). In 2004, it was found in France on some basil crops near Saint Tropez (Southern France) [4]. In the same year, it was found in Belgium, but there are no data about the first detection.

After those first European reports, the pathogen was rapidly spread through Europe. In summer 2009, it was detected in United Kingdom in *Agastache* (hyssop) plants (*Agastache mexicana* and *Agastache* sp.) at Wisley gardens (Surrey) and on the summer of 2010 in protected basil plants in south-east England [5, 6]. In 2010, a significant incidence of downy mildew was reported in Hungary at two plant stands at Budapest-Soroksár and Tordasal though a similar disease had been observed in 2003 in a greenhouse at Albertirsa [7]. It was reported from Czech Republic in 2012 as well as from Cyprus [8]. In 2014, it was found again in United Kingdom but in several plants of coleus (*Solenostemon scutellarioides* cv. 'Chocolate Mint') but in 2016 has been shown that the pathogen causing coleus downy mildew is *P. belbahrii* 

*sensu lato* [9]. In 2016, it has been reported from Spain on basil collected from the island of Tenerife (Islas Canarias) and afterwards was also noted that has been causing severe symptoms and economic losses in Almería, Andalucía [10].

In the United States, downy mildew of basil is considered as relatively new disease but the pathogen has been detected in October 2007 in South Florida [11]. Since its first detection in the United States, it has been observed on basil in at least 42 states [11, 12]. Interesting is founding in 2008 on basil plants produced in various nurseries in Sebastopol, Sonoma County because trace-back investigation revealed that the seeds had originated from Italy. This disease was also reported in Argentina in February 2008 [13] and Canada in 2011 [14]. In 2011, it was reported in Hawaii for the first time and in Mexico in 2015 [15].

First report in Asia was in Iran, where a severe outbreak of downy mildew was observed in sweet basil fields in 2006 [16]. A year later, in April 2007, it has been found in Japan on coleus plants cultivated in a greenhouse in Chiba Prefecture (Honshu) [17]. In the spring of 2009, it has been found in Taiwan in the field of Nantu and Yunlin [18]. In Israel, it was firstly found in December 2011 near Bet She'an, and in 2012 the disease has been spread throughout the country to all basil-growing areas [19]. Recently, it has been found in China in July 2014 on basil on the island of Hainan in Sanya City and in 2016 in the Shunyi and Daxing districts of Beijing which is concerned as first report in mainland China [20, 21]. Last Asian report is from Korea, where it has been first observed in November 2015 on sweet basil plants growing in plastic greenhouses in Gwangmyeong [22].

Until 2017, the disease was considered exotic to Australia, when it has been reported from South-east Queensland. Within 6 months, the disease was present along the eastern seaboard from north Queensland to Victoria, South Australia and the Northern Territory. In the scientific literature, Australia has been listed as a host country as early as 2015; however, no records of detections could be traced [23].

Finally, the first report of *P. belbahrii sensu lato* detection on coleus (*Plectranthus* spp.) in Brazil was reported in 2019 [24].

The first official report for Croatia was done in 2015 by Croatian Agency for Agriculture and Food based on symptoms and morphological characteristic not confirmed by molecular diagnostic [25]. In October 2015, as part of regular reporting reviews conducted by the Advisory Service, infected plants were found in greenhouses in the Varaždin County. The disease was spread soon after that founding on areas of four more counties (Krapina-Zagorje, Međimurje, Split-Dalmatian and Zagreb). Interestingly, in Dubrovnik-Neretva County, the disease was found on pot-plants imported from Italy. Up to date in Croatia, downy mildew is recorded only in production of basil in greenhouses. This chapter authors are currently investigating the occurrence on basil in the greenhouse 'Green friends' of eco-grower of culinary herbs and spices, situated in Rakovica in the Zagreb County. We confirmed determination of the *P. belbahrii* by molecular diagnostic (PCR sequence comparison of the ITS rDNA sequences and Cox2 region) (unpublished).

#### **2. Disease symptoms**

The first symptoms can be spotted on lower leaves, where infection starts and progresses upwards. The most noticeable symptom is yellowing (slightly chlorotic) of the leaves with the veins remaining green. The initial yellowing can be misinterpreted as a nutritional deficiency and so disease can go unrecognised. With time on upper surface of leaves, large chlorotic lesions with soft margins are developing. Chlorosis often involved the entire leaf surface. Since the pathogen is a biotroph, it causes dying of cells from which it absorbed nutrients and therefore necrotisation

**5**

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

*Brown growth of* Peronospora belbahrii *on abaxial side of basil leaf.*

occur after chlorosis and the central portion of a chlorotic lesion become necrotic. This can lead to slight curvature of leaves. Necrotic spots are variable in size and of irregular shape as they are limited by the main veins. In some cases, entire area of the leaf surface is affected. In humid conditions, necrotic regions can become dark brown to black in colour. On abaxial leaf surfaces, both in chlorotic and necrotic regions, a typical greyish to brown, furry or downy moulds could be observed giving the leaves a dirty appearance (**Figure 1**). Parasitisation results in shrinkage of

Disease can go asymptomatic under cool and dry conditions [26] and sometimes plants not showing symptoms at harvest can develop symptoms during transport [27]. In report from Taiwan, it was noted that the pathogen caused chlorosis and leaf shrinkage on basil in the field, but did not cause any symptom on coleus, Pai-tsai Chinese cabbage (*Brassica rapa*), leaf lettuce (*Lactuca sativa*) and melon

Sweet basil is the natural host of *P. belbahrii* and the majority of *P. belbahrii* findings have been on sweet basil. In 2009, Thines et al. concluded that coleus is also the natural host of this pathogen [28]. They also investigated the downy mildew of sage, but were unable to confirm that it is caused by *P. belbahrii* so did not considered sage as natural host. Coleus has been confirmed as host of *P. belbahrii* in Japan, United States, United Kingdom and Germany [29]. Species concept has been refined recently and pathogen causal of coleus downy mildew was specified as *P. belbahrii sensu lato* [9]. Moreover, an unidentified species of *Peronospora* sp. infects coleus in Israel [30]. Interestingly, Israeli isolates of *P. belbahrii* from sweet basil do not infect coleus although infects other *Lamiaceae* species: rosemary (*Rosmarinus officinalis*) Nepeta (*Nepeta curviflora*), Clinopodium (*Micromeria fruticosa*) and two species of sage (*Salvia pinnata* and *S. fruticosa*) [30]. Further, the conidia from mentioned species failed to infect sweet basil and therefore the role of these species in the epidemiology of basil downy mildew in Israel is unknown [30]. The *Peronospora* sp. on coleus was reported in 2005 Louisiana, New York

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

leaf and premature leaf fall.

(*Cucumis melo*) [18].

**3. Hosts range**

**Figure 1.**

*Downy Mildew of Basil: A New Destructive Disease Worldwide DOI: http://dx.doi.org/10.5772/intechopen.91903*

#### **Figure 1.**

*Plant Diseases-Current Threats and Management Trends*

for the first time and in Mexico in 2015 [15].

spp.) in Brazil was reported in 2019 [24].

*sensu lato* [9]. In 2016, it has been reported from Spain on basil collected from the island of Tenerife (Islas Canarias) and afterwards was also noted that has been causing severe symptoms and economic losses in Almería, Andalucía [10]. In the United States, downy mildew of basil is considered as relatively new disease but the pathogen has been detected in October 2007 in South Florida [11]. Since its first detection in the United States, it has been observed on basil in at least 42 states [11, 12]. Interesting is founding in 2008 on basil plants produced in various nurseries in Sebastopol, Sonoma County because trace-back investigation revealed that the seeds had originated from Italy. This disease was also reported in Argentina in February 2008 [13] and Canada in 2011 [14]. In 2011, it was reported in Hawaii

First report in Asia was in Iran, where a severe outbreak of downy mildew was observed in sweet basil fields in 2006 [16]. A year later, in April 2007, it has been found in Japan on coleus plants cultivated in a greenhouse in Chiba Prefecture (Honshu) [17]. In the spring of 2009, it has been found in Taiwan in the field of Nantu and Yunlin [18]. In Israel, it was firstly found in December 2011 near Bet She'an, and in 2012 the disease has been spread throughout the country to all basil-growing areas [19]. Recently, it has been found in China in July 2014 on basil on the island of Hainan in Sanya City and in 2016 in the Shunyi and Daxing districts of Beijing which is concerned as first report in mainland China [20, 21]. Last Asian report is from Korea, where it has been first observed in November 2015 on sweet

Until 2017, the disease was considered exotic to Australia, when it has been reported from South-east Queensland. Within 6 months, the disease was present along the eastern seaboard from north Queensland to Victoria, South Australia and the Northern Territory. In the scientific literature, Australia has been listed as a host country as early as 2015; however, no records of detections could be traced [23]. Finally, the first report of *P. belbahrii sensu lato* detection on coleus (*Plectranthus*

The first official report for Croatia was done in 2015 by Croatian Agency for Agriculture and Food based on symptoms and morphological characteristic not confirmed by molecular diagnostic [25]. In October 2015, as part of regular reporting reviews conducted by the Advisory Service, infected plants were found in greenhouses in the Varaždin County. The disease was spread soon after that founding on areas of four more counties (Krapina-Zagorje, Međimurje, Split-Dalmatian and Zagreb). Interestingly, in Dubrovnik-Neretva County, the disease was found on pot-plants imported from Italy. Up to date in Croatia, downy mildew is recorded only in production of basil in greenhouses. This chapter authors are currently investigating the occurrence on basil in the greenhouse 'Green friends' of eco-grower of culinary herbs and spices, situated in Rakovica in the Zagreb County. We confirmed determination of the *P. belbahrii* by molecular diagnostic (PCR sequence compari-

The first symptoms can be spotted on lower leaves, where infection starts and progresses upwards. The most noticeable symptom is yellowing (slightly chlorotic) of the leaves with the veins remaining green. The initial yellowing can be misinterpreted as a nutritional deficiency and so disease can go unrecognised. With time on upper surface of leaves, large chlorotic lesions with soft margins are developing. Chlorosis often involved the entire leaf surface. Since the pathogen is a biotroph, it causes dying of cells from which it absorbed nutrients and therefore necrotisation

basil plants growing in plastic greenhouses in Gwangmyeong [22].

son of the ITS rDNA sequences and Cox2 region) (unpublished).

**4**

**2. Disease symptoms**

*Brown growth of* Peronospora belbahrii *on abaxial side of basil leaf.*

occur after chlorosis and the central portion of a chlorotic lesion become necrotic. This can lead to slight curvature of leaves. Necrotic spots are variable in size and of irregular shape as they are limited by the main veins. In some cases, entire area of the leaf surface is affected. In humid conditions, necrotic regions can become dark brown to black in colour. On abaxial leaf surfaces, both in chlorotic and necrotic regions, a typical greyish to brown, furry or downy moulds could be observed giving the leaves a dirty appearance (**Figure 1**). Parasitisation results in shrinkage of leaf and premature leaf fall.

Disease can go asymptomatic under cool and dry conditions [26] and sometimes plants not showing symptoms at harvest can develop symptoms during transport [27]. In report from Taiwan, it was noted that the pathogen caused chlorosis and leaf shrinkage on basil in the field, but did not cause any symptom on coleus, Pai-tsai Chinese cabbage (*Brassica rapa*), leaf lettuce (*Lactuca sativa*) and melon (*Cucumis melo*) [18].

#### **3. Hosts range**

Sweet basil is the natural host of *P. belbahrii* and the majority of *P. belbahrii* findings have been on sweet basil. In 2009, Thines et al. concluded that coleus is also the natural host of this pathogen [28]. They also investigated the downy mildew of sage, but were unable to confirm that it is caused by *P. belbahrii* so did not considered sage as natural host. Coleus has been confirmed as host of *P. belbahrii* in Japan, United States, United Kingdom and Germany [29]. Species concept has been refined recently and pathogen causal of coleus downy mildew was specified as *P. belbahrii sensu lato* [9]. Moreover, an unidentified species of *Peronospora* sp. infects coleus in Israel [30]. Interestingly, Israeli isolates of *P. belbahrii* from sweet basil do not infect coleus although infects other *Lamiaceae* species: rosemary (*Rosmarinus officinalis*) Nepeta (*Nepeta curviflora*), Clinopodium (*Micromeria fruticosa*) and two species of sage (*Salvia pinnata* and *S. fruticosa*) [30]. Further, the conidia from mentioned species failed to infect sweet basil and therefore the role of these species in the epidemiology of basil downy mildew in Israel is unknown [30]. The *Peronospora* sp. on coleus was reported in 2005 Louisiana, New York

and Florida in U.S. [31, 32]. In 2015, *Peronospora* sp. on coleus was reported in Tennessee, and the morphological and molecular characteristics were consistent with Thines description of P*. belbahrii sensu lato* [9, 28]. So, Rivera et al. concluded that *P. belbahrii* can be described as complex of species likely defined by plant host [9]. Recently, coleus downy mildew causal pathogen is confirmed as *P. belbahrii sensu lato* host based on pathogenicity test in Brazil, and this is the first such report for the South America [24].

In 2009, the *Agastache* species (*Lamiaceae*) was also named as the new *P. belbahrii* host by Henricot et al. [6]. The host range is today broadened and as alternative hosts are considered culinary and ornamental varieties related to basil and coleus from *Lamiaceae* family and here are mint (*Mentha* spp.) and sage (*Salvia* spp.). All cultivars of sweet basil are hosts and as highly susceptible ones are cv. Genovese Nufar, Italian Large Leaf, Queenette, Superbo, Poppy Joe's and Milita [27]. Some of the exotic, spice and ornamental basils cultivars such as red types (*O. basilicum purpurescens* cv. Red Rubin, Red leaf), lemon basil (*O. citridiorum* cv. Lemon std., Mrs. Burn's Lemon, Lemona & Lime) and lime basil (*O. americanum* cv. Blue Spice, Spice & Blue Spice F1) have been found less susceptible or even resistant to downy mildew [9, 28, 33]. This chapter's authors detected downy mildew on spice cultivars of basil, and *P. belbahrii* was confirmed as causal pathogen by molecular analysis (unpublished).

#### **4. Description of pathogen**

The causal pathogen of basil's downy mildew is pseudofungus *Peronospora belbahrii* Thines and has been formally introduced under name *P. belbahrii* by Thines et al. in 2009 as dedication to Lassaard Belbahrii who first suggested that the pathogen on basil might be a distinct undescribed species and distinguished it from a different closely related species that parasitizes sage (*Salvia officinalis*) [28]. It is assumed that *P. belbahrii* is of African origin, as its host basil is native to this continent [28]. As oomycete it is classified in *Chromysta*, *Oomycota*, *Oomycetes*, *Peronosporales* and *Peronosporaceae*. The pathogen was molecularly determined in 2005 by Belbahri et al. [3] and showed through ITS sequencing that it is a newly occurring species on basil that differs from *P. lamii*, the only previously reported downy mildew on sweet basil and also differs from *Peronospora* species that is affecting lamiaceous hosts worldwide [1, 2]. Perhaps, previous findings of *Peronospora* sp. on sweet basil and coleus may be *P. belbahrii* but have been misidentified as *P. lamii* before sequence identification was carried out and before it was first described as a new species *P. belbahrii*. Confusion between species is likely to occur without sequence data; therefore, samples must be submitted to a competent testing laboratory for identification. Using morphological comparison and molecular phylogenetic reconstructions, Thines et al. also confirmed that *P. belbahrii* is not identical to *P. swingleii* on *Salvia reflexa* [28]*. P. belbahrii* on basil and coleus seems to be closely related yet; it has been shown that they are morphologically and genetically different [28]. Limited potential to infect basil has been reported for the isolates from coleus, as it was described earlier [30]. The significance of differences between causal pathogen of downy mildew on basil and coleus needs to be investigated further; but for now, the pathogen on coleus is determined as *P. belbahrii sensu lato.*

The growths on the underside of the symptomatic leaves in a form of a brown downy mould are asexual organs, sporangia bearing sporangiophores which emerge from leaf stomata. Microscopic observations will show that they are consistent with the characteristics of a genus *Peronospora*. The first descriptions of sporangia and sporangiophores on basil and coleus that were confirmed by molecular determination were provided by Thines et al. in 2009 [28]. The sporangia of genus *Peronospora*

**7**

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

are spore-like structures and they act as conidia and germinate into a germ-tube when they are near a leaf stoma. Therefore, the use of synonym conidia, or simply

Conidia are dark brown to olive in colour and pedicel is absent. They are rounded and egg-shaped with a length 24–29–30.8–33–36 μm on basil and 26–29–31.3–33–37 μm on coleus. They width are 20–23–24–26–29 μm on basil and 20–23–24.5–26–29 μm on coleus. Ratio of length and width is 1.1–1.2–1.29–1.4–1.5

Sporangiophores are colourless (hyaline) with a long, straight trunk and monopodially with a length 270–300–400–520–680 μm on basil and 330–380–466–570– 650 μm on coleus [28]. Numbers of ramifications were 3–4–4.9–5–7 per sporophore on basil and 4–5–5.2–6–7 μm on coleus. Ultimate branchlets were in pairs, curved, longer one in length 13–18–20.6–26–31 μm on basil and 12–13–18–22–31 μm on coleus while the shorter one in length 3.8–7.7–9.80–10–15 μm on basil and 5.1–7.7– 10.7–13–17 μm on coleus. Ratio of longer to shorter branches is 1.3–1.8–2.25–2.7–4 on basil and 1.1–1.6–1.71–1.9–2.5 on coleus. Ultimate branches end dichotomically and tips (sterigmata) are acute to subacute on both, basil and coleus. Tips are bear-

The shortest sporangiophores were reported in Iran and were 130–290 μm (avg. 194 μm) long and branched two to five times [16]. The longest sporangiophores were recorded in Hungary, and they were in length of 416–784 μm (avg. 572 μm)

There are two oospore detections published up to date, both from Israel, found in leaves of susceptible sweet basil cultivar 'Peri'. In 2013, Cohen et al. identified and described oospores as thick-walled, brown in colour, measuring of 46.2 ± 2.8 μm in diameter [34]. Oospores never occurred on the infected leaf surface, but inside the mesophyll [30]. In 2016, in walk-in tunnel experiments that simulated commercial production conditions, oospores were observed attached to the leaf surface, to older parts of the infection area, and also found to water washes of the leaf surface by Elad et al. [35]. Discovery of oospores suggests the potential for sexual reproduction, but little is known on *P. belbahrii* oospore formation or is it homothallic or heterothallic. Currently, only one mating type has been found [22], although it is already presumed that it is heterothallic [26, 36, 37]. The pathogenicity of oospores is investigated, but without positive infections [30, 38], and their role in the basil

The *P. belbahrii* thrives in warm, humid conditions and produce conidia that can infect in temperatures as low as 15°C (59°F) [26]. For example, downy mildew is present in Israeli basil-cropping regions where in the cooler season temperatures may reach minimum of 5–10°C at night and a maximum of 10–25°C during the day [35]. This corroborates with our observations. Pathogen can tolerate cold weather (10–15°C) but, like its host basil, cannot survive freezing winter temperatures at continental climate. Conidia cannot survive harsh winters and as pathogen is biotroph it needs living host. Therefore, in climates with harsh winters and with just one mating type of the *P. belbahrii* it can survive only on living plants in greenhouse production operations that produce basil year round. In mild winters and in warm, temperate regions where the host, basil will not freeze, the second overwintering inoculum are mycelium and conidia in infected plant buds, plant stems, leaf tissue and shoots. Congruently, the most devastating damage is often seen in warm and

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

spore, has become commonplace for sporangia.

on basil and 1.1–1.2–1.28–1.4–1.5 on coleus [28].

and monopodially branched five to seven times [7].

downy mildew epidemiology is not known.

humid conditions, late summer and in greenhouses.

**5. Disease cycle and dispersal**

ing single sporangia.

*Plant Diseases-Current Threats and Management Trends*

for the South America [24].

**4. Description of pathogen**

determined as *P. belbahrii sensu lato.*

and Florida in U.S. [31, 32]. In 2015, *Peronospora* sp. on coleus was reported in Tennessee, and the morphological and molecular characteristics were consistent with Thines description of P*. belbahrii sensu lato* [9, 28]. So, Rivera et al. concluded that *P. belbahrii* can be described as complex of species likely defined by plant host [9]. Recently, coleus downy mildew causal pathogen is confirmed as *P. belbahrii sensu lato* host based on pathogenicity test in Brazil, and this is the first such report

In 2009, the *Agastache* species (*Lamiaceae*) was also named as the new *P. belbahrii* host by Henricot et al. [6]. The host range is today broadened and as alternative hosts are considered culinary and ornamental varieties related to basil and coleus from *Lamiaceae* family and here are mint (*Mentha* spp.) and sage (*Salvia* spp.). All cultivars of sweet basil are hosts and as highly susceptible ones are cv. Genovese Nufar, Italian Large Leaf, Queenette, Superbo, Poppy Joe's and Milita [27]. Some of the exotic, spice and ornamental basils cultivars such as red types (*O. basilicum purpurescens* cv. Red Rubin, Red leaf), lemon basil (*O. citridiorum* cv. Lemon std., Mrs. Burn's Lemon, Lemona & Lime) and lime basil (*O. americanum* cv. Blue Spice, Spice & Blue Spice F1) have been found less susceptible or even resistant to downy mildew [9, 28, 33]. This chapter's authors detected downy mildew on spice cultivars of basil, and *P. belbahrii* was confirmed as causal pathogen by molecular analysis (unpublished).

The causal pathogen of basil's downy mildew is pseudofungus *Peronospora belbahrii* Thines and has been formally introduced under name *P. belbahrii* by Thines et al. in 2009 as dedication to Lassaard Belbahrii who first suggested that the pathogen on basil might be a distinct undescribed species and distinguished it from a different closely related species that parasitizes sage (*Salvia officinalis*) [28]. It is assumed that *P. belbahrii* is of African origin, as its host basil is native to this continent [28]. As oomycete it is classified in *Chromysta*, *Oomycota*, *Oomycetes*, *Peronosporales* and *Peronosporaceae*. The pathogen was molecularly determined in 2005 by Belbahri et al. [3] and showed through ITS sequencing that it is a newly occurring species on basil that differs from *P. lamii*, the only previously reported downy mildew on sweet basil and also differs from *Peronospora* species that is affecting lamiaceous hosts worldwide [1, 2]. Perhaps, previous findings of *Peronospora* sp. on sweet basil and coleus may be *P. belbahrii* but have been misidentified as *P. lamii* before sequence identification was carried out and before it was first described as a new species *P. belbahrii*. Confusion between species is likely to occur without sequence data; therefore, samples must be submitted to a competent testing laboratory for identification. Using morphological comparison and molecular phylogenetic reconstructions, Thines et al. also confirmed that *P. belbahrii* is not identical to *P. swingleii* on *Salvia reflexa* [28]*. P. belbahrii* on basil and coleus seems to be closely related yet; it has been shown that they are morphologically and genetically different [28]. Limited potential to infect basil has been reported for the isolates from coleus, as it was described earlier [30]. The significance of differences between causal pathogen of downy mildew on basil and coleus needs to be investigated further; but for now, the pathogen on coleus is

The growths on the underside of the symptomatic leaves in a form of a brown downy mould are asexual organs, sporangia bearing sporangiophores which emerge from leaf stomata. Microscopic observations will show that they are consistent with the characteristics of a genus *Peronospora*. The first descriptions of sporangia and sporangiophores on basil and coleus that were confirmed by molecular determination were provided by Thines et al. in 2009 [28]. The sporangia of genus *Peronospora*

**6**

are spore-like structures and they act as conidia and germinate into a germ-tube when they are near a leaf stoma. Therefore, the use of synonym conidia, or simply spore, has become commonplace for sporangia.

Conidia are dark brown to olive in colour and pedicel is absent. They are rounded and egg-shaped with a length 24–29–30.8–33–36 μm on basil and 26–29–31.3–33–37 μm on coleus. They width are 20–23–24–26–29 μm on basil and 20–23–24.5–26–29 μm on coleus. Ratio of length and width is 1.1–1.2–1.29–1.4–1.5 on basil and 1.1–1.2–1.28–1.4–1.5 on coleus [28].

Sporangiophores are colourless (hyaline) with a long, straight trunk and monopodially with a length 270–300–400–520–680 μm on basil and 330–380–466–570– 650 μm on coleus [28]. Numbers of ramifications were 3–4–4.9–5–7 per sporophore on basil and 4–5–5.2–6–7 μm on coleus. Ultimate branchlets were in pairs, curved, longer one in length 13–18–20.6–26–31 μm on basil and 12–13–18–22–31 μm on coleus while the shorter one in length 3.8–7.7–9.80–10–15 μm on basil and 5.1–7.7– 10.7–13–17 μm on coleus. Ratio of longer to shorter branches is 1.3–1.8–2.25–2.7–4 on basil and 1.1–1.6–1.71–1.9–2.5 on coleus. Ultimate branches end dichotomically and tips (sterigmata) are acute to subacute on both, basil and coleus. Tips are bearing single sporangia.

The shortest sporangiophores were reported in Iran and were 130–290 μm (avg. 194 μm) long and branched two to five times [16]. The longest sporangiophores were recorded in Hungary, and they were in length of 416–784 μm (avg. 572 μm) and monopodially branched five to seven times [7].

There are two oospore detections published up to date, both from Israel, found in leaves of susceptible sweet basil cultivar 'Peri'. In 2013, Cohen et al. identified and described oospores as thick-walled, brown in colour, measuring of 46.2 ± 2.8 μm in diameter [34]. Oospores never occurred on the infected leaf surface, but inside the mesophyll [30]. In 2016, in walk-in tunnel experiments that simulated commercial production conditions, oospores were observed attached to the leaf surface, to older parts of the infection area, and also found to water washes of the leaf surface by Elad et al. [35]. Discovery of oospores suggests the potential for sexual reproduction, but little is known on *P. belbahrii* oospore formation or is it homothallic or heterothallic. Currently, only one mating type has been found [22], although it is already presumed that it is heterothallic [26, 36, 37]. The pathogenicity of oospores is investigated, but without positive infections [30, 38], and their role in the basil downy mildew epidemiology is not known.

#### **5. Disease cycle and dispersal**

The *P. belbahrii* thrives in warm, humid conditions and produce conidia that can infect in temperatures as low as 15°C (59°F) [26]. For example, downy mildew is present in Israeli basil-cropping regions where in the cooler season temperatures may reach minimum of 5–10°C at night and a maximum of 10–25°C during the day [35]. This corroborates with our observations. Pathogen can tolerate cold weather (10–15°C) but, like its host basil, cannot survive freezing winter temperatures at continental climate. Conidia cannot survive harsh winters and as pathogen is biotroph it needs living host. Therefore, in climates with harsh winters and with just one mating type of the *P. belbahrii* it can survive only on living plants in greenhouse production operations that produce basil year round. In mild winters and in warm, temperate regions where the host, basil will not freeze, the second overwintering inoculum are mycelium and conidia in infected plant buds, plant stems, leaf tissue and shoots. Congruently, the most devastating damage is often seen in warm and humid conditions, late summer and in greenhouses.

Most of *Peronospora* species can reside in soil as soil-borne oospores that are formed in leaf tissue and may overwinter in leaf litter or may be released into the soil as leaves decay and considered as soil-borne inoculum. Any movement of soil particles with soil-borne oospores inoculum can spread it from infected plants to non-infected ones. Although *Peronospora* species are biotrophs, they can survive without host as oil-borne oospores because they are in dormancy and can be viable for few years depending on species. Until now, there are no reports about *P. belbahrii* soil-borne oospores even in cases when oospores were detected inside the mesophyll of the leaves [30, 35]. Large-scale experiments were conducted to elucidate the pathogenicity of oospores to basil plants. Soil was infested with oospores (10 oospores/5 g of soil/well) and three to four basil seeds were planted in each well. Plants were grown until the four-leaf stage, but none of the 2000 plants that developed showed symptoms of downy mildew or sporulation of *P. belbahrii* [30]. Also, the experiments conducted in the Israeli walk-in tunnels lead to a conclusion that oospores are minimally affected by high temperature, and therefore the high temperature presumably did not affect pathogen survival [35].

The life cycle of *P. belbahrii* is initiated as abundantly produced air-borne conidia which can readily be spread by moist wind [37]. The conidia can be carried by rain drops, by wind, and can be splashed by rain to wet leaves near the ground. It does not need a vector for dispersal. Survivability of conidia, contrary to oospores, are strongly affected by temperature and duration of exposure so, a longer exposure period and higher temperature weakened the infection capacity of the conidia. Wetted-dried conidia lost their activity after 55 h at 25°C, 20 h at 30°C and 9 h at 40°C [30]. Therefore, conidia are short lived and viable just for few days so, they will endanger only susceptible host within the conidia dispersal area. McGrath conducted an experiment with field-grown basil at the Long Island Horticultural Research and Extension Center (LIHREC) in Riverhead, NY and considered the primary source of initial inoculum in this area to be long-distance wind dispersed conidia from affected plants [39] although the distance is not specified. The possibility to use frozen conidia as inoculum was also tested and those collected from infected leaves frozen for 3 months at −20°C or 2 years at −80°C retain high germination capability [30]. In other trial, frozen conidia germinated at 25% in contrast to nearly 90% germination rate of freshly harvested conidia [40]. Their germination was favoured between 5 and 15°C on water agar *in vitro*. Inoculation of basil plants with frozen or fresh conidia (3 × 104 mL<sup>−</sup><sup>1</sup> ) resulted in high disease severity 14 days post inoculation [40].

Sporulation occurs in moisture saturated atmosphere at an appropriate temperature and often during the night, in the dark and in chlorotic lesions 5–15 days old [41]. In controlled greenhouse experiments, sporulation occurs 6–7 days post inoculations [37]. The sporulation starts when pathogen biomass in the leaf mesophyll reached a certain threshold and complete within 8–12 h from onset of darkness in optimal conditions (saturated atmosphere at 18°C). During the first 6 h, hyaline sporophores are formed and as they emerge from stomata gradually become dichotomously. In the subsequent 5 h, dark spores are produced on the tips of the sporophore branchlets (sterigmata) [41]. The light strongly inhibits spore formation, but not sporophore development and emergence through leaf stomata. Yet, sporophores formed under the light are abnormal and unable to form spores. Cohen et al. in 2013 discovered that lightning during the second half of the night inhibits spore formation, and narrow band led illumination showed that red light (λmax 625 nm) was most inhibitory to spore formation comparing to blue light (λmax 440 nm) while in other oomycetes is quite the opposite [41]. They speculate that probably *P. belbahrii* has a different photoreceptor sensitive to red light. The sporulation is greater when the portion of carbohydrates in the leaf is higher [41].

**9**

occur [30].

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

The carbohydrates accumulating during the day are hydrolysed to hexoses during the first half of the night which pathogen uses for formation of conidia during the rest of the night [41]. Therefore, the greater the accumulation of carbohydrates in infected leaves during the daytime contributes to the greater sporulation in the following dark, wet period of the night. This all suggests that the sporulation terminates with necrosis of leaf which obstructs assimilation as plant cells die because of

Conidia germinate in 3–5 days into one or two germ tubes and infect plant tissues via a germ-tube which penetrates through leaf stomata [28, 36] and it takes 3 h [35]. Germ tubes rarely form an appressoria-like structures prior infection. Developing hypha grows into intercellular spaces within the leaf mesophyll, proliferate and eventually invaginate the host cell plant cells through special globuse structures called haustoria (a hallmark oomycete structure) for nutrient acquisition [37]. Further branching and spreading of this initial hypha lead to forming of a cushion of intercellular mycelia just below the stomata. From this cushion, sporangiophores arise and emerge through stomata on sterigmata bearing sporangia. Conidia are produced simultaneously and are carried by wind and rain to new infection sites of the same or different plant. Leaf wetness of at least 6 h is required for conidial infection [42, 43]. Under favourable conditions, sporulation progresses in the polycyclic disease cycle leading to an epidemic of downy mildew disease.

*P. belbahrii* is also a seed-borne pathogen. Detection of *P. belbahrii* in several commercially produced basil seed batches confirmed that the pathogen is seedborne [3, 28, 44]. It is considered that infected seed act as primary inoculum source in basil production, and is so far considered to be the most important way of this pathogen spreading as it can explain the rapid global spread of *P. belbahrii.* Great example for the spreading of *P. belbahrii* with seed transport and seed-marketing to long distances is that the biotype that was detected for the first time in US in 2007 was genetically identical to the one reported in Switzerland in 2001 [27]. Also, the disease occurrence in US Sonoma County in 2008 was connected with the origin of the used seed that was introduced from Italy. Investigation conducted by Farahani-Kofoet and Römer detected *P. belbahrii* on 80–90% of randomly selected commercial seed stocks [45] and assumed that *P. belbahrii* can be spread by transport and

On contaminated seeds, *P. belbahrii* has been found in form of conidia and oospore [38, 45]. Until now, *P. belbahrii* was not reported inside the basil seed or embryo. Based on their observations, Farahani-Kofoet and Römer concluded that *P. belbahrii* is able to survive for several years on seeds [45]. Generally, oospores of *Peronospora* species can also be formed on seeds and infect the emerging seedling. Their oospores germinate in a way similar to that described for conidia and the infection process is similar. Investigation of *P. belbahrii* oospore infection of basil seeds was conducted, but plants developed from seeds planted in soil infested with oospores were symptomless and sporulation characteristic for *P. belbahrii* did not

It has not yet been clarified whether the pathogen infects the seed deeply and systematically or is just a contaminant. In some European investigations, systemic infections in seeds and in different plant parts (leaves, stems) even in a symptomless plant have been detected [44, 45]. Novel investigation of seed transmission conducted in Israel showed that *P. belbahrii* is seed-borne but not seed-transmitted, as seeds produced by infected plants in the field can be externally contaminated with conidia that were embedded in the surface, but not entirely [46]. Further, plants grown in growth chambers until 5–6 leaf stage from contaminated seeds did not show any symptom of downy mildew and did not carry latent infection. Also, systemic infections were rarely seen in the field. They confirmed systemic spread

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

pathogen absorbed all nutrients from it.

marketing of seed stocks.

#### *Downy Mildew of Basil: A New Destructive Disease Worldwide DOI: http://dx.doi.org/10.5772/intechopen.91903*

*Plant Diseases-Current Threats and Management Trends*

plants with frozen or fresh conidia (3 × 104

14 days post inoculation [40].

Most of *Peronospora* species can reside in soil as soil-borne oospores that are formed in leaf tissue and may overwinter in leaf litter or may be released into the soil as leaves decay and considered as soil-borne inoculum. Any movement of soil particles with soil-borne oospores inoculum can spread it from infected plants to non-infected ones. Although *Peronospora* species are biotrophs, they can survive without host as oil-borne oospores because they are in dormancy and can be viable

*P. belbahrii* soil-borne oospores even in cases when oospores were detected inside the mesophyll of the leaves [30, 35]. Large-scale experiments were conducted to elucidate the pathogenicity of oospores to basil plants. Soil was infested with oospores (10 oospores/5 g of soil/well) and three to four basil seeds were planted in each well. Plants were grown until the four-leaf stage, but none of the 2000 plants that developed showed symptoms of downy mildew or sporulation of *P. belbahrii* [30]. Also, the experiments conducted in the Israeli walk-in tunnels lead to a conclusion that oospores are minimally affected by high temperature, and therefore

for few years depending on species. Until now, there are no reports about

the high temperature presumably did not affect pathogen survival [35].

The life cycle of *P. belbahrii* is initiated as abundantly produced air-borne conidia which can readily be spread by moist wind [37]. The conidia can be carried by rain drops, by wind, and can be splashed by rain to wet leaves near the ground. It does not need a vector for dispersal. Survivability of conidia, contrary to oospores, are strongly affected by temperature and duration of exposure so, a longer exposure period and higher temperature weakened the infection capacity of the conidia. Wetted-dried conidia lost their activity after 55 h at 25°C, 20 h at 30°C and 9 h at 40°C [30]. Therefore, conidia are short lived and viable just for few days so, they will endanger only susceptible host within the conidia dispersal area. McGrath conducted an experiment with field-grown basil at the Long Island Horticultural Research and Extension Center (LIHREC) in Riverhead, NY and considered the primary source of initial inoculum in this area to be long-distance wind dispersed conidia from affected plants [39] although the distance is not specified. The possibility to use frozen conidia as inoculum was also tested and those collected from infected leaves frozen for 3 months at −20°C or 2 years at −80°C retain high germination capability [30]. In other trial, frozen conidia germinated at 25% in contrast to nearly 90% germination rate of freshly harvested conidia [40]. Their germination was favoured between 5 and 15°C on water agar *in vitro*. Inoculation of basil

mL<sup>−</sup><sup>1</sup>

Sporulation occurs in moisture saturated atmosphere at an appropriate temperature and often during the night, in the dark and in chlorotic lesions 5–15 days old [41]. In controlled greenhouse experiments, sporulation occurs 6–7 days post inoculations [37]. The sporulation starts when pathogen biomass in the leaf mesophyll reached a certain threshold and complete within 8–12 h from onset of darkness in optimal conditions (saturated atmosphere at 18°C). During the first 6 h, hyaline sporophores are formed and as they emerge from stomata gradually become dichotomously. In the subsequent 5 h, dark spores are produced on the tips of the sporophore branchlets (sterigmata) [41]. The light strongly inhibits spore formation, but not sporophore development and emergence through leaf stomata. Yet, sporophores formed under the light are abnormal and unable to form spores. Cohen et al. in 2013 discovered that lightning during the second half of the night inhibits spore formation, and narrow band led illumination showed that red light (λmax 625 nm) was most inhibitory to spore formation comparing to blue light (λmax 440 nm) while in other oomycetes is quite the opposite [41]. They speculate that probably *P. belbahrii* has a different photoreceptor sensitive to red light. The sporulation is greater when the portion of carbohydrates in the leaf is higher [41].

) resulted in high disease severity

**8**

The carbohydrates accumulating during the day are hydrolysed to hexoses during the first half of the night which pathogen uses for formation of conidia during the rest of the night [41]. Therefore, the greater the accumulation of carbohydrates in infected leaves during the daytime contributes to the greater sporulation in the following dark, wet period of the night. This all suggests that the sporulation terminates with necrosis of leaf which obstructs assimilation as plant cells die because of pathogen absorbed all nutrients from it.

Conidia germinate in 3–5 days into one or two germ tubes and infect plant tissues via a germ-tube which penetrates through leaf stomata [28, 36] and it takes 3 h [35]. Germ tubes rarely form an appressoria-like structures prior infection. Developing hypha grows into intercellular spaces within the leaf mesophyll, proliferate and eventually invaginate the host cell plant cells through special globuse structures called haustoria (a hallmark oomycete structure) for nutrient acquisition [37]. Further branching and spreading of this initial hypha lead to forming of a cushion of intercellular mycelia just below the stomata. From this cushion, sporangiophores arise and emerge through stomata on sterigmata bearing sporangia. Conidia are produced simultaneously and are carried by wind and rain to new infection sites of the same or different plant. Leaf wetness of at least 6 h is required for conidial infection [42, 43]. Under favourable conditions, sporulation progresses in the polycyclic disease cycle leading to an epidemic of downy mildew disease.

*P. belbahrii* is also a seed-borne pathogen. Detection of *P. belbahrii* in several commercially produced basil seed batches confirmed that the pathogen is seedborne [3, 28, 44]. It is considered that infected seed act as primary inoculum source in basil production, and is so far considered to be the most important way of this pathogen spreading as it can explain the rapid global spread of *P. belbahrii.* Great example for the spreading of *P. belbahrii* with seed transport and seed-marketing to long distances is that the biotype that was detected for the first time in US in 2007 was genetically identical to the one reported in Switzerland in 2001 [27]. Also, the disease occurrence in US Sonoma County in 2008 was connected with the origin of the used seed that was introduced from Italy. Investigation conducted by Farahani-Kofoet and Römer detected *P. belbahrii* on 80–90% of randomly selected commercial seed stocks [45] and assumed that *P. belbahrii* can be spread by transport and marketing of seed stocks.

On contaminated seeds, *P. belbahrii* has been found in form of conidia and oospore [38, 45]. Until now, *P. belbahrii* was not reported inside the basil seed or embryo. Based on their observations, Farahani-Kofoet and Römer concluded that *P. belbahrii* is able to survive for several years on seeds [45]. Generally, oospores of *Peronospora* species can also be formed on seeds and infect the emerging seedling. Their oospores germinate in a way similar to that described for conidia and the infection process is similar. Investigation of *P. belbahrii* oospore infection of basil seeds was conducted, but plants developed from seeds planted in soil infested with oospores were symptomless and sporulation characteristic for *P. belbahrii* did not occur [30].

It has not yet been clarified whether the pathogen infects the seed deeply and systematically or is just a contaminant. In some European investigations, systemic infections in seeds and in different plant parts (leaves, stems) even in a symptomless plant have been detected [44, 45]. Novel investigation of seed transmission conducted in Israel showed that *P. belbahrii* is seed-borne but not seed-transmitted, as seeds produced by infected plants in the field can be externally contaminated with conidia that were embedded in the surface, but not entirely [46]. Further, plants grown in growth chambers until 5–6 leaf stage from contaminated seeds did not show any symptom of downy mildew and did not carry latent infection. Also, systemic infections were rarely seen in the field. They confirmed systemic spread

of mycelium in the basil plants which corroborated with previous finding [45]. Systemically infected plants remained stunt and produced no seeds. Therefore, the Israeli investigators postulated that seed infections and seed transmission may occur in Europe, as it was reported [44, 45], and other locations with wetter summers, especially under prolonged wetness periods at the flowering and seed production.

Both investigations, European and Israeli, confirmed that contaminated seeds can be harvested from symptomless, latently infected plants and also, that contaminated seeds can give symptomless, latently infected plants [44–46].

*Peronospora belbahrii* can also be spread through vegetative materials like contaminated plant cuttings, transplants and fresh leaves. Novel Israeli investigation showed that *P. belbahrii* is spread systematically in basil plants [46]. Mycelium has been found to grow acropetally to the stem apex and basipetally to the cotyledons and hypocotyl and laterally to the axillar buds but, mycelium has never reached the roots. Especially in young basil plants, this pathogen systemically runs through tissue and causes plant stunt and fail to produce seeds.

#### **6. Management of basil downy mildew**

The control methods of the downy mildew pathogen today involve fungicides, seed treatment and breeding for resistance. In the greenhouses, they can be augmented with physical measures: nocturnal illumination, ventilation and daytime solar heating. The last one is also suitable for net-houses [47].

Current control measures rely mainly on fungicide application. In conventionally produced basil, it can be controlled in a preventive program with conventional foliar fungicides. The efficient once are based on mefenoxam, azoxystrobin, cyazofamid, mandipropamid, fluopicolide and fenamidone [11, 12, 26, 34, 38]. There are also phosphorous acid fungicides which are in most cases labelled and allowed in greenhouses. The best control of 98% was achieved with preventive fungicide application, before symptoms occurred, on a weekly schedule [38].

The *P. belbahrii* developed mefenoxam-resistance within 1 year of use in Israel and was reported in 2013 [34]. It was also detected in Italy were mefenoxam (metalaxyl-M) plus copper has been the most widely used and effective product against *P. belbahrii*, since its registration on basil in Italy in 2004 [48]. As the systemic fungicides are prone to the resistance development, ingredients with different modes of action are needed [32]. The novel fungicides with extremely high efficacy against oomycete including *P. belbahrii* are oxathiapiprolin [30] and valifenalate [49]. Oxathiapiprolin acts at multiple stages of the pathogen's asexual life cycle at extremely low concentrations and due to translaminar and acropetally systemic movement, it protects treated leaves and new leaves as they emerge and grow. In *P. belbahrii*, it inhibits sporangia germination and curatively, it stops mycelial growth within the host plant before visible lesions occur and inhibits further lesion expansion, offering protection at 1 and 2 days post-infection [50]. It was found to be effective against mefenoxam-resistant biotypes as well [30]. But, as it is a single-site inhibitor and its target is the oxysterol binding protein, the resistance to oxathiapiprolin assume to be medium to high and resistance management is required [51]. The soil application of mixture of oxathiapiprolin and benthiavalicarb or their single application against *P. belbahrii* was tested. Application to the root of 1 mg active ingredient per plant in the field experiment provided durable protection of up to 4 weeks against *P. belbahrii* [52]. The mixture performed better than single applications of those two compounds suggesting a synergistic interaction between them. The valifenalate is also a single-site inhibitor and acts as the inhibitor of cellulose

**11**

s −1

surface was exposed to light [41].

draw on an early, fast and specific detecting test [45].

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

to prevent favourable conditions for disease development.

synthesis in the Oomycete plant pathogens [49]. The resistance to valifenalate is

In organic farming, conventional fungicides are not allowed, so neem oil, potassium bicarbonate and hydrogen dioxide can be used for protection only, but they do not give satisfied protection [38]. Organic fungicides are contacts and do not go into plant tissue where is the pathogen and they are not able to translocate to abaxial side of leaves where sporulation occur. Therefore, their performance is not commercially acceptable and as they provide limited to no control, including when applied twice weekly on a preventive schedule to a moderately resistant variety [12, 38, 39]. As alternative, there are some bio-products based on *Bacillus amyloliquefaciens*, *Streptomyces lydicus* and the extract of *Reynoutria sachalinensis* [38]. Organic production should be in protected conditions and better transplanting then seeding as pathogen-free seed is not available. In greenhouse, it is important

Certain cultural practices which create less optimal conditions for the pathogen can be helpful in reducing the amount of infection. Such practices include providing good soil drainage and good air circulation among plants. Increasing plant spacing in the field or greenhouse prevents the creation of high-humidity conditions on plant surfaces and can inhibit infection as *P. belbahrii* requires humidity for sporulation as well as free leaf moisture for infection. The humidity should be keeping below 85% and this is crucial. In the greenhouse, the use of plastic mulch and drip irrigation is recommended instead of bare ground and overhead irrigation. Effective measure for reducing ambient relative humidity and avoids vapour deposition of leaves surface is ventilation [47]. In some experiments, combining daytime solar heating with nocturnal illumination without fungicide applications showed to be an effective control in organic farming [30, 47]. High temperature is detrimental to the *P. belbahrii* and exposure of infected plant of 35–45°C for 6–9 h suppressed survival of conidia and mycelia [47]. Subsequently, solar heating has been used to cure plants. In Israel, solar energy was captured by closing greenhouse windows or covering the house with a transparent IR polyethylene sheet during sunny hours of the days: best is to use three consecutive daily exposures of 3–4 h starting at 8 am [47]. Solar heating should be conducted cautiously to avoid plant heat damage [47]. Ensuring light during the night, especially red light should prevent sporulation. The protective effect of nocturnal illumination was determined in laboratory and greenhouse trials; but in Israel, field trials (net-houses) also demonstrated that light can be successfully used to supress downy mildew in field-grown basil [41]. The inhibitory effect of incandescent or CW fluorescent light of 3.5 or 6 μmoles.m2

on sporulation was 100% on lower leaf surface even when only the upper leaf

The rapid global spread of the downy mildew may be related to transmission of *P. belbahrii* by infected seeds and/or trade of basil cuttings and plants with latent infection [3, 38, 45, 46]*.* Infested seeds are a great risk for spreading the pathogen by transport and seed-marketing to long distances. Implementation of seed-certification schemes to exclude seed batches infested with *P. belbahrii* from marketing would be of great value for both seed-producing companies and growers [45]. Therefore, improving seed production; developing and implementing seed testing, certification protocols, and standards for the basil seed industry and strict following of import restriction may have halted *P. belbahrii* [38, 45]*.* To limit the spread of the pathogen by seed shipments, it is crucial for breeders and growers to

Seed should be tested on the presence of *P. belbahrii* and for that purpose realtime PCR have been designed [3]*.* Belbahri et al. have designed a specific primer pair (Bas-F/Bas-R) based on sequences within the unique genomic ribosomal DNA

.

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

assumed to be low to medium risk [51].

*Plant Diseases-Current Threats and Management Trends*

production.

of mycelium in the basil plants which corroborated with previous finding [45]. Systemically infected plants remained stunt and produced no seeds. Therefore, the Israeli investigators postulated that seed infections and seed transmission may occur in Europe, as it was reported [44, 45], and other locations with wetter summers, especially under prolonged wetness periods at the flowering and seed

Both investigations, European and Israeli, confirmed that contaminated seeds can be harvested from symptomless, latently infected plants and also, that contami-

*Peronospora belbahrii* can also be spread through vegetative materials like contaminated plant cuttings, transplants and fresh leaves. Novel Israeli investigation showed that *P. belbahrii* is spread systematically in basil plants [46]. Mycelium has been found to grow acropetally to the stem apex and basipetally to the cotyledons and hypocotyl and laterally to the axillar buds but, mycelium has never reached the roots. Especially in young basil plants, this pathogen systemically runs through

The control methods of the downy mildew pathogen today involve fungicides, seed treatment and breeding for resistance. In the greenhouses, they can be augmented with physical measures: nocturnal illumination, ventilation and daytime

Current control measures rely mainly on fungicide application. In conventionally produced basil, it can be controlled in a preventive program with conventional foliar fungicides. The efficient once are based on mefenoxam, azoxystrobin, cyazofamid, mandipropamid, fluopicolide and fenamidone [11, 12, 26, 34, 38]. There are also phosphorous acid fungicides which are in most cases labelled and allowed in greenhouses. The best control of 98% was achieved with preventive fungicide

The *P. belbahrii* developed mefenoxam-resistance within 1 year of use in Israel and was reported in 2013 [34]. It was also detected in Italy were mefenoxam (metalaxyl-M) plus copper has been the most widely used and effective product against *P. belbahrii*, since its registration on basil in Italy in 2004 [48]. As the systemic fungicides are prone to the resistance development, ingredients with different modes of action are needed [32]. The novel fungicides with extremely high efficacy against oomycete including *P. belbahrii* are oxathiapiprolin [30] and valifenalate [49]. Oxathiapiprolin acts at multiple stages of the pathogen's asexual life cycle at extremely low concentrations and due to translaminar and acropetally systemic movement, it protects treated leaves and new leaves as they emerge and grow. In *P. belbahrii*, it inhibits sporangia germination and curatively, it stops mycelial growth within the host plant before visible lesions occur and inhibits further lesion expansion, offering protection at 1 and 2 days post-infection [50]. It was found to be effective against mefenoxam-resistant biotypes as well [30]. But, as it is a single-site inhibitor and its target is the oxysterol binding protein, the resistance to oxathiapiprolin assume to be medium to high and resistance management is required [51]. The soil application of mixture of oxathiapiprolin and benthiavalicarb or their single application against *P. belbahrii* was tested. Application to the root of 1 mg active ingredient per plant in the field experiment provided durable protection of up to 4 weeks against *P. belbahrii* [52]. The mixture performed better than single applications of those two compounds suggesting a synergistic interaction between them. The valifenalate is also a single-site inhibitor and acts as the inhibitor of cellulose

nated seeds can give symptomless, latently infected plants [44–46].

tissue and causes plant stunt and fail to produce seeds.

solar heating. The last one is also suitable for net-houses [47].

application, before symptoms occurred, on a weekly schedule [38].

**6. Management of basil downy mildew**

**10**

synthesis in the Oomycete plant pathogens [49]. The resistance to valifenalate is assumed to be low to medium risk [51].

In organic farming, conventional fungicides are not allowed, so neem oil, potassium bicarbonate and hydrogen dioxide can be used for protection only, but they do not give satisfied protection [38]. Organic fungicides are contacts and do not go into plant tissue where is the pathogen and they are not able to translocate to abaxial side of leaves where sporulation occur. Therefore, their performance is not commercially acceptable and as they provide limited to no control, including when applied twice weekly on a preventive schedule to a moderately resistant variety [12, 38, 39]. As alternative, there are some bio-products based on *Bacillus amyloliquefaciens*, *Streptomyces lydicus* and the extract of *Reynoutria sachalinensis* [38]. Organic production should be in protected conditions and better transplanting then seeding as pathogen-free seed is not available. In greenhouse, it is important to prevent favourable conditions for disease development.

Certain cultural practices which create less optimal conditions for the pathogen can be helpful in reducing the amount of infection. Such practices include providing good soil drainage and good air circulation among plants. Increasing plant spacing in the field or greenhouse prevents the creation of high-humidity conditions on plant surfaces and can inhibit infection as *P. belbahrii* requires humidity for sporulation as well as free leaf moisture for infection. The humidity should be keeping below 85% and this is crucial. In the greenhouse, the use of plastic mulch and drip irrigation is recommended instead of bare ground and overhead irrigation. Effective measure for reducing ambient relative humidity and avoids vapour deposition of leaves surface is ventilation [47]. In some experiments, combining daytime solar heating with nocturnal illumination without fungicide applications showed to be an effective control in organic farming [30, 47]. High temperature is detrimental to the *P. belbahrii* and exposure of infected plant of 35–45°C for 6–9 h suppressed survival of conidia and mycelia [47]. Subsequently, solar heating has been used to cure plants. In Israel, solar energy was captured by closing greenhouse windows or covering the house with a transparent IR polyethylene sheet during sunny hours of the days: best is to use three consecutive daily exposures of 3–4 h starting at 8 am [47]. Solar heating should be conducted cautiously to avoid plant heat damage [47]. Ensuring light during the night, especially red light should prevent sporulation. The protective effect of nocturnal illumination was determined in laboratory and greenhouse trials; but in Israel, field trials (net-houses) also demonstrated that light can be successfully used to supress downy mildew in field-grown basil [41]. The inhibitory effect of incandescent or CW fluorescent light of 3.5 or 6 μmoles.m<sup>2</sup> . s −1 on sporulation was 100% on lower leaf surface even when only the upper leaf surface was exposed to light [41].

The rapid global spread of the downy mildew may be related to transmission of *P. belbahrii* by infected seeds and/or trade of basil cuttings and plants with latent infection [3, 38, 45, 46]*.* Infested seeds are a great risk for spreading the pathogen by transport and seed-marketing to long distances. Implementation of seed-certification schemes to exclude seed batches infested with *P. belbahrii* from marketing would be of great value for both seed-producing companies and growers [45]. Therefore, improving seed production; developing and implementing seed testing, certification protocols, and standards for the basil seed industry and strict following of import restriction may have halted *P. belbahrii* [38, 45]*.* To limit the spread of the pathogen by seed shipments, it is crucial for breeders and growers to draw on an early, fast and specific detecting test [45].

Seed should be tested on the presence of *P. belbahrii* and for that purpose realtime PCR have been designed [3]*.* Belbahri et al. have designed a specific primer pair (Bas-F/Bas-R) based on sequences within the unique genomic ribosomal DNA (ITS1) and the primer pair generates a single fragment of approximately 134 base pairs [3]*.* The PCR method proved to be very sensitive for direct detection of *P. belbahrii* on seeds and plant samples [45]. The PCR detection limit of *P. belbahrii* in artificially infested seeds corresponded to the DNA amount of a single spore per seed (3.4 pg of *P. belbahrii* genomic DNA extracted from a pure spore suspension at a density of 103 spores ml<sup>−</sup><sup>1</sup> using 1 μl as a template) [45]. Further, with this PCR protocol, *P. belbahrii* can be detected with high sensitivity in leaves and stems as well and not only at seeds, even if symptoms are not evident. Finding that latent systemic infection can result in the contamination of basil seed and vice versa supported the necessity to implement PCR-based detection in a seed-certification scheme [45]. Pathogen-free seed is most important for greenhouse crops plantings not expected to be exposed to wind dispersed spores [53]. It should be emphasised that the presence of pathogen DNA in seeds does not implicate spontaneous disease outbreaks because the PCR test cannot assess the viability of spores as specific fragments can also be generated from DNA material of dead spores. Moreover, the disease inception and development depends on host-pathogen interaction and existing environmental conditions. Yet, PCR test allows the testing of high numbers of samples within a short time and rapidly gives accurate information on *P. belbahrii* presence. Considering all, it is recommendable to be admitted in seed-certification schemes for routine testing of seed materials in order to inhibit marketing of infested seeds. As the PCR test can be used for detection in different plant parts, it can also be used for evaluation of procedures to control the downy mildew pathogen.

Seed treatments and at-seedling fungicides may have the potential for the good start of basil production [38]*.* There are no fungicides labelled for use on seed, but the at-seedling fungicides are available although not labelled for use on basil seed. Mefenoxam can be applied at seedling into the soil in field growing basil [38]*.* In novel trails, the root treatment of mixture of oxathiapiprolin and benthiavalicarb given to the young seedlings, growing in the multi-cell trays in the nursery, may be effective basil downy mildew measure [52]. The only organic fungicide labelled for ground application is based on the extract of *Reynoutria sachalinensis* [38].

Novel, non-chemical basil seed treatment is steam-air treatment and USA seed companies start to implement it [53]. Steam-air treatment of basil seeds against seed-borne fungi was tested in 1997 in Australia [54]. Steam-air treatment at 54–58°C for 30 min was successful in two-cylinder configuration in the steam-air machine. They noticed that this configuration against sixth-cylinders configuration provides extra steam velocity that prevents basil seed clumping which happened because when wet basil seed easily stick together as they have very thick gelatinous coat. Therefore, basil seeds are not amenable to hot-water treatment as the seed clumping makes the seed challenging to handle [53, 54].

The cultivation of resistant sweet basil cultivars also can be efficient control strategy. Highly resistant cultivars will be especially welcome in organic farming. Earlier, some cultivars of red types, lemon and lime basil have been found less susceptible [9, 30, 31, 33, 39]. New resistant basil varieties started to be marketed in USA in 2018 [53]. The first commercially available resistant variety is Eleonora. The Rutgers University basil breeding program released Devotion, Obsession, Passion, and Thunderstruck. They are marketed by VDF Specialty Seeds. Organically produced seed is available and marketed like Prosperais (Johnny's Selected Seeds), Emma and Everleaf (aka Basil Pesto Party and M4828Z) [53].

That accurate monitoring can be of great importance in field growing as well as in protected conditions as instrumentation for optimization of plant protection measures was shown by USA monitoring programme. It started in 2009 by McGrath and augmented knowledge of basil downy mildew [38]. It is an online

**13**

**Author details**

Zagreb, Croatia

Snježana Topolovec-Pintarić\* and Katarina Martinko

guideline, in the context of global trade with seeds.

\*Address all correspondence to: tpintaric@agr.hr

provided the original work is properly cited.

Faculty of Agriculture, Department of Plant Pathology, University of Zagreb,

© 2020 The Author(s). Licensee IntechOpen. This chapter is 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,

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

spreadsheet program set-up in Google Docs accessible by anyone. Until 2019, each year a spreadsheet page was set up for anyone to log and view occurrence reports [53]. In 2019, a new website was launched with a mapping program that mapped reports by county plus information about basil downy mildew. The growers need to be educated to accurately based on first symptoms recognise the diseases on time. In greenhouses, monitoring should be on daily basis as downy mildew can develop

'*One touch of nature makes the whole world kin*' Shakespeare wrote and this is so valid for the pandemic downy mildew agent like *P. belbahrii* from sweet basil. The only way to deal with this pathogen is knowledge. To fill the existing knowledge gaps research into various aspects of the pathogen will be needed. The dual identification according to the morphology and ITS sequence analysis is recognised and implemented. It would be of the most value to investigate *P. belbahrii* sexual reproduction and to identify mating types and mechanisms of their compatibility if it is heterothallic. Further, valuable will be to obtain knowledge of the *P. belbahrii* natural distribution range because its present distribution is due to human activity and trades. More research into aspects of *P. belbahrii* physiology, asymptomatic infections and oospore role in epidemiology. The seed transmission still needs to be elucidated and the question of whether the pathogen penetrates into the seed should be answered. Because it has been already spread worldwide, the *P. belbahrii* is not on the quarantine lists; although, it will be beneficial to follow the quarantine

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

very quickly [38, 41].

**7. Conclusion**

spreadsheet program set-up in Google Docs accessible by anyone. Until 2019, each year a spreadsheet page was set up for anyone to log and view occurrence reports [53]. In 2019, a new website was launched with a mapping program that mapped reports by county plus information about basil downy mildew. The growers need to be educated to accurately based on first symptoms recognise the diseases on time. In greenhouses, monitoring should be on daily basis as downy mildew can develop very quickly [38, 41].

### **7. Conclusion**

*Plant Diseases-Current Threats and Management Trends*

at a density of 103 spores ml<sup>−</sup><sup>1</sup>

pathogen.

(ITS1) and the primer pair generates a single fragment of approximately 134 base pairs [3]*.* The PCR method proved to be very sensitive for direct detection of *P. belbahrii* on seeds and plant samples [45]. The PCR detection limit of *P. belbahrii* in artificially infested seeds corresponded to the DNA amount of a single spore per seed (3.4 pg of *P. belbahrii* genomic DNA extracted from a pure spore suspension

protocol, *P. belbahrii* can be detected with high sensitivity in leaves and stems as well and not only at seeds, even if symptoms are not evident. Finding that latent systemic infection can result in the contamination of basil seed and vice versa supported the necessity to implement PCR-based detection in a seed-certification scheme [45]. Pathogen-free seed is most important for greenhouse crops plantings not expected to be exposed to wind dispersed spores [53]. It should be emphasised that the presence of pathogen DNA in seeds does not implicate spontaneous disease outbreaks because the PCR test cannot assess the viability of spores as specific fragments can also be generated from DNA material of dead spores. Moreover, the disease inception and development depends on host-pathogen interaction and existing environmental conditions. Yet, PCR test allows the testing of high numbers of samples within a short time and rapidly gives accurate information on *P. belbahrii* presence. Considering all, it is recommendable to be admitted in seed-certification schemes for routine testing of seed materials in order to inhibit marketing of infested seeds. As the PCR test can be used for detection in different plant parts, it can also be used for evaluation of procedures to control the downy mildew

Seed treatments and at-seedling fungicides may have the potential for the good start of basil production [38]*.* There are no fungicides labelled for use on seed, but the at-seedling fungicides are available although not labelled for use on basil seed. Mefenoxam can be applied at seedling into the soil in field growing basil [38]*.* In novel trails, the root treatment of mixture of oxathiapiprolin and benthiavalicarb given to the young seedlings, growing in the multi-cell trays in the nursery, may be effective basil downy mildew measure [52]. The only organic fungicide labelled for

Novel, non-chemical basil seed treatment is steam-air treatment and USA seed companies start to implement it [53]. Steam-air treatment of basil seeds against seed-borne fungi was tested in 1997 in Australia [54]. Steam-air treatment at 54–58°C for 30 min was successful in two-cylinder configuration in the steam-air machine. They noticed that this configuration against sixth-cylinders configuration provides extra steam velocity that prevents basil seed clumping which happened because when wet basil seed easily stick together as they have very thick gelatinous coat. Therefore, basil seeds are not amenable to hot-water treatment as the seed

The cultivation of resistant sweet basil cultivars also can be efficient control strategy. Highly resistant cultivars will be especially welcome in organic farming. Earlier, some cultivars of red types, lemon and lime basil have been found less susceptible [9, 30, 31, 33, 39]. New resistant basil varieties started to be marketed in USA in 2018 [53]. The first commercially available resistant variety is Eleonora. The Rutgers University basil breeding program released Devotion, Obsession, Passion, and Thunderstruck. They are marketed by VDF Specialty Seeds. Organically produced seed is available and marketed like Prosperais (Johnny's Selected Seeds),

That accurate monitoring can be of great importance in field growing as well as in protected conditions as instrumentation for optimization of plant protection measures was shown by USA monitoring programme. It started in 2009 by McGrath and augmented knowledge of basil downy mildew [38]. It is an online

ground application is based on the extract of *Reynoutria sachalinensis* [38].

clumping makes the seed challenging to handle [53, 54].

Emma and Everleaf (aka Basil Pesto Party and M4828Z) [53].

using 1 μl as a template) [45]. Further, with this PCR

**12**

'*One touch of nature makes the whole world kin*' Shakespeare wrote and this is so valid for the pandemic downy mildew agent like *P. belbahrii* from sweet basil. The only way to deal with this pathogen is knowledge. To fill the existing knowledge gaps research into various aspects of the pathogen will be needed. The dual identification according to the morphology and ITS sequence analysis is recognised and implemented. It would be of the most value to investigate *P. belbahrii* sexual reproduction and to identify mating types and mechanisms of their compatibility if it is heterothallic. Further, valuable will be to obtain knowledge of the *P. belbahrii* natural distribution range because its present distribution is due to human activity and trades. More research into aspects of *P. belbahrii* physiology, asymptomatic infections and oospore role in epidemiology. The seed transmission still needs to be elucidated and the question of whether the pathogen penetrates into the seed should be answered. Because it has been already spread worldwide, the *P. belbahrii* is not on the quarantine lists; although, it will be beneficial to follow the quarantine guideline, in the context of global trade with seeds.

### **Author details**

Snježana Topolovec-Pintarić\* and Katarina Martinko Faculty of Agriculture, Department of Plant Pathology, University of Zagreb, Zagreb, Croatia

\*Address all correspondence to: tpintaric@agr.hr

© 2020 The Author(s). Licensee IntechOpen. This chapter is 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.

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[11] Zhang S, Mersha Z, Roberts PD, Raid R. Downy Mildew of Basil in South Florida. Gainesville: University of Florida, IFAS Extension; 2009. Doc. PP271. Available from: http://edis. ifas.ufl.edu/pdffiles/PP/PP27100.pdf [Accessed: 15 September 2019]

[12] McGrath MT. Expect and Prepare for Downy Mildew in Basil. Ithaca, NY: Department of Plant Pathology, Cornell University; 2019. Available from: http:// vegetablemdonline.ppath.cornell. edu/NewsArticles/BasilDowny.html [Accessed: 24 November 2019]

[13] Saude C, Westerveld S, Filotas M, McDonald MR. First report of downy mildew caused by *Peronospora belbahrii* on Basil (*Ocimum* spp.) in Ontario. Plant Diseases. 2013;**97**(9):1248. DOI: 10.1094/PDIS-01-13-0026-PDN

[14] Ronco L, Rollan C, Choi YJ, Shin HD. Downy mildew of sweet basil (*Ocimum basilicum* ) caused by *Peronospora* sp. in Argentina. Plant Pathology. 2009;**58**:395. DOI: 10.1111/j.1365-3059.2008.02006.x

**15**

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

[23] Grice K, Sun G, Trevorrow P. Basil

Downy Mildew Management Options—Is it Seedborne? Agri-Science Queensland Innovation Opportunity. Brisbane: Department of Agriculture and Fisheries; 2018. Available from: http://era.daf.qld.gov. au/id/eprint/6386/7/H%26FS%20 -%20Basil%20Downy%20Mildew%20

%28Grice%29%20FINAL.pdf [Accessed: 18 November 2019]

Cruciol GCD, de Pieri C, Dovigo LH, Pavan M, et al. First Report of downy mildew on coleus (*Plectranthus* spp.) caused by *Peronospora belbahrii* sensu lato in Brazil. Plant Disease. 2020;**104**(1):294. DOI: 10.1094/

[24] Gorayeb E, Pieroni LP,

PDIS-07-19-1551-PDN

[25] Novak A, Sever Z, Ivić D, Čajkulić AM. Plamenjača Bosiljka (*Peronospora belbahrii*)—Destruktivna bolest u proizvodnji bosiljka, Hrvatski centar za poljoprivredu, hranu i selo— Zavod za zaštitu bilja. Glasilo Biljne

Zaštite. 2016;**16**(6):544-547

[26] Grabowski M. Basil Downy Mildew *Peronospora belbahrii*. Saint Paul: Regents of the University of Minnesota, University of Minnesota Extension; 2012. Available from: http:// www.extension.umn.edu/garden/ yard-garden/vegetables/basil-downymildew/docs/basil-downy-mildew-pub. pdf [Accessed: 06 September 2019]

[27] Roberts PD, Raid RN, Harmon PF,

Jordan SA, Palmateer AJ. First report of Downy Mildew caused by a *Peronospora* sp. on basil in Florida and the United States. Plant Disease. 2009;**93**:199. DOI: 10.1094/

[28] Thines M, Telle S, Ploch S, Runge F. Identity of the downy

mildew pathogens of basil, coleus, and sage with implications for quarantine measures. Mycological Research. 2009;**113**(5):532-540. DOI: 10.1016/j.

PDIS-93-2-0199B

mycres.2008.12.005

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

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Lefort F. First report of downy mildew caused by *Peronospora sp.* on basil in Northern Iran. Journal of Plant Pathology. 2007;**89**(3):S70

[17] Ito Y, Takeuchi T, Matsushita Y, Chikuo Y, Satou M. Downy mildew of coleus caused by *Peronospora belbahrii* in Japan. Journal of General Plant Pathology. 2015;**81**(4):328-330. DOI:

[18] Chen CH, Huang JH, Hsieh TF. First report of *Peronospora belbahrii* causing downy mildew on basil in Taiwan. Plant Pathology Bulletin. 2010;**19**:177-180

[19] Cohen Y, Galperin M, Vaknin M, Ben-Naim Y, Rubin AE, Silverman D, et al. Downy mildew in basil, a new disease in Israel. Phytoparasitica.

[20] Kong XY, Wang S, Wan SL, Xiao CL, Luo F, Liu Y. First report of downy mildew on basil (*Ocimum basilicum*) in China. Plant Disease. 2015;**99**:1642. DOI: 10.1094/ PDIS-01-15-0077-PDN

[21] Hu B, Li Z, Hu M, Sun H, Zheng J, Diao Y. Outbreak of downy mildew caused by *Peronospora belbahrii* on *Ocimum basilicum* var. polosum in China. New Disease Reports. 2018;**37**:1. DOI: 10.5197/j.2044-0588.2018.037.001

[22] Choi YJ, Choi IY, Lee KJ, Shin H. First report of downy mildew caused by *Peronospora belbahrii* on sweet basil (*Ocimum basilicum*) in Korea. Plant Disease. 2016;**100**(11):2335. DOI: 10.1094/PDIS-05-16-0771-PDN

10.1007/s10327-015-0601-3

2013;**41**(4):458

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10.1111/jph.12391

[16] Khateri H, Calmin G, Moarrefzadeh N, Belbahri L, *Downy Mildew of Basil: A New Destructive Disease Worldwide DOI: http://dx.doi.org/10.5772/intechopen.91903*

[15] Romero M, Amador BM, Picazo LS, Nieto-Gariby A. First report of *Peronospora belbahrii* on sweet basil in Baja California Sur Mexico. Journal of Phytopathology. 2015;**4**:1-5. DOI: 10.1111/jph.12391

[16] Khateri H, Calmin G, Moarrefzadeh N, Belbahri L, Lefort F. First report of downy mildew caused by *Peronospora sp.* on basil in Northern Iran. Journal of Plant Pathology. 2007;**89**(3):S70

[17] Ito Y, Takeuchi T, Matsushita Y, Chikuo Y, Satou M. Downy mildew of coleus caused by *Peronospora belbahrii* in Japan. Journal of General Plant Pathology. 2015;**81**(4):328-330. DOI: 10.1007/s10327-015-0601-3

[18] Chen CH, Huang JH, Hsieh TF. First report of *Peronospora belbahrii* causing downy mildew on basil in Taiwan. Plant Pathology Bulletin. 2010;**19**:177-180

[19] Cohen Y, Galperin M, Vaknin M, Ben-Naim Y, Rubin AE, Silverman D, et al. Downy mildew in basil, a new disease in Israel. Phytoparasitica. 2013;**41**(4):458

[20] Kong XY, Wang S, Wan SL, Xiao CL, Luo F, Liu Y. First report of downy mildew on basil (*Ocimum basilicum*) in China. Plant Disease. 2015;**99**:1642. DOI: 10.1094/ PDIS-01-15-0077-PDN

[21] Hu B, Li Z, Hu M, Sun H, Zheng J, Diao Y. Outbreak of downy mildew caused by *Peronospora belbahrii* on *Ocimum basilicum* var. polosum in China. New Disease Reports. 2018;**37**:1. DOI: 10.5197/j.2044-0588.2018.037.001

[22] Choi YJ, Choi IY, Lee KJ, Shin H. First report of downy mildew caused by *Peronospora belbahrii* on sweet basil (*Ocimum basilicum*) in Korea. Plant Disease. 2016;**100**(11):2335. DOI: 10.1094/PDIS-05-16-0771-PDN

[23] Grice K, Sun G, Trevorrow P. Basil Downy Mildew Management Options—Is it Seedborne? Agri-Science Queensland Innovation Opportunity. Brisbane: Department of Agriculture and Fisheries; 2018. Available from: http://era.daf.qld.gov. au/id/eprint/6386/7/H%26FS%20 -%20Basil%20Downy%20Mildew%20 %28Grice%29%20FINAL.pdf [Accessed: 18 November 2019]

[24] Gorayeb E, Pieroni LP, Cruciol GCD, de Pieri C, Dovigo LH, Pavan M, et al. First Report of downy mildew on coleus (*Plectranthus* spp.) caused by *Peronospora belbahrii* sensu lato in Brazil. Plant Disease. 2020;**104**(1):294. DOI: 10.1094/ PDIS-07-19-1551-PDN

[25] Novak A, Sever Z, Ivić D, Čajkulić AM. Plamenjača Bosiljka (*Peronospora belbahrii*)—Destruktivna bolest u proizvodnji bosiljka, Hrvatski centar za poljoprivredu, hranu i selo— Zavod za zaštitu bilja. Glasilo Biljne Zaštite. 2016;**16**(6):544-547

[26] Grabowski M. Basil Downy Mildew *Peronospora belbahrii*. Saint Paul: Regents of the University of Minnesota, University of Minnesota Extension; 2012. Available from: http:// www.extension.umn.edu/garden/ yard-garden/vegetables/basil-downymildew/docs/basil-downy-mildew-pub. pdf [Accessed: 06 September 2019]

[27] Roberts PD, Raid RN, Harmon PF, Jordan SA, Palmateer AJ. First report of Downy Mildew caused by a *Peronospora* sp. on basil in Florida and the United States. Plant Disease. 2009;**93**:199. DOI: 10.1094/ PDIS-93-2-0199B

[28] Thines M, Telle S, Ploch S, Runge F. Identity of the downy mildew pathogens of basil, coleus, and sage with implications for quarantine measures. Mycological Research. 2009;**113**(5):532-540. DOI: 10.1016/j. mycres.2008.12.005

**14**

*Plant Diseases-Current Threats and Management Trends*

downy mildew caused by *Peronospora belbahrii* on sweet basil (*Ocimum basilicum*) in Cyprus. Plant Disease.

2014;**98**:283. DOI: 10.1094/ PHYTO-02-15-0032-FI

PDIS-10-15-1120-PDN

[9] Rivera Y, Salgado-Salazar C, Windham AS, Crouch JA. Downy mildew on Coleus (*Plectranthus scutellarioides*) caused by *Peronospora belbahrii* sensu lato in Tennessee. Plant Disease. 2016;**100**:655. DOI: 10.1094/

[10] Gómez Tenorio MA, Lupión Rodríguez B, Boix Ruiz A, Ruiz Olmos C, Moreno Díaz A, Marín Guirao JI, et al. El mildiu nueva enfermedad de la albahaca en España. Phytoma España. 2016;**282**:48-52

[11] Zhang S, Mersha Z, Roberts PD, Raid R. Downy Mildew of Basil in South Florida. Gainesville: University of Florida, IFAS Extension; 2009. Doc. PP271. Available from: http://edis. ifas.ufl.edu/pdffiles/PP/PP27100.pdf [Accessed: 15 September 2019]

[12] McGrath MT. Expect and Prepare for Downy Mildew in Basil. Ithaca, NY: Department of Plant Pathology, Cornell University; 2019. Available from: http:// vegetablemdonline.ppath.cornell. edu/NewsArticles/BasilDowny.html [Accessed: 24 November 2019]

[13] Saude C, Westerveld S, Filotas M, McDonald MR. First report of downy mildew caused by *Peronospora belbahrii* on Basil (*Ocimum* spp.) in Ontario. Plant Diseases. 2013;**97**(9):1248. DOI: 10.1094/PDIS-01-13-0026-PDN

[14] Ronco L, Rollan C, Choi YJ, Shin HD. Downy mildew of sweet basil (*Ocimum basilicum* ) caused by *Peronospora* sp. in Argentina. Plant Pathology. 2009;**58**:395. DOI: 10.1111/j.1365-3059.2008.02006.x

[1] Hansford CG. Annual report of the mycologist. Review of Applied

[2] Hansford CG. Annual report of the mycologist. Review of Applied

[3] Belbahrii L, Calmin G, Pawlowski J, Lefort F. Phylogenetic analysis and real time PCR detection of a presumably undescribed *Peronospora* species on sweet basil and sage. EPPO Mycological Research. 2005;**109**:1276-1287. DOI:

[4] Garibaldi A, Minuto A, Gullino ML. First report of downy mildew caused by *Peronospora* sp. on basil (*Ocimum basilicum*) in France. Plant Disease. 2005;**89**(6):683. DOI: 10.1094/

[5] Webb K, Sansford C, MacLeod A, Matthews-Berry S. Rapid Assessment of the Need for a Detailed Pest Risk Analysis for *Peronospora belbahrii*. UK Risk Register Details for *Peronospora belbahrii*. London: UK Plant Health Risk Register (DEFRA); 2012. Available from: https://secure.fera. defra.gov.uk/phiw/riskRegister/ downloadExternalPra.cfm?id=3901 [Accessed: 06 November 2019]

[6] Henricot B, Denton J, Scrace J, Barnes AV, Lane CR. *Peronospora belbahrii* causing downy mildew disease on *Agastache* in the UK: A new host and location for the pathogen. New Disease Reports. 2009;**20**:26. DOI: 10.1111/j.1365-3059.2010.02264.x

[7] Nagy G, Horváth A. Occurrence of downy mildew caused by *Peronospora belbahrii* on sweet basil in Hungary. Plant Disease. 2011;**95**(8):1034. DOI:

[8] Kanetis L, Vasiliou A, Neophytou G, Samouel S, Tsaltas D. First report of

10.1094/PDIS-04-11-0329

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PD-89-0683C

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[30] Cohen Y, Ben Naim Y, Falach L, Avia E. Epidemiology of basil downy mildew. Phytopathology. 2017;**107**:1149-1160. DOI: 10.1094/ PHYTO-01-17-0017-FI

[31] Harlan B, Linderman S, Hyatt L, Hausbeck M. Research gives clues for preventing coleus downy mildew. Greenhouse Grower. 2015. Available from: https://www.greenhousegrower. com/production/crop-inputs/researchgives-clues-for-preventing-coleusdowny-mildew/[Accessed: 16 October 2019]

[32] Farr DF, Rossman AY. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA; 2015. Online Publication. Available from: http://nt.ars-grin.gov/ fungaldatabases/ [Accessed: 01 October 2019]

[33] Babadoost M. Downy mildew of basil. Department of Crop Science, University of Illinois. RPD No. 1216. 2016. Available from: http://extension. cropsciences.illinois.edu/fruitveg/ pdfs/1216.pdf [Accessed: 12 September 2019]

[34] Cohen Y, Vaknin M, Ben-Naim Y, Rubin AE, Galperin M, Silverman D, et al. First report of the occurrence and resistance to mefenoxam of *Peronospora belbahrii*, causal agent of downy mildew of basil (*Ocimum basilicum*) in Israel. Plant Disease. 2013;**97**(5):692. DOI: 10.1094/PDIS-12-12-1126-PDN

[35] Elad Y, Omer C, Nisan Z, Harari D, Goren H, Adler U, et al. Passive heat treatment of sweet basil crops

suppresses *Peronospora belbahrii* downy mildew. The Annals of Applied Biology. 2016;**168**:373-389. DOI: 10.1111/ aab.12269

[36] Zhang G, Thompson A, Schisler D, Johnson ET. Characterization of the infection process by *Peronospora belbahrii* on basil by scanning electron microscopy. Heliyon. 2019;**5**:e01117. DOI: 10.1016/j.heliyon.2019.e01117

[37] Pyne RM. Introgression of genetic resistance to downy mildew (*Peronospora belbahrii*) in a non-model plant species, sweet basil (*Ocimum basilicum*) [Thesis]. New Jersey, USA: Graduate School-New Brunswick Rutgers, The State University of New Jersey; 2017

[38] Wyenandt CA, Simon JE, Pyne RM, Homa K, McGrath MT, Zhang S, et al. Basil downy mildew (*Peronospora belbahrii*): Discoveries and challenges relative to its control. Disease Control and Pest Management. 2015;**105**(7):885-894. DOI: 10.1094/ PHYTO-02-15-0032-FI

[39] McGrath MT, LaMarsh KA. Evaluation of organic and conventional fungicide programs for downy mildew in basil. Plant Disease Management Reports. 2015;**9**:V026. DOI: 10.1094/ PHYTO-02-15-0032-FI

[40] Farahani-Kofoet D, Römero P, Grosch R. Selecting basil genotypes with resistance against downy mildew. Scientia Horticulturae. 2014;**179**(24):248-255. DOI: 10.1016/j. scienta.2014.09.036

[41] Cohen Y, Vaknin M, Ben-Naim Y, Rubin AE. Light suppresses sporulation and epidemics of *Peronospora belbahrii*. PLoS One. 2013;**8**(11):e81282. DOI: 10.1371/journal.pone.0081282

[42] Garibaldi A, Bertetti D, Gullino ML. Effect of leaf wetness duration

**17**

*Downy Mildew of Basil: A New Destructive Disease Worldwide*

Pathology. 2010;**11**(2):227-243. DOI: 10.1111/j.1364-3703.2009.00604.x

[50] Cohen Y, Rubin AE, Galperin M. Oxathiapiprolin-based fungicides provide enhanced control of tomato late blight induced by mefenoxaminsensitive *Phytophthora infestans*. PLoS One. 2018;**13**(9):e0204523. DOI:

[51] FRAC Code List ©\*. Fungal Control Agents Sorted by Cross Resistance Pattern and Mode of Action (Including

10.1371/journal.pone.0204523

FRAC Code Numbering). 2019. Available from: https://www.frac.info/ docs/default-source/publications/ frac-code-list/frac-code-list-2019-fina le0af2a2c512362eb9a1eff00004acf5d. pdf?sfvrsn=7d8c489a\_2 [Accessed: 25

[52] Cohen Y. Root treatment with oxathiapiprolin, benthiavalicarb or their mixture provides prolonged systemic protection against oomycete

[53] McGrath MT. Expect and Prepare for Downy Mildew in Basil. 2019. Available from: http://blogs.cornell. edu/livegpath/extension/basildowny-mildew/where-in-the-usa-isbasildowny-mildew/ [Accessed: 16

foliar pathogens. PLoS One. 2020;**15**(1):e0227556. DOI: 10.1371/

[54] Mebalds M, Henderson B, Hepworth G. Development of Steam-Air Treatment to Control Seed-Borne Diseases of Vegetables and Flowers. HRDC Project No. NY536. Gordon, Australia: Horticultural Research and Development Corporation; 1997. Available from: https://www. greenlifeindustry.com.au/Attachment? Action=Download&Attachment\_ id=1773 [Accessed: 16 December 2019]

journal.pone.0227556

December 2019]

November 2019]

*DOI: http://dx.doi.org/10.5772/intechopen.91903*

and temperature on infection of downy mildew (*Peronospora* sp.) of basil. Journal of Plant Diseases and Protection. 2007;**114**:6-8. DOI: 10.1007/

[43] Cohen Y, Ben-Naim Y. Nocturnal fanning suppresses downy mildew epidemics in sweet basil. PLoS One. 2016;**11**:e0155330. DOI: 10.1371/journal.

[44] Garibaldi A, Minuto G, Bertetti D, Gullino ML. Seed transmission of *Peronospora* sp. of basil. Journal of Plant Diseases and Protection.

[45] Farahani-Kofoet RD, Römer P, Grosch R. Systemic spread of downy mildew in basil plants and detection of the pathogen in seed and plant samples. Mycological Progress. 2012;**11**:961-966. DOI: 10.1007/s11557-012-0816-z

[46] Falach-Block L, Ben-Naim Y, Cohen Y. Investigation of seed transmission in *Peronospora belbahrii*: The causal agent of basil downy mildew. Agronomy. 2019;**9**(205):1-11. DOI:

[47] Cohen Y, Rubin AE. Daytime solar heating controls downy mildew *Peronospora belbahrii* in sweet basil. PLoS One. 2015;**10**(5):e0126103. DOI:

10.1371/journal.pone.0126103

[48] Collina M, Merighi M, Turan C, Pirondi A, Minuto G, Brunelli A. First report of resistance of *Peronospora belbahrii*, causal agent of downy mildew of basil, to Mefenoxam in Italy. Plant Disease. 2016;**100**(8):1787. DOI: 10.1094/PDIS-02-16-0237-PDN

[49] Blum M, Boehler M, Randall E, Young V, Csukai M, Kraus S, et al. Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, *Phytophthora infestans*. Molecular Plant

10.3390/agronomy9040205

BF03356196

pone.0155330

2004;**111**:465-469

*Downy Mildew of Basil: A New Destructive Disease Worldwide DOI: http://dx.doi.org/10.5772/intechopen.91903*

and temperature on infection of downy mildew (*Peronospora* sp.) of basil. Journal of Plant Diseases and Protection. 2007;**114**:6-8. DOI: 10.1007/ BF03356196

*Plant Diseases-Current Threats and Management Trends*

suppresses *Peronospora belbahrii* downy mildew. The Annals of Applied Biology.

[36] Zhang G, Thompson A, Schisler D, Johnson ET. Characterization of the infection process by *Peronospora belbahrii* on basil by scanning electron microscopy. Heliyon. 2019;**5**:e01117. DOI: 10.1016/j.heliyon.2019.e01117

2016;**168**:373-389. DOI: 10.1111/

[37] Pyne RM. Introgression of genetic resistance to downy mildew (*Peronospora belbahrii*) in a non-model plant species, sweet basil (*Ocimum basilicum*) [Thesis]. New Jersey, USA: Graduate School-New Brunswick Rutgers, The State University of New

[38] Wyenandt CA, Simon JE, Pyne RM, Homa K, McGrath MT, Zhang S, et al. Basil downy mildew (*Peronospora belbahrii*): Discoveries and challenges relative to its control. Disease Control and Pest Management. 2015;**105**(7):885-894. DOI: 10.1094/

PHYTO-02-15-0032-FI

PHYTO-02-15-0032-FI

scienta.2014.09.036

[39] McGrath MT, LaMarsh KA.

[40] Farahani-Kofoet D, Römero P, Grosch R. Selecting basil genotypes with resistance against downy mildew. Scientia Horticulturae. 2014;**179**(24):248-255. DOI: 10.1016/j.

[41] Cohen Y, Vaknin M, Ben-Naim Y, Rubin AE. Light suppresses sporulation and epidemics of *Peronospora belbahrii*. PLoS One. 2013;**8**(11):e81282. DOI: 10.1371/journal.pone.0081282

[42] Garibaldi A, Bertetti D, Gullino ML.

Effect of leaf wetness duration

Evaluation of organic and conventional fungicide programs for downy mildew in basil. Plant Disease Management Reports. 2015;**9**:V026. DOI: 10.1094/

aab.12269

Jersey; 2017

[29] Harlan BR, Hausbeck MK. Diseases of coleus. In: McGovern R, Elmer W, editors. Handbook of Florists' Crops

Diseases. Handbook of Plant Disease Management. Cham: Springer; 2018. pp. 911-925. DOI: 10.1007/978-3-319-39670-5\_32

mildew. Phytopathology.

PHYTO-01-17-0017-FI

2019]

2019]

2019]

[30] Cohen Y, Ben Naim Y, Falach L, Avia E. Epidemiology of basil downy

2017;**107**:1149-1160. DOI: 10.1094/

[31] Harlan B, Linderman S, Hyatt L, Hausbeck M. Research gives clues for preventing coleus downy mildew. Greenhouse Grower. 2015. Available from: https://www.greenhousegrower. com/production/crop-inputs/researchgives-clues-for-preventing-coleusdowny-mildew/[Accessed: 16 October

[32] Farr DF, Rossman AY. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA; 2015. Online Publication. Available from: http://nt.ars-grin.gov/ fungaldatabases/ [Accessed: 01 October

[33] Babadoost M. Downy mildew of basil. Department of Crop Science, University of Illinois. RPD No. 1216. 2016. Available from: http://extension. cropsciences.illinois.edu/fruitveg/ pdfs/1216.pdf [Accessed: 12 September

[34] Cohen Y, Vaknin M, Ben-Naim Y, Rubin AE, Galperin M, Silverman D, et al. First report of the occurrence and resistance to mefenoxam of *Peronospora belbahrii*, causal agent of downy mildew of basil (*Ocimum basilicum*) in Israel. Plant Disease. 2013;**97**(5):692. DOI: 10.1094/PDIS-12-12-1126-PDN

[35] Elad Y, Omer C, Nisan Z, Harari D, Goren H, Adler U, et al. Passive heat treatment of sweet basil crops

**16**

[43] Cohen Y, Ben-Naim Y. Nocturnal fanning suppresses downy mildew epidemics in sweet basil. PLoS One. 2016;**11**:e0155330. DOI: 10.1371/journal. pone.0155330

[44] Garibaldi A, Minuto G, Bertetti D, Gullino ML. Seed transmission of *Peronospora* sp. of basil. Journal of Plant Diseases and Protection. 2004;**111**:465-469

[45] Farahani-Kofoet RD, Römer P, Grosch R. Systemic spread of downy mildew in basil plants and detection of the pathogen in seed and plant samples. Mycological Progress. 2012;**11**:961-966. DOI: 10.1007/s11557-012-0816-z

[46] Falach-Block L, Ben-Naim Y, Cohen Y. Investigation of seed transmission in *Peronospora belbahrii*: The causal agent of basil downy mildew. Agronomy. 2019;**9**(205):1-11. DOI: 10.3390/agronomy9040205

[47] Cohen Y, Rubin AE. Daytime solar heating controls downy mildew *Peronospora belbahrii* in sweet basil. PLoS One. 2015;**10**(5):e0126103. DOI: 10.1371/journal.pone.0126103

[48] Collina M, Merighi M, Turan C, Pirondi A, Minuto G, Brunelli A. First report of resistance of *Peronospora belbahrii*, causal agent of downy mildew of basil, to Mefenoxam in Italy. Plant Disease. 2016;**100**(8):1787. DOI: 10.1094/PDIS-02-16-0237-PDN

[49] Blum M, Boehler M, Randall E, Young V, Csukai M, Kraus S, et al. Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, *Phytophthora infestans*. Molecular Plant

Pathology. 2010;**11**(2):227-243. DOI: 10.1111/j.1364-3703.2009.00604.x

[50] Cohen Y, Rubin AE, Galperin M. Oxathiapiprolin-based fungicides provide enhanced control of tomato late blight induced by mefenoxaminsensitive *Phytophthora infestans*. PLoS One. 2018;**13**(9):e0204523. DOI: 10.1371/journal.pone.0204523

[51] FRAC Code List ©\*. Fungal Control Agents Sorted by Cross Resistance Pattern and Mode of Action (Including FRAC Code Numbering). 2019. Available from: https://www.frac.info/ docs/default-source/publications/ frac-code-list/frac-code-list-2019-fina le0af2a2c512362eb9a1eff00004acf5d. pdf?sfvrsn=7d8c489a\_2 [Accessed: 25 November 2019]

[52] Cohen Y. Root treatment with oxathiapiprolin, benthiavalicarb or their mixture provides prolonged systemic protection against oomycete foliar pathogens. PLoS One. 2020;**15**(1):e0227556. DOI: 10.1371/ journal.pone.0227556

[53] McGrath MT. Expect and Prepare for Downy Mildew in Basil. 2019. Available from: http://blogs.cornell. edu/livegpath/extension/basildowny-mildew/where-in-the-usa-isbasildowny-mildew/ [Accessed: 16 December 2019]

[54] Mebalds M, Henderson B, Hepworth G. Development of Steam-Air Treatment to Control Seed-Borne Diseases of Vegetables and Flowers. HRDC Project No. NY536. Gordon, Australia: Horticultural Research and Development Corporation; 1997. Available from: https://www. greenlifeindustry.com.au/Attachment? Action=Download&Attachment\_ id=1773 [Accessed: 16 December 2019]

**19**

**Chapter 2**

**Abstract**

**1. Introduction**

Nanophytovirology: An Emerging

Nanotechnology positions as a new armament in our collection against the increasing challenges in disease management and plant/human health. The application of nanotechnology in plant/human disease administration, diagnosis, and genetic transformations is still in its early stages. Apart from the scope of this chapter, there is also a mounting collection of new tools and techniques where nanoparticles are employed as delivery vehicles for genetic material in plants. Due to their nanoscale dimensions, nanoparticles may knockout virus particles and thus may open a novel arena of virus control in plants/humans. Our aim is to enlighten and enthuse researchers about the swiftly expanding prospects of nanotechnology

**Keywords:** nanoparticles, plant pathology, human pathology, virology, disease diagnosis, disease management, plant protection, case study

Food security has always been the principal apprehension for mankind [1]. Food losses because of crop infections by pathogens like bacteria, fungus and viruses are known as obstinate issues in agriculture since centuries around the globe [2]. Even countries, societies and their administrations have been facing this problem a long time. Quarantine strategies employed for crops and ornamental plants a requite effective in preventing harmful diseases and arthropod pest epidemics from being imported and getting spread in the purchasing country [3]. Plants are infected by a number bacterial, fungal and virus species [4–8]. Viruses are considered as the minutest known microbes to the mankind and yet they reason for the most significant losses in agriculture sector [9], thus putting the plants under stress [10]. Same holds true for humans as well. Many a time, the finest recognized treatment for viruses is the innate immunological resistance system of host; else, the initial prevention of viral infection is the only substitute [11]. Consequently, diagnosing host for viruses at earliest is the prime approach toward controlling and eliminating harmful virus [12, 13]. The starter of a novel class of nanoscale particles with numerous exceptional properties and functions has flashed a series of innovative applications [14]. Engineered nano-materials (nanoparticles) range from 1 to 100 nm in size [15]. Engineered nanoparticles can be synthesized to precise dimensions and intended in numerous composite arrays, making their function and efficacy applicable in many fields. Suitable sensors and good delivery systems might

Field for Disease Management

*Avinash Marwal and R.K. Gaur*

in plant pathology i.e., "nanophytovirology."

help infighting viruses and other crop pathogens.

#### **Chapter 2**

## Nanophytovirology: An Emerging Field for Disease Management

*Avinash Marwal and R.K. Gaur*

#### **Abstract**

Nanotechnology positions as a new armament in our collection against the increasing challenges in disease management and plant/human health. The application of nanotechnology in plant/human disease administration, diagnosis, and genetic transformations is still in its early stages. Apart from the scope of this chapter, there is also a mounting collection of new tools and techniques where nanoparticles are employed as delivery vehicles for genetic material in plants. Due to their nanoscale dimensions, nanoparticles may knockout virus particles and thus may open a novel arena of virus control in plants/humans. Our aim is to enlighten and enthuse researchers about the swiftly expanding prospects of nanotechnology in plant pathology i.e., "nanophytovirology."

**Keywords:** nanoparticles, plant pathology, human pathology, virology, disease diagnosis, disease management, plant protection, case study

#### **1. Introduction**

Food security has always been the principal apprehension for mankind [1]. Food losses because of crop infections by pathogens like bacteria, fungus and viruses are known as obstinate issues in agriculture since centuries around the globe [2]. Even countries, societies and their administrations have been facing this problem a long time. Quarantine strategies employed for crops and ornamental plants a requite effective in preventing harmful diseases and arthropod pest epidemics from being imported and getting spread in the purchasing country [3]. Plants are infected by a number bacterial, fungal and virus species [4–8]. Viruses are considered as the minutest known microbes to the mankind and yet they reason for the most significant losses in agriculture sector [9], thus putting the plants under stress [10]. Same holds true for humans as well. Many a time, the finest recognized treatment for viruses is the innate immunological resistance system of host; else, the initial prevention of viral infection is the only substitute [11]. Consequently, diagnosing host for viruses at earliest is the prime approach toward controlling and eliminating harmful virus [12, 13]. The starter of a novel class of nanoscale particles with numerous exceptional properties and functions has flashed a series of innovative applications [14]. Engineered nano-materials (nanoparticles) range from 1 to 100 nm in size [15]. Engineered nanoparticles can be synthesized to precise dimensions and intended in numerous composite arrays, making their function and efficacy applicable in many fields. Suitable sensors and good delivery systems might help infighting viruses and other crop pathogens.

Nanoparticles might employ an important integrity in future plant and human disease management that might range from disease diagnosis to disease treatment [16]. In recent past several nanoparticles has been synthesized across the globe by eminent scientists in various forms [17, 18]. Like quantum dots, metalloids, metallic oxides, nonmetals, carbon nanomaterials [19], dendrimers, liposomes [20], Virus-based nanoparticles (VNPs) are few examples of this category [21, 22]. Nanoparticles greatest advantage lies in their small size, greater surface area and strong reactivity: such efficient activity favors for vast application in plant and human pathology [23]. Nanoparticles can be synthesized either by chemical route or by green synthesis method taking in account the top down or bottom up approach, whichever better feasible. This can be further categorized into chemical, reduction, microemulsion, colloidal, sonochemical, electrochemical, microwave, solvothermal and microbial synthesis of nanoparticles [24]. The present study focuses and centric towards the above said aspects of nanoparticles vs. plant virology ("nanophytovirology"), thus summarizing the available scattered literature at one place for the common audience.

#### **2. Pre-era of "nanophytovirology"**

Earlier several methods have been given by pioneers for virus detection in the host plant (crops, ornamental plants, weeds) [25]. Therefore, techniques for recognition and detection of viruses, equally in crops and carrier vectors, participate for a decisive role in virus disease management. All of them are listed as: electron microscopy [26], symptoms determination [27, 28], biotest [29, 30], mechanical transmission [31], seed transmission [32], serological techniques [enzyme-linked immunosorbent assay, phage display, tissue blot immunoassay (TIBA), lateral flow devices, immunocapture transmission electron microscopy (ICTEM)] [33, 34], restriction fragment length polymorphism (RFLP) [35], thermostable amplification based methods [PCR and reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR/RT-PCR, immunocapture PCR (IC-PCR), immuno-precipitation PCR (IP-PCR), nested PCR, multiplex nested PCR, real time PCR (qPCR), multiplex real time PCR, Co-operational PCR (Co-PCR)] [36–39], isothermal amplification based methods [helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), nucleic acid sequence base amplification (NASBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA)] [40, 41], nucleic acid sequence hybridization techniques [in situ hybridization, microarray, lateral flow microarrays] [42, 43], next generation sequencing (NGS), recombinant DNA approach [44]. Accessibility of the few above-mentioned diagnostic methods endow with a superior elasticity, increased sensitivity, and specificity for quick judgment of virus diseases. The accurate and reliable detection of the associated virus pathogens therefore forms the first line of defense in management of these diseases.

Likewise, number of methods has been devised in viral disease management [45–48]. It is conceivable that destruction/killing arthropod vectors, either via biological control or with traditional methods, helpful in reducing the viral populations. *Scymnus offmanni*, *Coccinella septempunctata*, *Propylaea japonica*, *Euseius cutalis*, etc. are some natural predators of whiteflies. Chemiecological technique employs honeydews excreta of whitefly, which work as a kairomone to attract natural prey, i.e., *Encarsia formosa*. Such approach has been successfully used in the Mediterranean regions against whiteflies vector. Even plant age is also crucial in vector population controlling. Field trial has been successful where young plants were covered with plastic bag (yellow polyethylene film) or grown under green

**21**

*Nanophytovirology: An Emerging Field for Disease Management*

house until maturing phase, limits the contact with arthropods vectors. Similarly masking the crop with living ground covers of perennial peanuts, cinquillo and coriander plants condensed the impact of incoming whitefly adults. An old age practice helps in diminishing the virus populations by destroying the weeds growing in the near vicinity of the crop fields, or even sowing the seeds a little later when the vector populations flourishes. Both help in viral disease management [49].

Further the use of insecticides against various arthropod vectors is also helpful to a certain extent. Few of them are Neonicotinoids, Buprofezin, Thiamethoxam 70 WS, Imidacloprid 600 FS, Imidacloprid 70WS and Carbosulfan 25 DS,

Triazophos, Ethion, Imidacloprid, Acephate 95 SG, Spirotetramat, Diafenthiuron, Nitroguanidines, Thiamethoxam, Ryanodine and Pymetrozine, quite lethal against whiteflies [50]. Two remarkable technology has come up as one the best solution against plant viruses, i.e., interference RNA (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR). RNAi-mediated virus resistance was reported against potato virus Y (PVY) in transgenic tobacco plants, against African cassava mosaic virus (ACMV), croton yellow vein mosaic virus (CYVMV) and many more [51–53]. RNAi is usually associated with methylation of nuclear DNA corresponding to the transcribed region of the target RNA despite transcription levels of the transgene remains unaffected [54]. CRISPR-Cas (CRISPR Associated Systems) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA based on sequence complementarity. Virus based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing cause mutations in target genome locations and resulted in transgenic plants showing resistance

"Nanophytovirology" is a front-line science which customs nanotechnology in diagnosis, detection and management of plant viral diseases and their pathogens especially arthropods at an initial phase, helping in plant protection from the epidemic diseases [56]. Among the various plant diseases, the diseases caused by viruses are the most difficult to manage [57], as one must stop the spread of the disease by the vectors. Nanotechnological-based disease diagnosis and management for virus infecting crop plants is attaining magnitude with the increased spread of viruses and threats of their epidemics [58]. Therefore, there is a demand for an improved management of viruses employed by a series of strategies [59, 60]; in-fact such practices relied on the ecology of the virus. Many approaches have been used to decrease crop losses due to viruses, only a few are effective in their management. Even understanding the plant mediated interactions between viruses and their carrier vector is quite important to tackle epidemiology of viral diseases [59, 60]. Developments in nanofabrication and nanotechnology endow a crucial part in plant viral disease detection, simplicity in handling and are cost-effective as compared to

Earlier fluorescent dyes were used straight away for staining the viruses, now nanoparticles and quantum dots (QDs) have been developed which helps in carrying the detection tags (dyes or anti-viral antibodies) and are quite efficient in identifying the viruses, which are also helpful as labeling and imaging agents. Such fluorescent tags are easily detected in flow cytometry enabled devices. In yet another instance nano-biosensor was developed against plasmodiophoromycete *Polymyxa betae* which is responsible for the carrier of beet necrotic yellow vein virus (BNYVV) and caused the deadly disease rhizomania in sugarcane plants. The authors used specific antibodies against conjugated with Cadmium-Telluride

*DOI: http://dx.doi.org/10.5772/intechopen.86653*

against viruses [55].

**3. Epoch of "nanophytovirology"**

other plant viral diagnostic methods.

*Nanophytovirology: An Emerging Field for Disease Management DOI: http://dx.doi.org/10.5772/intechopen.86653*

*Plant Diseases-Current Threats and Management Trends*

one place for the common audience.

**2. Pre-era of "nanophytovirology"**

defense in management of these diseases.

Nanoparticles might employ an important integrity in future plant and human disease management that might range from disease diagnosis to disease treatment [16]. In recent past several nanoparticles has been synthesized across the globe by eminent scientists in various forms [17, 18]. Like quantum dots, metalloids, metallic oxides, nonmetals, carbon nanomaterials [19], dendrimers, liposomes [20], Virus-based nanoparticles (VNPs) are few examples of this category [21, 22]. Nanoparticles greatest advantage lies in their small size, greater surface area and strong reactivity: such efficient activity favors for vast application in plant and human pathology [23]. Nanoparticles can be synthesized either by chemical route or by green synthesis method taking in account the top down or bottom up approach, whichever better feasible. This can be further categorized into chemical, reduction, microemulsion, colloidal, sonochemical, electrochemical, microwave, solvothermal and microbial synthesis of nanoparticles [24]. The present study focuses and centric towards the above said aspects of nanoparticles vs. plant virology ("nanophytovirology"), thus summarizing the available scattered literature at

Earlier several methods have been given by pioneers for virus detection in the host plant (crops, ornamental plants, weeds) [25]. Therefore, techniques for recognition and detection of viruses, equally in crops and carrier vectors, participate for a decisive role in virus disease management. All of them are listed as: electron microscopy [26], symptoms determination [27, 28], biotest [29, 30], mechanical transmission [31], seed transmission [32], serological techniques [enzyme-linked immunosorbent assay, phage display, tissue blot immunoassay (TIBA), lateral flow devices, immunocapture transmission electron microscopy (ICTEM)] [33, 34], restriction fragment length polymorphism (RFLP) [35], thermostable amplification based methods [PCR and reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR/RT-PCR, immunocapture PCR (IC-PCR), immuno-precipitation PCR (IP-PCR), nested PCR, multiplex nested PCR, real time PCR (qPCR), multiplex real time PCR, Co-operational PCR (Co-PCR)] [36–39], isothermal amplification based methods [helicase dependent amplification (HDA), recombinase polymerase amplification (RPA), nucleic acid sequence base amplification (NASBA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA)] [40, 41], nucleic acid sequence hybridization techniques [in situ hybridization, microarray, lateral flow microarrays] [42, 43], next generation sequencing (NGS), recombinant DNA approach [44]. Accessibility of the few above-mentioned diagnostic methods endow with a superior elasticity, increased sensitivity, and specificity for quick judgment of virus diseases. The accurate and reliable detection of the associated virus pathogens therefore forms the first line of

Likewise, number of methods has been devised in viral disease management [45–48]. It is conceivable that destruction/killing arthropod vectors, either via biological control or with traditional methods, helpful in reducing the viral populations. *Scymnus offmanni*, *Coccinella septempunctata*, *Propylaea japonica*, *Euseius cutalis*, etc. are some natural predators of whiteflies. Chemiecological technique employs honeydews excreta of whitefly, which work as a kairomone to attract natural prey, i.e., *Encarsia formosa*. Such approach has been successfully used in the Mediterranean regions against whiteflies vector. Even plant age is also crucial in vector population controlling. Field trial has been successful where young plants were covered with plastic bag (yellow polyethylene film) or grown under green

**20**

house until maturing phase, limits the contact with arthropods vectors. Similarly masking the crop with living ground covers of perennial peanuts, cinquillo and coriander plants condensed the impact of incoming whitefly adults. An old age practice helps in diminishing the virus populations by destroying the weeds growing in the near vicinity of the crop fields, or even sowing the seeds a little later when the vector populations flourishes. Both help in viral disease management [49].

Further the use of insecticides against various arthropod vectors is also helpful to a certain extent. Few of them are Neonicotinoids, Buprofezin, Thiamethoxam 70 WS, Imidacloprid 600 FS, Imidacloprid 70WS and Carbosulfan 25 DS, Triazophos, Ethion, Imidacloprid, Acephate 95 SG, Spirotetramat, Diafenthiuron, Nitroguanidines, Thiamethoxam, Ryanodine and Pymetrozine, quite lethal against whiteflies [50]. Two remarkable technology has come up as one the best solution against plant viruses, i.e., interference RNA (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR). RNAi-mediated virus resistance was reported against potato virus Y (PVY) in transgenic tobacco plants, against African cassava mosaic virus (ACMV), croton yellow vein mosaic virus (CYVMV) and many more [51–53]. RNAi is usually associated with methylation of nuclear DNA corresponding to the transcribed region of the target RNA despite transcription levels of the transgene remains unaffected [54]. CRISPR-Cas (CRISPR Associated Systems) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA based on sequence complementarity. Virus based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing cause mutations in target genome locations and resulted in transgenic plants showing resistance against viruses [55].

#### **3. Epoch of "nanophytovirology"**

"Nanophytovirology" is a front-line science which customs nanotechnology in diagnosis, detection and management of plant viral diseases and their pathogens especially arthropods at an initial phase, helping in plant protection from the epidemic diseases [56]. Among the various plant diseases, the diseases caused by viruses are the most difficult to manage [57], as one must stop the spread of the disease by the vectors. Nanotechnological-based disease diagnosis and management for virus infecting crop plants is attaining magnitude with the increased spread of viruses and threats of their epidemics [58]. Therefore, there is a demand for an improved management of viruses employed by a series of strategies [59, 60]; in-fact such practices relied on the ecology of the virus. Many approaches have been used to decrease crop losses due to viruses, only a few are effective in their management. Even understanding the plant mediated interactions between viruses and their carrier vector is quite important to tackle epidemiology of viral diseases [59, 60]. Developments in nanofabrication and nanotechnology endow a crucial part in plant viral disease detection, simplicity in handling and are cost-effective as compared to other plant viral diagnostic methods.

Earlier fluorescent dyes were used straight away for staining the viruses, now nanoparticles and quantum dots (QDs) have been developed which helps in carrying the detection tags (dyes or anti-viral antibodies) and are quite efficient in identifying the viruses, which are also helpful as labeling and imaging agents. Such fluorescent tags are easily detected in flow cytometry enabled devices. In yet another instance nano-biosensor was developed against plasmodiophoromycete *Polymyxa betae* which is responsible for the carrier of beet necrotic yellow vein virus (BNYVV) and caused the deadly disease rhizomania in sugarcane plants. The authors used specific antibodies against conjugated with Cadmium-Telluride

QDs against the glutathione-S-transferase protein's (GST). The developed nanobiosensor showed enough fluorescence resonance energy transfer (FRET) to detect the plasmodiophoromycete *Polymyxa betae*. Cadmium-Telluride QDs conjugated to antibodies were also developed against citrus tristeza virus (CTV) using the similar approach [61].

Surface plasmon resonance (SPR) is also an optical based technique which employs diagnosis of viruses by change in refractive index on a metal surface. In this gold nanoparticles are conjugated with anti-viral antibodies adsorbed on a glass substrate and are sensed by SPR. Quartz-crystal microbalance (QCM) is a well-known, commercially accessible mass sensor technique generally employed in quantitative measurement of the thickness of thin films. The principal is that the exterior part of the quartz-crystal device (microchip: dimensions in nanometer) is typically coated with anti-viral antibodies against the targeted plant viruses. Now when a virus is encountered on the quartz-crystal surface there results an increase in the mass, thereby resonant frequency decreases, the change in the frequency before and after of the chip is measured subsequently. SPR and QCM based nanobiosensor has been manufactured successfully for the detection of orchid viruses, tobacco mosaic virus (TMV), cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), etc. [62].

Microcantilevers are in the micrometer range, but their tip end is in the nanometer scale and is widely used for various biosensing applications. Microcantilevers works in two different modes, i.e., straining and resonating mode, both are helpful in identifying viruses. Resonating mode is like QCM, whereas straining mode relies on the changes in electrical resistance whenever a virus particle bound to the surface. The major limitation is the low performance of device in the liquid medium, hence the sample need drying before application. For virus particle detection nanowire employed transistors have been devised. An immuno-biosensor was developed for the detection of Plum pox virus in plum (*Prunus domestica*) and tobacco (*Nicotiana benthamiana*) leaves sap, where gold electrodes were modified with 1,6-hexanedithiol, gold nanoparticles, anti-PPV IgG polyclonal antibody and BSA. Nanowires can be engaged against the target virus, and when encountering a charged virus capsid, a depletion or gain of charge in the nanowires is thus recorded as a simple conductance change. In a similar instance a lithographically patterned nanowire electrodeposition (LPNE) technique was used to develop a label-free chemiresistive sensors based on a polypyrrole (PPy) nanoribbon conjugated with anti-viral antibodies against cucumber mosaic virus (CMV) [63].

Nanotechnology benefits agriculture sectors and diminish environmental pollution. This is carried out by manufacturing of pesticides and chemical fertilizers using nanoparticles and nano-capsules and has the capability to control or delayed the delivery and absorption of pesticides and chemical fertilizers with lower dose. "Nano-5" is a marketed product pesticide to control several plant viruses. It was found effective at dilution of 1:500 against Mosaic, ringspot, transitory yellowing, tristeza virus, exocortis viroid by spraying "Nano-5" onto the surface of leaves and apply to the roots once every 3 days. It was reported that chitosan nanoparticles have the ability to induce resistance in host crops against few viruses, for example mosaic virus of alfalfa, snuff, peanut, potato, and cucumber were targeted. Similarly, gold nanoparticles showed antiviral effects against Bean mild mosaic virus in beans, barley yellow mosaic virus in barley and tobacco necrosis virus in tobacco plants. Peoples also claimed silver nanoparticles application made the host plant resistance against sun-hemp rosette virus (infecting bean plants) and bean yellow mosaic virus (causing disease in faba bean crops) [64].

RNAi technique were also employed in coupling with nanotechnology, a remarkable study was carried out to show resistance against cucumber mosaic virus (CMV)

**23**

*Nanophytovirology: An Emerging Field for Disease Management*

**4. Challenges adjoining "nanophytovirology"**

and pepper mild mottle virus (PMMoV). In this wonderful approach dsRNA was loaded onto LDH (Layered double hydroxides) nanoparticles, called BioClay and were sprayed on the challenged plants. Plants showed resistance against the abovementioned viruses for 20 days as compared to controlled ones [65]. It is clear from the above discussion that "nanophytovirology" represent an attractive advancement, owing to their potential advantages for the plant disease management against

The application of nanoparticles, particularly in plant disease management, need specific structural and physicochemical features, and any slight variations to their planned properties can hinder the function and performance of designed nanoparticle conjugate [66]. Hence, numerous factors related to nanoparticle synthesis are quite important for the development of an effective virus detection assay. Further, the methods employed in making/synthesizing nanoparticles seem easy and quick for large scale production but getting the final product in uniformity (shape and size) remains challenging. Nanoparticles itself cannot detect the viruses solely and thus need additional biomolecule specific in sensing the pathogen. In comparison to nanoparticles, biomolecules are quite delicate to severe chemical and physical alterations (high temperature, high salt concentrations, reducing agents) which might can harmfully affect their reactivity and specificity [67]. Therefore, such procedures demand for proper optimization steps aimed at in detecting

But when employing/using nanoparticles and its conjugates for the application in virus disease management, their biosafety and toxicity on human health and environment is yet another a major challenge. For example, nano-pesticides might get inhaled by the workers during treatment process. Similarly, other nano-composites might get deposit on the leaves or flowers, can affect animals, birds, honey bees, etc. They may clog the stomatal pores and might hinder the penetration of pollen grains on stigma. If get inside the plant system, nanoparticle might affect plant metabolism and can cause similar effect on humans as well. Cellular toxicity can be induced by nanoparticles (NPs) that lead to toxic side effects such as enhanced ROS generation, disruption of redox homeostasis, lipid peroxidation, impaired mitochondrial function, and membrane damage. Due to their long persistence and greater reactivity nano-pesticides may contaminate water and soil system [68]. Regardless of these developments in nanotechnology, there are some unsolved problems concerning the detection of many plant viruses due to their low titer in the plants, their uneven distribution, the existence of latent infection and lack of

Virologists need complete knowledge about viral infection and of effects on host plants so that correct control procedures can be implemented [69]. Specificity of viruses varies greatly [70]. Some of them can colonize different species and some are specific or interact to specific cell machinery. There is more focus on reduction of crop loss by controlling pathogen movement from infected plants to healthy plants rather than treating the infected plants [71]. The work on the development of nanoparticles done by the pioneers in the field is particularly significant and beneficial for the humans and to the agriculture sector which supports the lives

*DOI: http://dx.doi.org/10.5772/intechopen.86653*

deadly crop viruses.

viruses.

validated sampling protocols.

**5. Conclusion**

*Plant Diseases-Current Threats and Management Trends*

approach [61].

ringspot virus (ORSV), etc. [62].

QDs against the glutathione-S-transferase protein's (GST). The developed nanobiosensor showed enough fluorescence resonance energy transfer (FRET) to detect the plasmodiophoromycete *Polymyxa betae*. Cadmium-Telluride QDs conjugated to antibodies were also developed against citrus tristeza virus (CTV) using the similar

Surface plasmon resonance (SPR) is also an optical based technique which employs diagnosis of viruses by change in refractive index on a metal surface. In this gold nanoparticles are conjugated with anti-viral antibodies adsorbed on a glass substrate and are sensed by SPR. Quartz-crystal microbalance (QCM) is a well-known, commercially accessible mass sensor technique generally employed in quantitative measurement of the thickness of thin films. The principal is that the exterior part of the quartz-crystal device (microchip: dimensions in nanometer) is typically coated with anti-viral antibodies against the targeted plant viruses. Now when a virus is encountered on the quartz-crystal surface there results an increase in the mass, thereby resonant frequency decreases, the change in the frequency before and after of the chip is measured subsequently. SPR and QCM based nanobiosensor has been manufactured successfully for the detection of orchid viruses, tobacco mosaic virus (TMV), cymbidium mosaic virus (CymMV), odontoglossum

Microcantilevers are in the micrometer range, but their tip end is in the nanometer scale and is widely used for various biosensing applications. Microcantilevers works in two different modes, i.e., straining and resonating mode, both are helpful in identifying viruses. Resonating mode is like QCM, whereas straining mode relies on the changes in electrical resistance whenever a virus particle bound to the surface. The major limitation is the low performance of device in the liquid medium, hence the sample need drying before application. For virus particle detection nanowire employed transistors have been devised. An immuno-biosensor was developed for the detection of Plum pox virus in plum (*Prunus domestica*) and tobacco (*Nicotiana benthamiana*) leaves sap, where gold electrodes were modified with 1,6-hexanedithiol, gold nanoparticles, anti-PPV IgG polyclonal antibody and BSA. Nanowires can be engaged against the target virus, and when encountering a charged virus capsid, a depletion or gain of charge in the nanowires is thus recorded as a simple conductance change. In a similar instance a lithographically patterned nanowire electrodeposition (LPNE) technique was used to develop a label-free chemiresistive sensors based on a polypyrrole (PPy) nanoribbon conjugated with

Nanotechnology benefits agriculture sectors and diminish environmental pollution. This is carried out by manufacturing of pesticides and chemical fertilizers using nanoparticles and nano-capsules and has the capability to control or delayed the delivery and absorption of pesticides and chemical fertilizers with lower dose. "Nano-5" is a marketed product pesticide to control several plant viruses. It was found effective at dilution of 1:500 against Mosaic, ringspot, transitory yellowing, tristeza virus, exocortis viroid by spraying "Nano-5" onto the surface of leaves and apply to the roots once every 3 days. It was reported that chitosan nanoparticles have the ability to induce resistance in host crops against few viruses, for example mosaic virus of alfalfa, snuff, peanut, potato, and cucumber were targeted. Similarly, gold nanoparticles showed antiviral effects against Bean mild mosaic virus in beans, barley yellow mosaic virus in barley and tobacco necrosis virus in tobacco plants. Peoples also claimed silver nanoparticles application made the host plant resistance against sun-hemp rosette virus (infecting bean plants) and bean

RNAi technique were also employed in coupling with nanotechnology, a remarkable study was carried out to show resistance against cucumber mosaic virus (CMV)

anti-viral antibodies against cucumber mosaic virus (CMV) [63].

yellow mosaic virus (causing disease in faba bean crops) [64].

**22**

and pepper mild mottle virus (PMMoV). In this wonderful approach dsRNA was loaded onto LDH (Layered double hydroxides) nanoparticles, called BioClay and were sprayed on the challenged plants. Plants showed resistance against the abovementioned viruses for 20 days as compared to controlled ones [65]. It is clear from the above discussion that "nanophytovirology" represent an attractive advancement, owing to their potential advantages for the plant disease management against deadly crop viruses.

#### **4. Challenges adjoining "nanophytovirology"**

The application of nanoparticles, particularly in plant disease management, need specific structural and physicochemical features, and any slight variations to their planned properties can hinder the function and performance of designed nanoparticle conjugate [66]. Hence, numerous factors related to nanoparticle synthesis are quite important for the development of an effective virus detection assay. Further, the methods employed in making/synthesizing nanoparticles seem easy and quick for large scale production but getting the final product in uniformity (shape and size) remains challenging. Nanoparticles itself cannot detect the viruses solely and thus need additional biomolecule specific in sensing the pathogen. In comparison to nanoparticles, biomolecules are quite delicate to severe chemical and physical alterations (high temperature, high salt concentrations, reducing agents) which might can harmfully affect their reactivity and specificity [67]. Therefore, such procedures demand for proper optimization steps aimed at in detecting viruses.

But when employing/using nanoparticles and its conjugates for the application in virus disease management, their biosafety and toxicity on human health and environment is yet another a major challenge. For example, nano-pesticides might get inhaled by the workers during treatment process. Similarly, other nano-composites might get deposit on the leaves or flowers, can affect animals, birds, honey bees, etc. They may clog the stomatal pores and might hinder the penetration of pollen grains on stigma. If get inside the plant system, nanoparticle might affect plant metabolism and can cause similar effect on humans as well. Cellular toxicity can be induced by nanoparticles (NPs) that lead to toxic side effects such as enhanced ROS generation, disruption of redox homeostasis, lipid peroxidation, impaired mitochondrial function, and membrane damage. Due to their long persistence and greater reactivity nano-pesticides may contaminate water and soil system [68]. Regardless of these developments in nanotechnology, there are some unsolved problems concerning the detection of many plant viruses due to their low titer in the plants, their uneven distribution, the existence of latent infection and lack of validated sampling protocols.

#### **5. Conclusion**

Virologists need complete knowledge about viral infection and of effects on host plants so that correct control procedures can be implemented [69]. Specificity of viruses varies greatly [70]. Some of them can colonize different species and some are specific or interact to specific cell machinery. There is more focus on reduction of crop loss by controlling pathogen movement from infected plants to healthy plants rather than treating the infected plants [71]. The work on the development of nanoparticles done by the pioneers in the field is particularly significant and beneficial for the humans and to the agriculture sector which supports the lives

of growing population [72, 73]. Nanoparticles affect the pathogens in a similar way as the chemical pesticides do at a very low concentration [74]. Nanomaterials have been used as carrier of active ingredients of pesticides, host defense inducing chemicals, etc. [75, 76], to target the viral pathogens. These nano-based diagnostic kits not only increase the speed of detection but also increase the power of the detection. Thus, finding nanotechnology-based solutions, will enable researchers to explore better management practices against viruses in a better way for the plants which are constantly challenged in the natural conditions.

### **Conflict of interest**

The authors have no conflict of action to declare.

### **Author contributions**

A.M. and R.K.G. drafted the manusxxcript. A.M. and R.K.G. contributed to acquisition of literature data. All authors read and approved the final version of the manuscript.

### **Author details**

Avinash Marwal1 and R.K. Gaur2 \*

1 Department of Biotechnology, Mohanlal Sukhadia University, Udaipur, Rajasthan, India

2 Department of Biosciences, School of Sciences, Mody University of Science and Technology, Lakshmangarh, Sikar, Rajasthan, India

\*Address all correspondence to: gaurrajarshi@hotmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

**25**

*Nanophytovirology: An Emerging Field for Disease Management*

Agro-Ecological Perspectives.

Singapore: Springer; 2017. pp. 255-273. DOI: 10.1007/978-981-10-5813-4\_13

[9] Marwal A, Prajapat R, Gaur RK. First

[10] Marwal A, Mishra M, Sekhsaria C, Gaur RK. Computational analysis and predicting ligand binding site in the rose leaf curl virus and its Betasatellite proteins: A step forward for antiviral agent designing. In: Saxena S, Tiwari AK, editors. Begomoviruses: Occurrence and Management in Asia and Africa. Singapore: Springer; 2017. pp. 157-168. DOI: 10.1007/978-981-10-5984-1\_9

[11] Marwal A, Sahu A, Gaur RK. New insights in the functional genomics of plants responding to abiotic stress. In: Gaur RK, Sharma P, editors. Molecular Approaches in Plant Abiotic Stress. Boca Raton, Florida, US: CRC Press, Science Publishers, Taylor & Francis Group; 2014. pp. 158-180. DOI:

[12] Quatrini L, Wieduwild E, Escaliere B, Filtjens J, Chasson L, Laprie C, et al. Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells. Nature Immunology. 2018;**19**:54-962. DOI:

10.1038/s41590-018-0185-0

report of recombination analysis of Betasatellite and Aplhasatellite sequence isolated from an ornamental plant Marigold in India: An in silico approach. International Journal of Virology. 2016;**12**:10-17. DOI: 10.3923/

[8] Marwal A, Sahu AK, Gaur RK. Molecular characterization of begomoviruses and DNA satellites associated with a new host Spanish flag (*Lantana camara*) in India. ISRN Virology. 2013;**2013**:5. DOI:

10.5402/2013/915703

ijv.2016.10.17

10.1201/b15538

*DOI: http://dx.doi.org/10.5772/intechopen.86653*

[1] Kleve S, Booth S, Davidson ZE, Palermo C. Walking the food security tightrope—Exploring the experiences of low-to-middle income Melbourne households. International Journal of Environmental Research and Public Health. 2018;**15**:2206. DOI: 10.3390/

[2] Gaur RK, Prajapat R, Marwal A, Sahu A, Rathore MS. First report of a Begomovirus infecting *Mimosa pudica* in India. Journal of Plant Pathology. 2011;**93**:S4.80. DOI: 10.4454/jpp.

[3] Almeida RPP. Emerging plant disease epidemics: Biological research is key but not enough. PLoS Biology. 2018;**16**:e2007020. DOI: 10.1371/journal.

[4] Marwal A, Prajapat R, Sahu A, Gaur RK. Current status of Geminivirus in India: RNAi technology, a challenging

cure. Asian Journal of Biological Sciences. 2012;**5**:273-293. DOI: 10.3923/

[5] Prajapat R, Marwal A, Jha PN. *Erwinia carotovora* associated with potato: A critical appraisal with respect to Indian perspective. International Journal of Current Microbiology and Applied

[6] Mahmood S, Lakra N, Marwal A, Sudheep NM, Anwar K. Crop genetic engineering: An approach to improve fungal resistance in plant system. In: Singh DP, editor. Plant-Microbe Interactions in Agro-Ecological Perspectives. Singapore: Springer; 2017. pp. 581-591. DOI: 10.1007/978-981-10-6593-4\_23

[7] Sudheep NM, Marwal A, Lakra N, Anwar K, Mahmood S. Fascinating fungal endophytes role and possible applications. In: Singh DP, editor. Plant-Microbe Interactions in

**References**

ijerph15102206

v93i4.2394

pbio.2007020

ajbs.2012.273.293

Sciences. 2013;**2**:83-89

*Nanophytovirology: An Emerging Field for Disease Management DOI: http://dx.doi.org/10.5772/intechopen.86653*

#### **References**

*Plant Diseases-Current Threats and Management Trends*

which are constantly challenged in the natural conditions.

The authors have no conflict of action to declare.

**Conflict of interest**

**Author contributions**

manuscript.

**Author details**

Avinash Marwal1

India

and R.K. Gaur2

Technology, Lakshmangarh, Sikar, Rajasthan, India

provided the original work is properly cited.

\*Address all correspondence to: gaurrajarshi@hotmail.com

\*

1 Department of Biotechnology, Mohanlal Sukhadia University, Udaipur, Rajasthan,

2 Department of Biosciences, School of Sciences, Mody University of Science and

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

of growing population [72, 73]. Nanoparticles affect the pathogens in a similar way as the chemical pesticides do at a very low concentration [74]. Nanomaterials have been used as carrier of active ingredients of pesticides, host defense inducing chemicals, etc. [75, 76], to target the viral pathogens. These nano-based diagnostic kits not only increase the speed of detection but also increase the power of the detection. Thus, finding nanotechnology-based solutions, will enable researchers to explore better management practices against viruses in a better way for the plants

A.M. and R.K.G. drafted the manusxxcript. A.M. and R.K.G. contributed to acquisition of literature data. All authors read and approved the final version of the

**24**

[1] Kleve S, Booth S, Davidson ZE, Palermo C. Walking the food security tightrope—Exploring the experiences of low-to-middle income Melbourne households. International Journal of Environmental Research and Public Health. 2018;**15**:2206. DOI: 10.3390/ ijerph15102206

[2] Gaur RK, Prajapat R, Marwal A, Sahu A, Rathore MS. First report of a Begomovirus infecting *Mimosa pudica* in India. Journal of Plant Pathology. 2011;**93**:S4.80. DOI: 10.4454/jpp. v93i4.2394

[3] Almeida RPP. Emerging plant disease epidemics: Biological research is key but not enough. PLoS Biology. 2018;**16**:e2007020. DOI: 10.1371/journal. pbio.2007020

[4] Marwal A, Prajapat R, Sahu A, Gaur RK. Current status of Geminivirus in India: RNAi technology, a challenging cure. Asian Journal of Biological Sciences. 2012;**5**:273-293. DOI: 10.3923/ ajbs.2012.273.293

[5] Prajapat R, Marwal A, Jha PN. *Erwinia carotovora* associated with potato: A critical appraisal with respect to Indian perspective. International Journal of Current Microbiology and Applied Sciences. 2013;**2**:83-89

[6] Mahmood S, Lakra N, Marwal A, Sudheep NM, Anwar K. Crop genetic engineering: An approach to improve fungal resistance in plant system. In: Singh DP, editor. Plant-Microbe Interactions in Agro-Ecological Perspectives. Singapore: Springer; 2017. pp. 581-591. DOI: 10.1007/978-981-10-6593-4\_23

[7] Sudheep NM, Marwal A, Lakra N, Anwar K, Mahmood S. Fascinating fungal endophytes role and possible applications. In: Singh DP, editor. Plant-Microbe Interactions in

Agro-Ecological Perspectives. Singapore: Springer; 2017. pp. 255-273. DOI: 10.1007/978-981-10-5813-4\_13

[8] Marwal A, Sahu AK, Gaur RK. Molecular characterization of begomoviruses and DNA satellites associated with a new host Spanish flag (*Lantana camara*) in India. ISRN Virology. 2013;**2013**:5. DOI: 10.5402/2013/915703

[9] Marwal A, Prajapat R, Gaur RK. First report of recombination analysis of Betasatellite and Aplhasatellite sequence isolated from an ornamental plant Marigold in India: An in silico approach. International Journal of Virology. 2016;**12**:10-17. DOI: 10.3923/ ijv.2016.10.17

[10] Marwal A, Mishra M, Sekhsaria C, Gaur RK. Computational analysis and predicting ligand binding site in the rose leaf curl virus and its Betasatellite proteins: A step forward for antiviral agent designing. In: Saxena S, Tiwari AK, editors. Begomoviruses: Occurrence and Management in Asia and Africa. Singapore: Springer; 2017. pp. 157-168. DOI: 10.1007/978-981-10-5984-1\_9

[11] Marwal A, Sahu A, Gaur RK. New insights in the functional genomics of plants responding to abiotic stress. In: Gaur RK, Sharma P, editors. Molecular Approaches in Plant Abiotic Stress. Boca Raton, Florida, US: CRC Press, Science Publishers, Taylor & Francis Group; 2014. pp. 158-180. DOI: 10.1201/b15538

[12] Quatrini L, Wieduwild E, Escaliere B, Filtjens J, Chasson L, Laprie C, et al. Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells. Nature Immunology. 2018;**19**:54-962. DOI: 10.1038/s41590-018-0185-0

[13] Prajapat R, Marwal A, Goyal M. In silico characterization of hemagglutinin protein of influenza a virus [A/ canine/Beijing/cau9/2009(H1N1)] of H1N1 subtype. Journal of Advances in Biotechnology. 2014;**1**:40-47. DOI: 10.24297/jbt.v1i1.1786

[14] Prajapat R, Marwal A, Shaikh Z, Gaur RK. Geminivirus database (GVDB): First database of family Geminiviridae and its genera Begomovirus. Pakistan Journal of Biological Sciences. 2012;**15**:702-706. DOI: 10.3923/pjbs.2012.702.706

[15] Das AK, Marwal A, Sain D, Pareek V. One-step green synthesis and characterization of plant protein-coated mercuric oxide (HgO) nanoparticles: Antimicrobial studies. International Nano Letters. 2015;**5**:125-132. DOI: 10.1007/s40089-015-0144-9

[16] Das AK, Marwal A, Sain D. One-step green synthesis and characterization of flower extractmediated mercuric oxide (HgO) nanoparticles from Callistemon viminalis. Research and Reviews: Journal of Pharmaceutics and Nanotechnology. 2014;**2**:25-28

[17] Antonacci A, Arduini F, Moscone D, Palleschi G, Scognamiglio V. Nanostructured (bio)sensors for smart agriculture. TrAC Trends in Analytical Chemistry. 2018;**98**:95-103. DOI: 10.1016/j.trac.2017.10.022

[18] Das AK, Marwal A, Verma R. Datura inoxia leaf extract mediated one step green synthesis and characterization of magnetite (Fe3O4) nanoparticles. Research and Reviews: Journal of Pharmaceutics and Nanotechnology. 2014;**2**:21-24

[19] Das S, Debnath N, Cui Y, Unrine J, Palli SR. Chitosan, carbon quantum dot, and silica nanoparticle mediated dsRNA delivery for gene silencing in Aedes aegypti: A comparative analysis. ACS Applied Materials & Interfaces. 2015;**7**:19530-19535. DOI: 10.1021/ acsami.5b05232

[20] Das AK, Marwal A, Pareek V. Nanoparticles-protein hybrid based magnetic liposome. World Academy of Science, Engineering and Technology, International Journal of Chemical, Nuclear, Materials and Metallurgical Engineering. 2015;**9**:230-233

[21] Das AK, Marwal A, Verma R. Preparation and characterization of nano-bio hybrid based magneto liposome. International Journal of Pharmaceutical Sciences and Research. 2015;**6**:367-375. DOI: 10.13040/ IJPSR.0975-8232.6(1).367-75

[22] Guenther RH, Lommel SA, Opperman CH, Sit TL. Plant virusbased nanoparticles for the delivery of agronomic compounds as a suspension concentrate. Methods in Molecular Biology. 2018;**1776**:203-214. DOI: 10.1007/978-1-4939-7808-3\_13

[23] Das AK, Marwal A, Sain D. One-step green synthesis and characterization of flower extractmediated mercuric oxide (HgO) nanoparticles from *Callistemon viminalis*. In: Bhoop BS, Sharma A, Mehta SK, Tripathi SK, editors. Nanotechnology: Novel Perspectives and Prospects. New Delhi, India: Tata-McGraw Hill; 2014. pp. 466-472

[24] Das AK, Marwal A, Pareek V, Joshi Y, Apoorva. Surface Engineering of Magnetite Nanoparticles by Plant Protein: Investigation into Magnetic Properties. Nano Hybrid and Composites. Vol. 11. Trans Tech Publication; 2016. pp. 38-44. DOI: 10.4028/www.scientific.net/NHC.11.38

[25] Prajapat R, Marwal A, Sahu AK, Gaur RK. First report of Begomovirus infecting *Sonchus asper* in India. Science International. 2013;**1**:108-110. DOI: 10.5567/sciintl.2013.108.110

**27**

*Nanophytovirology: An Emerging Field for Disease Management*

betasatellite in *Rosa indica* and in India. Australasian Plant Disease Notes.

[33] Nehra C, Verma RK, Mishra M, Marwal A, Sharma P, Gaur RK. Papaya

identified begomovirus infecting *Carica papaya* from the Indian subcontinent. The Journal of Horticultural Science & Biotechnology. 2019;**94**:475-480. DOI: 10.1080/14620316.2019.1570827

yellow leaf curl virus: A newly

[34] Marwal A, Sahu A, Gaur RK. Molecular marker: Tools for genetic analysis. In: Verma AS, Singh A, editors. Animal Biotechnology: Models in Discovery and Translation. Waltham, MA, US: Academic Press, Elsevier; 2014. pp. 289-305. DOI: 10.1016/ B978-0-12-416002-6.00016-X

[35] Chen J, Adams MJ. A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Archives of Virology. 2001;**146**:757-766. DOI: 10.1007/

[36] Marwal A, Sahu AK, Gaur RK. First report of molecular characterization of begomovirus infecting an ornamental plant *Tecoma stans* and a medicinal plant *Justicia adhatoda*. Science International.

[37] Nehra C, Sahu AK, Marwal A, Gaur RK. Natural occurrence of Clerodendron yellow mosaic virus on Bougainvillea in India. New Diseases Report BSPP. 2014;**30**:19. DOI: 10.5197/j.2044-0588.2014.030.019

[38] Zhang S, Ravelonandro M, Russell P, McOwen N, Briard P, Bohannon S, et al. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods. 2014;**207**:114-120. DOI: 10.1016/j.jviromet.2014.06.026

s007050170144

2013;**25**:837-839

2014;**9**:147

*DOI: http://dx.doi.org/10.5772/intechopen.86653*

[26] Marwal A, Sahu AK, Prajapat R, Choudhary DK, Gaur RK. Molecular and recombinational characterization of Begomovirus infecting an ornamental plant *Alternanthera sessilis*: A new host of tomato leaf curl Kerala virus reported in India. Science International. 2013;**1**:51- 56. DOI: 10.17311/sciintl.2013.51.56

[27] Marwal A, Sahu AK, Gaur RK. Evidence of the association of

10.24297/jab.v1i1.1554

DOI: 10.24297/jbt.v1i1.5055

Research. 2014;**52**:339-356

begomovirus and its betasatellite with the yellow vein disease of an ornamental plant *Calendula officinalis* (pot marigold) in Rajasthan, India: Molecular, sequence and recombination analysis. Journal of Advances in Biology. 2013;**1**:29-44. DOI:

[28] Prajapat R, Marwal A, Gaur RK. Datura leaf curl betasatellite associated for the first time with *Datura inoxia* leaf curl syndrome in India. Journal of Advances in Biotechnology. 2014;**1**:1-7.

[29] Marwal A, Sahu AK, Gaur RK. Association of begomovirus and an alphasatellite with the leaf curl disease of ornamental plant *Vinca alba* in Punjab, India. Journal of Agricultural

[30] Marwal A, Sahu AK, Gaur RK. First report on the association of a begomovirus with *Chrysanthemum indicum* exhibiting yellowing of leaf vein disease characterized by molecular

studies. Journal of Horticultural Research. 2013;**21**:17-21. DOI: 10.2478/

[31] Sahu AK, Nehra C, Marwal A, Gaur RK. First report of a begomovirus associated with betasatellites infecting new host spinach (*Spinacia oleracea*) in India. Journal of General Plant Pathology. 2015;**81**:146-150. DOI: 10.1007/s10327-014-0576-5

[32] Sahu AK, Marwal A, Nehra C, Shahid MS, Gaur RK. First report of a begomovirus and associated

johr-2013-0017

*Nanophytovirology: An Emerging Field for Disease Management DOI: http://dx.doi.org/10.5772/intechopen.86653*

[26] Marwal A, Sahu AK, Prajapat R, Choudhary DK, Gaur RK. Molecular and recombinational characterization of Begomovirus infecting an ornamental plant *Alternanthera sessilis*: A new host of tomato leaf curl Kerala virus reported in India. Science International. 2013;**1**:51- 56. DOI: 10.17311/sciintl.2013.51.56

*Plant Diseases-Current Threats and Management Trends*

ACS Applied Materials & Interfaces. 2015;**7**:19530-19535. DOI: 10.1021/

[20] Das AK, Marwal A, Pareek V. Nanoparticles-protein hybrid based magnetic liposome. World Academy of Science, Engineering and Technology, International Journal of Chemical, Nuclear, Materials and Metallurgical

Engineering. 2015;**9**:230-233

[21] Das AK, Marwal A, Verma R. Preparation and characterization of nano-bio hybrid based magneto liposome. International Journal of Pharmaceutical Sciences and Research.

2015;**6**:367-375. DOI: 10.13040/ IJPSR.0975-8232.6(1).367-75

[22] Guenther RH, Lommel SA, Opperman CH, Sit TL. Plant virusbased nanoparticles for the delivery of agronomic compounds as a suspension concentrate. Methods in Molecular Biology. 2018;**1776**:203-214. DOI: 10.1007/978-1-4939-7808-3\_13

[23] Das AK, Marwal A, Sain D. One-step green synthesis and characterization of flower extractmediated mercuric oxide (HgO) nanoparticles from *Callistemon viminalis*. In: Bhoop BS, Sharma A, Mehta SK, Tripathi SK, editors. Nanotechnology: Novel Perspectives and Prospects. New Delhi, India: Tata-McGraw Hill; 2014. pp. 466-472

[24] Das AK, Marwal A, Pareek V, Joshi Y, Apoorva. Surface Engineering

[25] Prajapat R, Marwal A, Sahu AK, Gaur RK. First report of Begomovirus infecting *Sonchus asper* in India. Science International. 2013;**1**:108-110. DOI:

10.5567/sciintl.2013.108.110

of Magnetite Nanoparticles by Plant Protein: Investigation into Magnetic Properties. Nano Hybrid and Composites. Vol. 11. Trans Tech Publication; 2016. pp. 38-44. DOI: 10.4028/www.scientific.net/NHC.11.38

acsami.5b05232

[13] Prajapat R, Marwal A, Goyal M. In silico characterization of hemagglutinin

[14] Prajapat R, Marwal A, Shaikh Z, Gaur RK. Geminivirus database (GVDB): First database of family Geminiviridae and its genera Begomovirus. Pakistan Journal of Biological Sciences. 2012;**15**:702-706. DOI: 10.3923/pjbs.2012.702.706

[15] Das AK, Marwal A, Sain D,

[16] Das AK, Marwal A, Sain D. One-step green synthesis and characterization of flower extractmediated mercuric oxide (HgO) nanoparticles from Callistemon viminalis. Research and Reviews: Journal of Pharmaceutics and Nanotechnology. 2014;**2**:25-28

Pareek V. One-step green synthesis and characterization of plant protein-coated mercuric oxide (HgO) nanoparticles: Antimicrobial studies. International Nano Letters. 2015;**5**:125-132. DOI: 10.1007/s40089-015-0144-9

[17] Antonacci A, Arduini F, Moscone D,

Nanostructured (bio)sensors for smart agriculture. TrAC Trends in Analytical Chemistry. 2018;**98**:95-103. DOI:

[18] Das AK, Marwal A, Verma R. Datura inoxia leaf extract mediated one step green synthesis and characterization of magnetite (Fe3O4) nanoparticles. Research and Reviews: Journal of Pharmaceutics and Nanotechnology.

[19] Das S, Debnath N, Cui Y, Unrine J, Palli SR. Chitosan, carbon quantum dot, and silica nanoparticle mediated dsRNA delivery for gene silencing in Aedes aegypti: A comparative analysis.

Palleschi G, Scognamiglio V.

10.1016/j.trac.2017.10.022

protein of influenza a virus [A/ canine/Beijing/cau9/2009(H1N1)] of H1N1 subtype. Journal of Advances in Biotechnology. 2014;**1**:40-47. DOI:

10.24297/jbt.v1i1.1786

**26**

2014;**2**:21-24

[27] Marwal A, Sahu AK, Gaur RK. Evidence of the association of begomovirus and its betasatellite with the yellow vein disease of an ornamental plant *Calendula officinalis* (pot marigold) in Rajasthan, India: Molecular, sequence and recombination analysis. Journal of Advances in Biology. 2013;**1**:29-44. DOI: 10.24297/jab.v1i1.1554

[28] Prajapat R, Marwal A, Gaur RK. Datura leaf curl betasatellite associated for the first time with *Datura inoxia* leaf curl syndrome in India. Journal of Advances in Biotechnology. 2014;**1**:1-7. DOI: 10.24297/jbt.v1i1.5055

[29] Marwal A, Sahu AK, Gaur RK. Association of begomovirus and an alphasatellite with the leaf curl disease of ornamental plant *Vinca alba* in Punjab, India. Journal of Agricultural Research. 2014;**52**:339-356

[30] Marwal A, Sahu AK, Gaur RK. First report on the association of a begomovirus with *Chrysanthemum indicum* exhibiting yellowing of leaf vein disease characterized by molecular studies. Journal of Horticultural Research. 2013;**21**:17-21. DOI: 10.2478/ johr-2013-0017

[31] Sahu AK, Nehra C, Marwal A, Gaur RK. First report of a begomovirus associated with betasatellites infecting new host spinach (*Spinacia oleracea*) in India. Journal of General Plant Pathology. 2015;**81**:146-150. DOI: 10.1007/s10327-014-0576-5

[32] Sahu AK, Marwal A, Nehra C, Shahid MS, Gaur RK. First report of a begomovirus and associated

betasatellite in *Rosa indica* and in India. Australasian Plant Disease Notes. 2014;**9**:147

[33] Nehra C, Verma RK, Mishra M, Marwal A, Sharma P, Gaur RK. Papaya yellow leaf curl virus: A newly identified begomovirus infecting *Carica papaya* from the Indian subcontinent. The Journal of Horticultural Science & Biotechnology. 2019;**94**:475-480. DOI: 10.1080/14620316.2019.1570827

[34] Marwal A, Sahu A, Gaur RK. Molecular marker: Tools for genetic analysis. In: Verma AS, Singh A, editors. Animal Biotechnology: Models in Discovery and Translation. Waltham, MA, US: Academic Press, Elsevier; 2014. pp. 289-305. DOI: 10.1016/ B978-0-12-416002-6.00016-X

[35] Chen J, Adams MJ. A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Archives of Virology. 2001;**146**:757-766. DOI: 10.1007/ s007050170144

[36] Marwal A, Sahu AK, Gaur RK. First report of molecular characterization of begomovirus infecting an ornamental plant *Tecoma stans* and a medicinal plant *Justicia adhatoda*. Science International. 2013;**25**:837-839

[37] Nehra C, Sahu AK, Marwal A, Gaur RK. Natural occurrence of Clerodendron yellow mosaic virus on Bougainvillea in India. New Diseases Report BSPP. 2014;**30**:19. DOI: 10.5197/j.2044-0588.2014.030.019

[38] Zhang S, Ravelonandro M, Russell P, McOwen N, Briard P, Bohannon S, et al. Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP(®) using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods. 2014;**207**:114-120. DOI: 10.1016/j.jviromet.2014.06.026

[39] Nehra C, Marwal A, Verma RK, Gaur RK. Molecular characterization of Begomoviruses DNA-A and associated beta satellites with new host *Ocimum sanctum* in India. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. 2018. DOI: 10.1007/s40011-018-1006-9

[40] Marwal A, Sahu AK, Gaur RK. Molecular characterization of a begomovirus infecting a new host Golden Duranta (*Duranta erecta*) in India. International Journal of Current Microbiology and Applied Sciences. 2013;**2**:45-48

[41] Lamptey JNL, Osei MK, Mochiah MB, Osei K, Berchie JN, Bolfrey-Arku G, et al. Serological detection of tobacco mosaic virus and cucumber mosaic virus infecting tomato (*Solanum Lycopersicum*) using a lateral flow immunoassay technique. Journal of Agricultural Studies. 2013;**1**:102-113. DOI: 10.5296/jas.v1i2.3768

[42] Elbeaino T, Digiaro M, Uppala M, Sudini H. Deep sequencing of pigeonpea sterility mosaic virus discloses five RNA segments related to emaraviruses. Virus Research. 2014;**188**:27-31. DOI: 10.1016/j.virusres.2014.03.022

[43] Marwal A, Kumar R, Khurana SMP, Gaur RK. Complete nucleotide sequence of a new geminivirus isolated from *Vitis vinifera* in India: A symptomless host of grapevine red blotch virus. Virus Disease. 2018;**30**:106-111. DOI: 10.1007/ s13337-018-0477-x

[44] Boonham N, Glover R, Tomlinson J, Mumford R. Exploiting generic platform technologies for the detection and identification of plant pathogens. In: Collinge DB, Munk L, Cooke BM, editors. Sustainable Disease Management in a European Context. Dordrecht: Springer; 2008. pp. 355-363. DOI: 10.1007/978-1-4020-8780-6\_15

[45] Marwal A, Prajapat R, Gaur RK. Prediction of binding site in eight

protein molecules of Begomovirus and its satellite components i.e., Betasatellite and Alphasatellite isolated from infected ornamental plant. Plant Pathology Journal. 2016;**15**:1-4. DOI: 10.3923/ ppj.2016.1.4

[46] Marwal A, Mishra M, Verma RK, Prajapat R, Gaur RK. In silico study of the Geminiviruses infecting ornamental plants. In: Choudhary DK, Kumar M, Prasad R, Kumar V, editors. In Silico Approach for Sustainable Agriculture. Singapore: Springer; 2018. pp. 69-90. DOI: 10.1007/978-981-13-0347-0\_4

[47] Prajapat R, Marwal A, Sahu A, Gaur RK. Phylogenetics and in silico docking studies between coat protein of Mimosa yellow vein virus and whey α-lactalbumin. American Journal of Biochemistry and Molecular Biology. 2011;**1**:265-274. DOI: 10.3923/ ajbmb.2011.265.274

[48] Makkouk KM. A study on tomato viruses in the Jordan valley with special emphasis on tomato yellow leaf curl. Plant Disease Report. 1978;**64**:259-262. DOI: 10.1007/BF02979527

[49] Kayser H, Kaufmann L, Schürmann F. Pymetrozine (CGA 215¢944): A novel compound for aphid and whitefly control. In: An Overview of Its Mode of Action. Proceedings of Brighton Crop Protection Conference– Pests and Diseases. Vol. 2; Brighton. 1994. pp. 737-742

[50] Sanjaya VVS, Prasad V, Kirthi N, Maiya SP, Savithri HS, Sita GL. Development of cotton transgenics with antisense AV2 gene for resistance against cotton leaf curl virus (CLCuD) via agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture. 2005;**81**: 55-63. DOI: 10.1007/s11240-004-2777-7

[51] Sahu AK, Marwal A, Nehra C, Choudhary DK, Sharma P, Gaur RK. RNAi mediated gene silencing against betasatellite associated with Croton

**29**

*Nanophytovirology: An Emerging Field for Disease Management*

Singapore: Springer; 2017. pp. 489-499. DOI: 10.1007/978-981-10-5813-4\_24

[59] Nehra C, Marwal A, Gaur RK. Diversity and phylogeny of

Begomovirus populations and their managements. Acta Microbiologica

[60] Vardhan H, Marwal A, Mathur P, Prajapat R. In silico characterization of hemagglutinin protein of H1N1 subtype. International Journal of Biological

[61] Marwal A, Sahu A, Sharma P, Gaur RK. Transmission and host interaction of Geminivirus in weeds. In: Gaur RK, Hohn T, Sharma P, editors. Plant Virus-Host Interaction: Molecular Approaches

[62] Shojaei TR, Salleh MAM, Sijam K, Rahim RA, Mohsenifar A, Safarnejad R, et al. Fluorometric immunoassay for detecting the plant virus Citrus tristeza using carbon nanoparticles acting as quenchers and antibodies labeled with CdTe quantum dots. Microchimica Acta. 2016;**183**:2277-2287. DOI: 10.1007/

[63] Hayden O, Bindeus R, Haderspock C, Mann KJ, Wirl B, Dickert FL. Mass sensitive detection of cells, viruses, and enzymes with artificial receptors. Sensors and Actuators B: Chemical. 2003;**91**:316-319. DOI: 10.1016/ S0925-4005(03)00093-5

[64] Chartuprayoon N, Rheem Y, Ng JCK, Nam J, Chend W, Myung NV. Polypyrrole nanoribbon based

[65] Elbeshehy EKF, Elazzazy AM, Aggelis G. Silver nanoparticles synthesis

10.1039/C3AY40371H

chemiresistive immunosensors for viral plant pathogen detection. Analytical Methods. 2013;**5**:3497-3502. DOI:

Bulgarica. 2016;**32**:108-113

Technology. 2012;**3**:62-66

and Viral Evolution. Waltham, MA, US: Academic Press, Elsevier; 2014. pp. 143-161. DOI: 10.1016/ B978-0-12-411584-2.00007-X

s00604-016-1867-7

*DOI: http://dx.doi.org/10.5772/intechopen.86653*

Molecular Biology Reports. 2014;**41**:7631- 7638. DOI: 10.1007/s11033-014-3653-0

[52] Marwal A, Sahu AK, Prajapat R, Choudhary DK, Gaur RK. RNA silencing suppressor encoded by Betasatellite DNA associated with Croton yellow vein mosaic virus. Open Access Scientific Reports. 2012;**1**:153. DOI: 10.4172/scientificreports.153

[53] Das S, Marwal A, Choudhary DK, Gupta VK, Gaur RK. Mechanism of RNA interference (RNAi): Current concept. In: International Proceedings

Environmental Engineering; Singapore.

[54] Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM. CRISPR/ Cas9-mediated viral interference in plants. Genome Biology. 2015;**16**:238. DOI: 10.1186/s13059-015-0799-6

[55] Worrall EA, Hamid A, Mody KT, Mitter N, Pappu HR. Nanotechnology for plant disease management. Agronomy. 2018;**8**:285. DOI: 10.3390/

[56] Prajapat R, Marwal A, Gaur RK. Evidence of the Association of Solanum leaf curl Lakshmangarh virus with a weed plant *Solanum nigrum* in Rajasthan, India. Science International. 2013;**1**:379- 383. DOI: 10.17311/sciintl.2013.379.383

[57] Khurana SMP, Marwal A. Recent developments towards detection & diagnosis for management of plant viruses. Indian Phytopathology.

[58] Marwal A, Gaur RK. Understanding functional genomics of PTGS silencing mechanisms for tobacco streak virus and other Ilarviruses mediated by RNAi and VIGS. In: Singh DP, editor. Plant-Microbe Interactions in Agro-Ecological Perspectives. Volume 1: Fundamental Mechanisms, Methods and Functions.

of Chemical, Biological and

Vol. 9. 2011. pp. 240-245

agronomy8120285

2016;**69**:30-34

yellow vein mosaic begomovirus.

*Nanophytovirology: An Emerging Field for Disease Management DOI: http://dx.doi.org/10.5772/intechopen.86653*

yellow vein mosaic begomovirus. Molecular Biology Reports. 2014;**41**:7631- 7638. DOI: 10.1007/s11033-014-3653-0

*Plant Diseases-Current Threats and Management Trends*

protein molecules of Begomovirus and its satellite components i.e., Betasatellite and Alphasatellite isolated from infected ornamental plant. Plant Pathology Journal. 2016;**15**:1-4. DOI: 10.3923/

[46] Marwal A, Mishra M, Verma RK, Prajapat R, Gaur RK. In silico study of the Geminiviruses infecting ornamental plants. In: Choudhary DK, Kumar M, Prasad R, Kumar V, editors. In Silico Approach for Sustainable Agriculture. Singapore: Springer; 2018. pp. 69-90. DOI: 10.1007/978-981-13-0347-0\_4

[47] Prajapat R, Marwal A, Sahu A, Gaur RK. Phylogenetics and in silico docking studies between coat protein of Mimosa yellow vein virus and whey α-lactalbumin. American Journal of Biochemistry and Molecular Biology. 2011;**1**:265-274. DOI: 10.3923/

[48] Makkouk KM. A study on tomato viruses in the Jordan valley with special emphasis on tomato yellow leaf curl. Plant Disease Report. 1978;**64**:259-262.

[50] Sanjaya VVS, Prasad V, Kirthi N, Maiya SP, Savithri HS, Sita GL. Development of cotton transgenics with antisense AV2 gene for resistance against cotton leaf curl virus (CLCuD) via agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture. 2005;**81**: 55-63. DOI: 10.1007/s11240-004-2777-7

[51] Sahu AK, Marwal A, Nehra C, Choudhary DK, Sharma P, Gaur RK. RNAi mediated gene silencing against betasatellite associated with Croton

ajbmb.2011.265.274

DOI: 10.1007/BF02979527

1994. pp. 737-742

[49] Kayser H, Kaufmann L, Schürmann F. Pymetrozine (CGA 215¢944): A novel compound for aphid and whitefly control. In: An Overview of Its Mode of Action. Proceedings of Brighton Crop Protection Conference– Pests and Diseases. Vol. 2; Brighton.

ppj.2016.1.4

[39] Nehra C, Marwal A, Verma RK, Gaur RK. Molecular characterization of Begomoviruses DNA-A and associated beta satellites with new host *Ocimum sanctum* in India. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. 2018. DOI: 10.1007/s40011-018-1006-9

[40] Marwal A, Sahu AK, Gaur RK. Molecular characterization of a begomovirus infecting a new host Golden Duranta (*Duranta erecta*) in India. International Journal of Current Microbiology and Applied Sciences.

[41] Lamptey JNL, Osei MK, Mochiah MB, Osei K, Berchie JN, Bolfrey-Arku G, et al. Serological detection of tobacco mosaic virus and cucumber mosaic virus infecting tomato (*Solanum Lycopersicum*)

technique. Journal of Agricultural Studies. 2013;**1**:102-113. DOI: 10.5296/jas.v1i2.3768

[42] Elbeaino T, Digiaro M, Uppala M, Sudini H. Deep sequencing of pigeonpea sterility mosaic virus discloses five RNA segments related to emaraviruses. Virus Research. 2014;**188**:27-31. DOI:

[43] Marwal A, Kumar R, Khurana SMP, Gaur RK. Complete nucleotide sequence of a new geminivirus isolated from *Vitis vinifera* in India: A symptomless host of grapevine red blotch virus. Virus Disease. 2018;**30**:106-111. DOI: 10.1007/

[44] Boonham N, Glover R, Tomlinson J,

platform technologies for the detection and identification of plant pathogens. In: Collinge DB, Munk L, Cooke BM, editors. Sustainable Disease Management in a European Context. Dordrecht: Springer; 2008. pp. 355-363. DOI: 10.1007/978-1-4020-8780-6\_15

[45] Marwal A, Prajapat R, Gaur RK. Prediction of binding site in eight

Mumford R. Exploiting generic

using a lateral flow immunoassay

10.1016/j.virusres.2014.03.022

s13337-018-0477-x

2013;**2**:45-48

**28**

[52] Marwal A, Sahu AK, Prajapat R, Choudhary DK, Gaur RK. RNA silencing suppressor encoded by Betasatellite DNA associated with Croton yellow vein mosaic virus. Open Access Scientific Reports. 2012;**1**:153. DOI: 10.4172/scientificreports.153

[53] Das S, Marwal A, Choudhary DK, Gupta VK, Gaur RK. Mechanism of RNA interference (RNAi): Current concept. In: International Proceedings of Chemical, Biological and Environmental Engineering; Singapore. Vol. 9. 2011. pp. 240-245

[54] Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM. CRISPR/ Cas9-mediated viral interference in plants. Genome Biology. 2015;**16**:238. DOI: 10.1186/s13059-015-0799-6

[55] Worrall EA, Hamid A, Mody KT, Mitter N, Pappu HR. Nanotechnology for plant disease management. Agronomy. 2018;**8**:285. DOI: 10.3390/ agronomy8120285

[56] Prajapat R, Marwal A, Gaur RK. Evidence of the Association of Solanum leaf curl Lakshmangarh virus with a weed plant *Solanum nigrum* in Rajasthan, India. Science International. 2013;**1**:379- 383. DOI: 10.17311/sciintl.2013.379.383

[57] Khurana SMP, Marwal A. Recent developments towards detection & diagnosis for management of plant viruses. Indian Phytopathology. 2016;**69**:30-34

[58] Marwal A, Gaur RK. Understanding functional genomics of PTGS silencing mechanisms for tobacco streak virus and other Ilarviruses mediated by RNAi and VIGS. In: Singh DP, editor. Plant-Microbe Interactions in Agro-Ecological Perspectives. Volume 1: Fundamental Mechanisms, Methods and Functions.

Singapore: Springer; 2017. pp. 489-499. DOI: 10.1007/978-981-10-5813-4\_24

[59] Nehra C, Marwal A, Gaur RK. Diversity and phylogeny of Begomovirus populations and their managements. Acta Microbiologica Bulgarica. 2016;**32**:108-113

[60] Vardhan H, Marwal A, Mathur P, Prajapat R. In silico characterization of hemagglutinin protein of H1N1 subtype. International Journal of Biological Technology. 2012;**3**:62-66

[61] Marwal A, Sahu A, Sharma P, Gaur RK. Transmission and host interaction of Geminivirus in weeds. In: Gaur RK, Hohn T, Sharma P, editors. Plant Virus-Host Interaction: Molecular Approaches and Viral Evolution. Waltham, MA, US: Academic Press, Elsevier; 2014. pp. 143-161. DOI: 10.1016/ B978-0-12-411584-2.00007-X

[62] Shojaei TR, Salleh MAM, Sijam K, Rahim RA, Mohsenifar A, Safarnejad R, et al. Fluorometric immunoassay for detecting the plant virus Citrus tristeza using carbon nanoparticles acting as quenchers and antibodies labeled with CdTe quantum dots. Microchimica Acta. 2016;**183**:2277-2287. DOI: 10.1007/ s00604-016-1867-7

[63] Hayden O, Bindeus R, Haderspock C, Mann KJ, Wirl B, Dickert FL. Mass sensitive detection of cells, viruses, and enzymes with artificial receptors. Sensors and Actuators B: Chemical. 2003;**91**:316-319. DOI: 10.1016/ S0925-4005(03)00093-5

[64] Chartuprayoon N, Rheem Y, Ng JCK, Nam J, Chend W, Myung NV. Polypyrrole nanoribbon based chemiresistive immunosensors for viral plant pathogen detection. Analytical Methods. 2013;**5**:3497-3502. DOI: 10.1039/C3AY40371H

[65] Elbeshehy EKF, Elazzazy AM, Aggelis G. Silver nanoparticles synthesis mediated by new isolates of Bacillus spp., nanoparticle characterization and their activity against bean yellow mosaic virus and human pathogens. Frontiers in Microbiology. 2015;**6**:453. DOI: 10.3389/ fmicb.2015.00453

[66] Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants. 2017;**3**:16207. DOI: 10.1038/nplants.2016.207

[67] Spicer CD, Jumeaux C, Gupta B, Stevens MM. Peptide and protein nanoparticle conjugates: Versatile platforms for biomedical applications. Chemical Society Reviews. 2018;**47**:3574. DOI: 10.1039/ C7CS00877E

[68] Jazayeri MH, Amani H, Pourfatollah AA, Toroudi HP, Sedighimoghaddam B. Various methods of gold nanoparticles (GNPs). Conjugation to antibodies. Sensing and Bio-Sensing Research. 2016;**9**:17-22. DOI: 10.1016/j. sbsr.2016.04.002

[69] McGee CF, Storey S, Clipson N, Doyle E. Soil microbial community responses to contamination with silver, aluminium oxide and silicon dioxide nanoparticles. Ecotoxicology. 2017;**26**:449-458. DOI: 10.1007/ s10646-017-1776-5

[70] Marwal A, Gaur RK, Khurana SMP. Possible approaches for developing different strategies to prevent transmission of Geminiviruses to important crops. In: Gaur RK, Khurana SMP, Dorokhov Y, editors. Plant Viruses: Diversity, Interaction and Management. Boca Raton, Florida, US: CRC Press, Taylor & Francis Group; 2018. pp. 301-319

[71] Sanfaçon H. Grand challenge in plant virology: Understanding the impact of plant viruses in model plants, in agricultural crops, and in

complex ecosystems. Frontiers in Microbiology. 2017;**8**:860. DOI: 10.3389/ fmicb.2017.00860

[72] Marwal A, Verma RK, Khurana SMP, Gaur RK. Molecular interactions between plant viruses and their biological vectors. In: Gaur RK, Khurana SMP, Dorokhov Y, editors. Plant Viruses: Diversity, Interaction and Management. Boca Raton, Florida, US: CRC Press, Taylor & Francis Group; 2018. pp. 205-216

[73] Das AK, Marwal A, Verma R. Plant protein-mediated green synthesis and characterization of magnetite (Fe3O4) Bionano hybrid. In: Bhoop BS, Sharma A, Mehta SK, Tripathi SK, editors. Nanotechnology: Novel Perspectives and Prospects. New Delhi, India: Tata-McGraw Hill; 2014. pp. 136-143

[74] Arts JH, Hadi M, Keene AM, Kreiling R, Lyon D, Maier M, et al. A critical appraisal of existing concepts for the grouping of nanomaterials. Regulatory Toxicology and Pharmacology. 2014;**70**:492-506. DOI: 10.1016/j.yrtph.2014.07.025

[75] San Miguel K, Scott JG. The next generation of insecticides: DsRNA is stable as a foliar-applied insecticide. Pest Management Science. 2016;**72**: 801-809. DOI: 10.1002/ps.4056

[76] Ghosh SKB, Hunter WB, Park AL, Gundersen-Rindal DE. Double strand RNA delivery system for plant-sap-feeding insects. PLoS ONE. 2017;**12**:e0171861. DOI: 10.1371/journal. pone.0171861

**31**

**1. Introduction**

**Chapter 3**

**Abstract**

Agriculture

Aspects in *Tobamovirus*

*Elisheva Smith and Aviv Dombrovsky*

Management in Intensive

In the recent years, disease spread of old and newly evolved tobamoviruses has occurred worldwide, affecting production of various vegetable and ornamental crops. The tobamoviruses are highly stable plant viruses that could cause severe disease symptoms. The well-known tobamovirus *Cucumber green mottle mosaic virus* (CGMMV) has recently caused severe damages in the cucumber, melon, and watermelon cucurbitaceous crops, worldwide. Similarly, a recent widespread of the newly identified tobamoviruses, *Tomato mottle mosaic virus* (ToMMV) and *Tomato brown rugose fruit virus* (ToBRFV), has reduced the solanaceous crop production. The primary route of tobamoviral infection is through mechanical means. These viruses adhere to agricultural facilities, contaminate the soil, infect seeds, and spread via beneficial pollinators and irrigation water. Mechanical plant injury suffices to initiate viral infection. Practicing hygiene by plant growers and in nurseries is currently the main strategy for mitigation of tobamoviral infection. Promoting the production of solanaceous vegetable crops genetically resistant to ToMMV and ToBRFV infection is a promising approach. However, CGMMV-resistant sources of cucurbitaceous vegetable crops are scarce. Conferring resistance to rootstocks and cross-protection strategies are newly implemented approaches that could alleviate

tobamovirus disease spread in both solanaceous and cucurbitaceous crops.

**Keywords:** Solanaceae, Cucurbitaceae, primary infection, secondary spread,

In the recent years, there has been a growing concern regarding disease damages and losses occurring in vegetable crop production. Plant viruses constitute the major causal factor for the diseases. Tomato plants, belonging to the Solanaceae family, and cucumber, melon, and watermelon plants, belonging to the Cucurbitaceae family, have shown the most severe disease symptoms. These symptoms are primarily attributed to infections by viruses belonging to the *Tobamovirus* genus, in the *Virgaviridae* family. The prevalent route of tobamovirus infection is via mechanical plant manipulations [1]. The tobamoviruses are highly stable and kept infectious in soil containing buried virus-contaminated plants [2], on various agricultural facility tool surfaces, in seeds [3], and upon adhering to beneficial pollinator body parts [4, 5]. Two tobamovirus species that had a conspicuous effect on vegetable crop production in various countries and caused severe disease symptoms in host plants are the *Tomato brown* 

strobilurins, resistant rootstocks, cross-protection

#### **Chapter 3**

*Plant Diseases-Current Threats and Management Trends*

complex ecosystems. Frontiers in Microbiology. 2017;**8**:860. DOI: 10.3389/

Francis Group; 2018. pp. 205-216

[74] Arts JH, Hadi M, Keene AM, Kreiling R, Lyon D, Maier M, et al. A critical appraisal of existing concepts for the grouping of nanomaterials.

Pharmacology. 2014;**70**:492-506. DOI:

[75] San Miguel K, Scott JG. The next generation of insecticides: DsRNA is stable as a foliar-applied insecticide. Pest Management Science. 2016;**72**: 801-809. DOI: 10.1002/ps.4056

[76] Ghosh SKB, Hunter WB, Park AL,

Gundersen-Rindal DE. Double strand RNA delivery system for plant-sap-feeding insects. PLoS ONE. 2017;**12**:e0171861. DOI: 10.1371/journal.

pone.0171861

Regulatory Toxicology and

10.1016/j.yrtph.2014.07.025

[73] Das AK, Marwal A, Verma R. Plant protein-mediated green synthesis and characterization of magnetite (Fe3O4) Bionano hybrid. In: Bhoop BS, Sharma A, Mehta SK, Tripathi SK, editors. Nanotechnology: Novel Perspectives and Prospects. New Delhi, India: Tata-McGraw Hill; 2014. pp. 136-143

[72] Marwal A, Verma RK, Khurana SMP, Gaur RK. Molecular interactions between plant viruses and their biological vectors. In: Gaur RK, Khurana SMP, Dorokhov Y, editors. Plant Viruses: Diversity, Interaction and Management. Boca Raton, Florida, US: CRC Press, Taylor &

fmicb.2017.00860

mediated by new isolates of Bacillus spp., nanoparticle characterization and their activity against bean yellow mosaic virus and human pathogens. Frontiers in Microbiology. 2015;**6**:453. DOI: 10.3389/

[66] Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants. 2017;**3**:16207. DOI: 10.1038/nplants.2016.207

[67] Spicer CD, Jumeaux C, Gupta B,

[68] Jazayeri MH, Amani H, Pourfatollah AA, Toroudi HP, Sedighimoghaddam B. Various methods of gold nanoparticles (GNPs). Conjugation to antibodies. Sensing and Bio-Sensing Research. 2016;**9**:17-22. DOI: 10.1016/j.

[69] McGee CF, Storey S, Clipson N, Doyle E. Soil microbial community responses to contamination with silver, aluminium oxide and silicon dioxide nanoparticles. Ecotoxicology. 2017;**26**:449-458. DOI: 10.1007/

[70] Marwal A, Gaur RK, Khurana SMP. Possible approaches for developing different strategies to prevent transmission of Geminiviruses to important crops. In: Gaur RK, Khurana SMP, Dorokhov Y, editors. Plant Viruses: Diversity, Interaction and Management. Boca Raton, Florida, US: CRC Press, Taylor & Francis Group; 2018.

[71] Sanfaçon H. Grand challenge in plant virology: Understanding the impact of plant viruses in model plants, in agricultural crops, and in

Stevens MM. Peptide and protein nanoparticle conjugates: Versatile platforms for biomedical applications. Chemical Society Reviews. 2018;**47**:3574. DOI: 10.1039/

C7CS00877E

sbsr.2016.04.002

s10646-017-1776-5

fmicb.2015.00453

**30**

pp. 301-319

## Aspects in *Tobamovirus* Management in Intensive Agriculture

*Elisheva Smith and Aviv Dombrovsky*

#### **Abstract**

In the recent years, disease spread of old and newly evolved tobamoviruses has occurred worldwide, affecting production of various vegetable and ornamental crops. The tobamoviruses are highly stable plant viruses that could cause severe disease symptoms. The well-known tobamovirus *Cucumber green mottle mosaic virus* (CGMMV) has recently caused severe damages in the cucumber, melon, and watermelon cucurbitaceous crops, worldwide. Similarly, a recent widespread of the newly identified tobamoviruses, *Tomato mottle mosaic virus* (ToMMV) and *Tomato brown rugose fruit virus* (ToBRFV), has reduced the solanaceous crop production. The primary route of tobamoviral infection is through mechanical means. These viruses adhere to agricultural facilities, contaminate the soil, infect seeds, and spread via beneficial pollinators and irrigation water. Mechanical plant injury suffices to initiate viral infection. Practicing hygiene by plant growers and in nurseries is currently the main strategy for mitigation of tobamoviral infection. Promoting the production of solanaceous vegetable crops genetically resistant to ToMMV and ToBRFV infection is a promising approach. However, CGMMV-resistant sources of cucurbitaceous vegetable crops are scarce. Conferring resistance to rootstocks and cross-protection strategies are newly implemented approaches that could alleviate tobamovirus disease spread in both solanaceous and cucurbitaceous crops.

**Keywords:** Solanaceae, Cucurbitaceae, primary infection, secondary spread, strobilurins, resistant rootstocks, cross-protection

#### **1. Introduction**

In the recent years, there has been a growing concern regarding disease damages and losses occurring in vegetable crop production. Plant viruses constitute the major causal factor for the diseases. Tomato plants, belonging to the Solanaceae family, and cucumber, melon, and watermelon plants, belonging to the Cucurbitaceae family, have shown the most severe disease symptoms. These symptoms are primarily attributed to infections by viruses belonging to the *Tobamovirus* genus, in the *Virgaviridae* family. The prevalent route of tobamovirus infection is via mechanical plant manipulations [1]. The tobamoviruses are highly stable and kept infectious in soil containing buried virus-contaminated plants [2], on various agricultural facility tool surfaces, in seeds [3], and upon adhering to beneficial pollinator body parts [4, 5]. Two tobamovirus species that had a conspicuous effect on vegetable crop production in various countries and caused severe disease symptoms in host plants are the *Tomato brown* 

*rugose fruit virus* (ToBRFV) that infected solanaceous plants [6, 7] and *Cucumber green mottle mosaic virus* (CGMMV) that infected cucurbitaceous plants [8]. An important strategy to reduce viral infection of cultivated crops is to practice hygiene during planting and to divide the planting procedures between workers. The use of appropriate chemicals for disinfection of trellising ropes, planting trays in nurseries, and the various agricultural tools, before planting, is highly recommended [9]. Importantly, the applications of highly sensitive methods to disclose virus-infected seeds [6, 10] increase the probability to sow virus-free seeds. The various maneuvers currently available for tobamoviral disease management and future strategies to alleviate tobamoviral infections are discussed below.

#### **2. Tobamovirus worldwide spread**

Viruses belonging to the *Tobamovirus* genus are positive-sense single-stranded RNA viruses that infect a wide range of plant species. *Tobacco mosaic virus* (TMV), first described by Mayer in 1886 [11], is the prototype of this genus, in the *Virgaviridae* family. Tobamoviruses infect vegetable crops mostly solanaceous and cucurbitaceous plants, ornamental plants, weeds, and medicinal plants. In the recent years, the spread of tobamoviruses that infect two major cultivated vegetable crops, the solanaceous and cucurbitaceous plants, has increased. The *Tomato mottle mosaic virus* (ToMMV) that infected tomato plants (*Solanum lycopersicum*) had spread in America and Spain [12, 13]. In the Middle East, ToBRFV had broken the highly durable resistance-conferring allele *Tm*-22 [6] that was introgressed into *Lycopersicon esculentum* from *L. peruvianum* [14]. Phylogenetic tree analysis showed that ToBRFV and ToMMV were clustered in separate clades [6]. ToBRFV infection of tomato plants has recently occurred in Mexico [15], Germany [16], and the USA [17]. A worldwide infection of the cucurbitaceous plants has occurred due to the spread of the tobamovirus CGMMV, first reported by Ainsworth in 1935 [8, 18]. Excluding few reports on CGMMV-resistant plants, commercial cultivars resistant to CGMMV are scarce [19, 20].

#### **3. Genome organization**

The genome organization of the tobamoviruses ToBRFV and CGMMV resembles in general that of TMV [21, 22]. The virus single-stranded RNA genome encodes four known proteins: short (126 or 129 kDa) and long (183 or 186 kDa) replicase-associated proteins. The long component is the outcome of a translational read-through of a termination codon of the short component. In addition, a movement protein (MP) of ~30 kDa and a coat protein (CP) of ~17 kDa are translated from sub-genomic RNA. A putative fifth 54 kDa protein resides between the two replicase-associated proteins [23]. Recently, in Solanaceae-infecting tobamoviruses, a sixth protein of 4–5 kDa has been identified, which is encoded by a region in the genome overlapping the open reading frames (ORFs) of the MP and the CP [24–26].

#### **4. Particle pathogenicity and systemic disease spread**

ToBRFV infection of solanaceous plants induced pathogenic systemic symptoms of narrowing leaves and yellow and brown spotted fruits. CGMMV infection of cucurbitaceous plants resulted in systemic mottle mosaic leaves and fruits as well as yellowing fruit flesh combined with necrotic peduncles (**Figure 1**). Increased severity of the symptoms could occur due to a variety of mixed infections. For example,

**33**

**Figure 1.**

*Pythium spinosum.*

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

the solanaceous tomato plants infected by both ToBRFV and the abundant tospovirus *Tomato spotted wilt virus* (TSWV) showed severe fruit necrosis (**Figure 1d**), and the cucurbitaceous cucumber plants infected by both CGMMV and the *Pythium*

The virulence factors that caused the severe symptoms occurring upon tobamovirus infection have not been established yet excluding the virulence factor of TMV upon infection of *Nicotiana benthamiana* that was identified as the orf6-expressed protein, which occurs in Solanaceae-infecting tobamoviruses [24]. Similarly, the

The viral MP is the avirulence factor recognized by the plant resistance-conferring

However, the MP modifications that have occurred during the evolvement of ToBRFV are still unknown, although in bioinformatics approach several potential mutations were identified in the MP of ToBRFV. In the Cucurbitaceae-infecting CGMMV, a single amino acid substitution at the replicase site resulted in symptom attenuation [30], conferring a role for the replicase in viral virulence mechanism.

*Tomato brown rugose fruit virus (ToBRFV) and Cucumber green mottle mosaic virus (CGMMV) infected vegetables. (a–d) ToBRFV-infected plants; (e–j) CGMMV-infected plants. (a) Narrowing tomato leaves with mosaic pattern. (b, c) Yellow and brown spotted tomato fruits. (d) Brown spotted and necrotic tomato fruits infected by both ToBRFV and Tomato spotted wilt virus. (e) Mottle mosaic pattern on watermelon leaves. (f) Yellowing and necrotic watermelons. (g) Necrotic peduncle. (h) Mosaic pattern on melon leaves. (i) Various manifestations of mottle mosaic melons. (j) Collapse of cucumber plants infected by both CGMMV and* 

resistance breaking by ToBRFV has not been discovered yet.

allele [29].

[28]. Mutational analysis of the MP revealed that a change of two

species *P. spinosum* showed plant wilting and collapse [27] (**Figure 1j**).

amino acids could overcome the resistance conferred by the *Tm*-2<sup>2</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

mechanism of *Tm*-2<sup>2</sup>

protein *Tm*-2<sup>2</sup>

#### *Aspects in* Tobamovirus *Management in Intensive Agriculture DOI: http://dx.doi.org/10.5772/intechopen.87101*

*Plant Diseases-Current Threats and Management Trends*

alleviate tobamoviral infections are discussed below.

**2. Tobamovirus worldwide spread**

resistant to CGMMV are scarce [19, 20].

**3. Genome organization**

*rugose fruit virus* (ToBRFV) that infected solanaceous plants [6, 7] and *Cucumber green mottle mosaic virus* (CGMMV) that infected cucurbitaceous plants [8]. An important strategy to reduce viral infection of cultivated crops is to practice hygiene during planting and to divide the planting procedures between workers. The use of appropriate chemicals for disinfection of trellising ropes, planting trays in nurseries, and the various agricultural tools, before planting, is highly recommended [9]. Importantly, the applications of highly sensitive methods to disclose virus-infected seeds [6, 10] increase the probability to sow virus-free seeds. The various maneuvers currently available for tobamoviral disease management and future strategies to

Viruses belonging to the *Tobamovirus* genus are positive-sense single-stranded RNA viruses that infect a wide range of plant species. *Tobacco mosaic virus* (TMV), first described by Mayer in 1886 [11], is the prototype of this genus, in the *Virgaviridae* family. Tobamoviruses infect vegetable crops mostly solanaceous and cucurbitaceous plants, ornamental plants, weeds, and medicinal plants. In the recent years, the spread of tobamoviruses that infect two major cultivated vegetable crops, the solanaceous and cucurbitaceous plants, has increased. The *Tomato mottle mosaic virus* (ToMMV) that infected tomato plants (*Solanum lycopersicum*) had spread in America and Spain [12, 13]. In the Middle East, ToBRFV had broken the highly durable resistance-conferring allele

 [6] that was introgressed into *Lycopersicon esculentum* from *L. peruvianum* [14]. Phylogenetic tree analysis showed that ToBRFV and ToMMV were clustered in separate clades [6]. ToBRFV infection of tomato plants has recently occurred in Mexico [15], Germany [16], and the USA [17]. A worldwide infection of the cucurbitaceous plants has occurred due to the spread of the tobamovirus CGMMV, first reported by Ainsworth in 1935 [8, 18]. Excluding few reports on CGMMV-resistant plants, commercial cultivars

The genome organization of the tobamoviruses ToBRFV and CGMMV resembles in general that of TMV [21, 22]. The virus single-stranded RNA genome encodes four known proteins: short (126 or 129 kDa) and long (183 or 186 kDa) replicase-associated proteins. The long component is the outcome of a translational read-through of a termination codon of the short component. In addition, a movement protein (MP) of ~30 kDa and a coat protein (CP) of ~17 kDa are translated from sub-genomic RNA. A putative fifth 54 kDa protein resides between the two replicase-associated proteins [23]. Recently, in Solanaceae-infecting tobamoviruses, a sixth protein of 4–5 kDa has been identified, which is encoded by a region in the genome overlapping the open reading frames (ORFs) of the MP and the CP [24–26].

ToBRFV infection of solanaceous plants induced pathogenic systemic symptoms

of narrowing leaves and yellow and brown spotted fruits. CGMMV infection of cucurbitaceous plants resulted in systemic mottle mosaic leaves and fruits as well as yellowing fruit flesh combined with necrotic peduncles (**Figure 1**). Increased severity of the symptoms could occur due to a variety of mixed infections. For example,

**4. Particle pathogenicity and systemic disease spread**

**32**

*Tm*-22

the solanaceous tomato plants infected by both ToBRFV and the abundant tospovirus *Tomato spotted wilt virus* (TSWV) showed severe fruit necrosis (**Figure 1d**), and the cucurbitaceous cucumber plants infected by both CGMMV and the *Pythium* species *P. spinosum* showed plant wilting and collapse [27] (**Figure 1j**).

The virulence factors that caused the severe symptoms occurring upon tobamovirus infection have not been established yet excluding the virulence factor of TMV upon infection of *Nicotiana benthamiana* that was identified as the orf6-expressed protein, which occurs in Solanaceae-infecting tobamoviruses [24]. Similarly, the mechanism of *Tm*-2<sup>2</sup> resistance breaking by ToBRFV has not been discovered yet. The viral MP is the avirulence factor recognized by the plant resistance-conferring protein *Tm*-2<sup>2</sup> [28]. Mutational analysis of the MP revealed that a change of two amino acids could overcome the resistance conferred by the *Tm*-2<sup>2</sup> allele [29]. However, the MP modifications that have occurred during the evolvement of ToBRFV are still unknown, although in bioinformatics approach several potential mutations were identified in the MP of ToBRFV. In the Cucurbitaceae-infecting CGMMV, a single amino acid substitution at the replicase site resulted in symptom attenuation [30], conferring a role for the replicase in viral virulence mechanism.

#### **Figure 1.**

*Tomato brown rugose fruit virus (ToBRFV) and Cucumber green mottle mosaic virus (CGMMV) infected vegetables. (a–d) ToBRFV-infected plants; (e–j) CGMMV-infected plants. (a) Narrowing tomato leaves with mosaic pattern. (b, c) Yellow and brown spotted tomato fruits. (d) Brown spotted and necrotic tomato fruits infected by both ToBRFV and Tomato spotted wilt virus. (e) Mottle mosaic pattern on watermelon leaves. (f) Yellowing and necrotic watermelons. (g) Necrotic peduncle. (h) Mosaic pattern on melon leaves. (i) Various manifestations of mottle mosaic melons. (j) Collapse of cucumber plants infected by both CGMMV and Pythium spinosum.*

The tobamovirus CP molecules constitute the capsid of the virion, which is ~300 nm long and 18 nm wide. For viral RNA encapsidation, *ca*. 2000 CP subunits form a right-handed helix in which each subunit binds three nucleotides. There are electrostatic interactions between charged amino acid residues that contribute to CP subunit interactions and particle stability, which is strengthened by hydrophobic contacts in the capsid [31] and carboxylate interactions between subunits [32]. Tobamovirus particles can survive 90°C heating and years of storage [31, 33].

Tobamovirus encapsidation is necessary for long-distance movement of the virus in the plant but is dispensable for cell-to-cell movement [34, 35]. Since viruses are localized in the symplast, it is necessary to maneuver cell-to-cell movement. Viral MP binds RNA and increases the plasmodesmata size exclusion limit [36]. The virus could then be transmitted via the widened cytoplasmic continuity that was formed between cells [37]. In addition to the MP effect on viral cell-to-cell movement, a role for the short replicase-associated protein in cell-to-cell viral movement was also observed, although the mechanism of the replicase effect is still unclear [38].

Tobamovirus CP is required for long-distance viral dissemination [34, 39]. It is required for viral movement across the boundary between vascular parenchyma and companion cells [40]. It is not quite clear, however, whether the CP is necessary for interactions with host factors [39]. In the phloem, virus particles follow photoassimilate transportation [41]. However, mechanisms of entry and egress from the phloem differ [42, 43]. For example, egress from the phloem involves the activity of the host plant pectin methylesterase [44].

Viral genome replication that occurs in the epidermis and mesophyll cells induces plant resistance programs such as the RNA silencing process [45, 46]. Plant RNA silencing is triggered by viral double-stranded RNA precursors, which are processed by RNase III Dicer-like protein to small interfering RNA duplexes (siRNA), 21–24 nucleotide long [47, 48]. The siRNAs are stabilized by HUA ENHANCER1 (HEN1), which catalyzes methylation at the 3′ end, generating 2′-*O*-methylated siRNAs [49]. The methylation prevents uridylation and degradation of the siRNA duplexes [49, 50]. Single-stranded siRNAs direct ARGONAUTE (AGO) protein residing in RNA-induced silencing complex (RISC) to silence posttranscriptionally complementary RNA by endonucleolytic activity [51]. Importantly, small RNA duplexes that are formed by the plant silencing mechanism function also as silencing signals that spread via the plasmodesmata between cells and systemically through the phloem [52, 53]. Establishing silencing process systemically can lead to degradation of newly infecting viruses prior to viral replication [54]. Viruses counteract the plant silencing process by the expression of silencing suppressors [55, 56]. The tobamovirus short protein associated with the replicase, such as the 126 kDa protein of TMV [57] or ToMV [58] interferes with the methylation of the siRNA duplexes catalyzed by HEN1 and thereby induces degradation of the siRNAs [54]. Viral RNA silencing suppressors are therefore positive factors in viral long-distance movement [39]. However, the contribution of the replicase-associated protein to viral entry into the phloem is not clear yet but could not be attributed to suppression of RNA silencing [33].

#### **5. Modes of infection**

#### **5.1 Primary infections**

#### *5.1.1 Seeds*

Tobamoviruses are seed-borne viruses, although the average of reported seed to seedling transmission ratios was only 4.1% [3]. Low viral transmission ratios in

**35**

*5.1.2 Soil*

*5.1.3 Beneficial pollinators*

**5.2 Secondary disease spread**

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

to seedling transmission, and seed coat contamination of the soil.

Tobamovirus soil contamination primarily occurs due to buried plant debris originated from tobamovirus-infected crops [63]. Using a serological method for ToMV detection, a high primary infection ratio, of up to 80%, apparently occurred in the tomato plants grown in the contaminated soil [63]. Under field conditions as well, ToMV was detected in soil containing tomato debris of crops originated from a previous year planting [2]. ToMV was also recovered from forest soil in which the mineral fraction had more virus than the organic fraction [64]. Clay in the soil adsorbs a high fraction of tobamovirus particles [65], which could be visualized by scanning electron microscopy. Similarly, CGMMV inoculum buried in the ground for overwintering contaminated the soil [66]. Soil contamination was apparent by CGMMV detection in the soil supernatant, by inoculating uninfected cucurbit plants with the soil supernatant and by planting uninfected cucurbit plants in the contaminated soil [66]. Various soil types mediate CGMMV dispersal in various efficiencies,

which could be attributed to root damage in the case of rock containing soil.

Bumblebees (*Bombus terrestris*) are essential beneficial pollinators of tomato crop cultivation. Bumblebee hives that were placed in ToBRFV-infected tomato-growing areas were ToBRFV contaminated [5]. The virus was detected in the hive components: the comb, the enveloping cotton, and the nectar. ToBRFV in the hives was infectious as studied by inoculating the laboratory test plant *Nicotiana tabacum* cv. Samsun with virus purified from the hive comb. ToBRFV adhered to the bumblebee body parts, primarily the abdomen, suggesting that ToBRFV could be transmitted by buzz pollination. Importantly, bumblebee hives from ToBRFV-infected tomato greenhouses placed in a new greenhouse of uninfected tomato plants constituted carriers of a primary infectious inoculum. ToBRFV infection ratios of the newly infected tomato plants were 12–60%. The ToBRFV infection ratios were positively correlated with bumblebee activity [5].

Tobamovirus disease spread is abiotic. Mechanical manipulations during crop cultivation and the commonly associated plant injury constitute a major route for

grow-out experiments are commonly indicative of uninfected embryos. Seed coat contamination could occur due to physical attachment between the seeds and the fruit flesh. However, there were also reports on ToMV infecting the endosperm of tomato plant seeds [1]. The tobamovirus passage through the maternal seed coat to the endosperm, which originates from both maternal and paternal sources, is enigmatic in the face of the uninfected embryos [3]. The consequence of endosperm infection is problematic, in particular, for considerations of the appropriate seed disinfection procedures. CGMMV-infected cucurbitaceous seeds are the most challenging for disinfection practices. Seeds from symptomatic CGMMV-infected cucurbit plants showed tobamoviral infection of both the seed coat and the perispermendosperm (PE) envelope underlying the seed coat [59, 60], which is characteristic to cucurbits [61, 62]. Importantly, the PE envelope is comprised of endospermic cells on top of which noncellular lipid and callose layers were formed [61, 62]. A similar question is raised therefore regarding CGMMV occurrence in the PE envelope in the face of the uninfected embryos [59, 60]. Tobamoviral dispersal emerging from infected seeds could occur via physical manipulations of the seeds upon sowing, seed

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

#### *Aspects in* Tobamovirus *Management in Intensive Agriculture DOI: http://dx.doi.org/10.5772/intechopen.87101*

grow-out experiments are commonly indicative of uninfected embryos. Seed coat contamination could occur due to physical attachment between the seeds and the fruit flesh. However, there were also reports on ToMV infecting the endosperm of tomato plant seeds [1]. The tobamovirus passage through the maternal seed coat to the endosperm, which originates from both maternal and paternal sources, is enigmatic in the face of the uninfected embryos [3]. The consequence of endosperm infection is problematic, in particular, for considerations of the appropriate seed disinfection procedures. CGMMV-infected cucurbitaceous seeds are the most challenging for disinfection practices. Seeds from symptomatic CGMMV-infected cucurbit plants showed tobamoviral infection of both the seed coat and the perispermendosperm (PE) envelope underlying the seed coat [59, 60], which is characteristic to cucurbits [61, 62]. Importantly, the PE envelope is comprised of endospermic cells on top of which noncellular lipid and callose layers were formed [61, 62]. A similar question is raised therefore regarding CGMMV occurrence in the PE envelope in the face of the uninfected embryos [59, 60]. Tobamoviral dispersal emerging from infected seeds could occur via physical manipulations of the seeds upon sowing, seed to seedling transmission, and seed coat contamination of the soil.

#### *5.1.2 Soil*

*Plant Diseases-Current Threats and Management Trends*

the host plant pectin methylesterase [44].

The tobamovirus CP molecules constitute the capsid of the virion, which is ~300 nm long and 18 nm wide. For viral RNA encapsidation, *ca*. 2000 CP subunits form a right-handed helix in which each subunit binds three nucleotides. There are electrostatic interactions between charged amino acid residues that contribute to CP subunit interactions and particle stability, which is strengthened by hydrophobic contacts in the capsid [31] and carboxylate interactions between subunits [32]. Tobamovirus particles can survive 90°C heating and years of storage [31, 33].

Tobamovirus encapsidation is necessary for long-distance movement of the virus in the plant but is dispensable for cell-to-cell movement [34, 35]. Since viruses are localized in the symplast, it is necessary to maneuver cell-to-cell movement. Viral MP binds RNA and increases the plasmodesmata size exclusion limit [36]. The virus could then be transmitted via the widened cytoplasmic continuity that was formed between cells [37]. In addition to the MP effect on viral cell-to-cell movement, a role for the short replicase-associated protein in cell-to-cell viral movement was also observed, although the mechanism of the replicase effect is still unclear [38].

Tobamovirus CP is required for long-distance viral dissemination [34, 39]. It is required for viral movement across the boundary between vascular parenchyma and companion cells [40]. It is not quite clear, however, whether the CP is necessary for interactions with host factors [39]. In the phloem, virus particles follow photoassimilate transportation [41]. However, mechanisms of entry and egress from the phloem differ [42, 43]. For example, egress from the phloem involves the activity of

Viral genome replication that occurs in the epidermis and mesophyll cells induces

plant resistance programs such as the RNA silencing process [45, 46]. Plant RNA silencing is triggered by viral double-stranded RNA precursors, which are processed by RNase III Dicer-like protein to small interfering RNA duplexes (siRNA), 21–24 nucleotide long [47, 48]. The siRNAs are stabilized by HUA ENHANCER1 (HEN1), which catalyzes methylation at the 3′ end, generating 2′-*O*-methylated siRNAs [49]. The methylation prevents uridylation and degradation of the siRNA duplexes [49, 50]. Single-stranded siRNAs direct ARGONAUTE (AGO) protein residing in RNA-induced silencing complex (RISC) to silence posttranscriptionally complementary RNA by endonucleolytic activity [51]. Importantly, small RNA duplexes that are formed by the plant silencing mechanism function also as silencing signals that spread via the plasmodesmata between cells and systemically through the phloem [52, 53]. Establishing silencing process systemically can lead to degradation of newly infecting viruses prior to viral replication [54]. Viruses counteract the plant silencing process by the expression of silencing suppressors [55, 56]. The tobamovirus short protein associated with the replicase, such as the 126 kDa protein of TMV [57] or ToMV [58] interferes with the methylation of the siRNA duplexes catalyzed by HEN1 and thereby induces degradation of the siRNAs [54]. Viral RNA silencing suppressors are therefore positive factors in viral long-distance movement [39]. However, the contribution of the replicase-associated protein to viral entry into the phloem is not clear yet but could not be attributed to suppression of RNA silencing [33].

Tobamoviruses are seed-borne viruses, although the average of reported seed to seedling transmission ratios was only 4.1% [3]. Low viral transmission ratios in

**34**

*5.1.1 Seeds*

**5. Modes of infection**

**5.1 Primary infections**

Tobamovirus soil contamination primarily occurs due to buried plant debris originated from tobamovirus-infected crops [63]. Using a serological method for ToMV detection, a high primary infection ratio, of up to 80%, apparently occurred in the tomato plants grown in the contaminated soil [63]. Under field conditions as well, ToMV was detected in soil containing tomato debris of crops originated from a previous year planting [2]. ToMV was also recovered from forest soil in which the mineral fraction had more virus than the organic fraction [64]. Clay in the soil adsorbs a high fraction of tobamovirus particles [65], which could be visualized by scanning electron microscopy. Similarly, CGMMV inoculum buried in the ground for overwintering contaminated the soil [66]. Soil contamination was apparent by CGMMV detection in the soil supernatant, by inoculating uninfected cucurbit plants with the soil supernatant and by planting uninfected cucurbit plants in the contaminated soil [66]. Various soil types mediate CGMMV dispersal in various efficiencies, which could be attributed to root damage in the case of rock containing soil.

#### *5.1.3 Beneficial pollinators*

Bumblebees (*Bombus terrestris*) are essential beneficial pollinators of tomato crop cultivation. Bumblebee hives that were placed in ToBRFV-infected tomato-growing areas were ToBRFV contaminated [5]. The virus was detected in the hive components: the comb, the enveloping cotton, and the nectar. ToBRFV in the hives was infectious as studied by inoculating the laboratory test plant *Nicotiana tabacum* cv. Samsun with virus purified from the hive comb. ToBRFV adhered to the bumblebee body parts, primarily the abdomen, suggesting that ToBRFV could be transmitted by buzz pollination. Importantly, bumblebee hives from ToBRFV-infected tomato greenhouses placed in a new greenhouse of uninfected tomato plants constituted carriers of a primary infectious inoculum. ToBRFV infection ratios of the newly infected tomato plants were 12–60%. The ToBRFV infection ratios were positively correlated with bumblebee activity [5].

#### **5.2 Secondary disease spread**

Tobamovirus disease spread is abiotic. Mechanical manipulations during crop cultivation and the commonly associated plant injury constitute a major route for tobamovirus disease spread. Low concentrations of tobamovirus contamination could establish the disease spread in a growing area due to mechanical manipulations [1]. For effective tobamovirus disease transmission, leaf or root injury seems imperative. Although the most common way of tobamovirus disease spread is via physical attachment, root-to-root viral transmission ratios in tobamoviruscontaminated soil are low [67]. Similarly, seeds sown in tobamovirus-contaminated soil showed low infection ratios when compared to seedling planting, which could involve plant injury [68]. However, high concentrations of the contaminating tobamoviruses and repeated exposure to the infectious source reduce the impact of injury as a necessary determinant in tobamovirus disease spread [63, 65].

#### *5.2.1 Irrigation water*

Humidity preserves tobamovirus particle viability in soil. Infectious tobamovirus particles of TMV and ToMV were found in environmental waters such as ponds and streams. The occurrence of the tobamoviruses was visualized by electron microscopy, and the infectivity of the tobamovirus particles was examined in a biological assay on laboratory test plants [69]. A quantitative and sensitive approach to detect the tobamoviruses in environmental waters was also applied using sensitive reverse-transcription real-time PCR analysis [70]. Apparently, there were environmental water samples that tested positive for ToMV without the usual sample concentration step. Dispersal of the Cucurbitaceae-infecting tobamovirus CGMMV via irrigation water was tested, for example, in laboratory facilities in the Volcani Center, Israel. In the middle of a planting tray of cucumber (*Cucumis sativus*) plants, one plant was sap-inoculated with the virus and was then separated from adjacent plants by an open plastic vessel to prevent any mechanical transmission of the virus via any other way than that of the irrigation dripping system that was applied. The results inspected a month later showed that CGMMV infection ratios ranged between 36 and 91%, while the control plants were CGMMV free (Dombrovsky and Darzi, unpublished data). CGMMV transmission efficiency via dripping and flooding irrigation systems was examined in a glasshouse experiment. The distances of CGMMV infection of watermelon (*Citrullus lanatus* Thunb.) plants by dripping and flooding irrigation systems were 1.9 and 2.3 m, respectively [71]. CGMMV was also detected in a river close to a farm of CGMMV-infected muskmelon (*Cucumis melo*) and watermelon plants [72].

#### *5.2.2 Plant manipulations*

Contaminated hands, pruning shears, knives, trellising ropes, and grafting procedures are the most common means for effective tobamovirus transmission. Attempts to quantitate the contribution of mechanical contact to tobamovirus disease spread revealed that a high number of repeated contacts between TMVinfected tobacco leaves and uninfected plants were positively correlated with increased tobamovirus transmission efficiency [63]. Interestingly, there was no correlation between the TMV quantity in the source leaves and the efficiency of viral transmission. Several characteristics of the source of infection could also affect transmission efficiency, such as leaf age and the viral source, whether it was the outcome of systemic spread or it was the primary inoculated material. However, the effects of these parameters on TMV disease spread were not conclusive [73]. Quantitating the disease transmission ratios of the Cucurbitaceae-infecting CGMMV in cucumber plants was conducted, for example, by touching the plants with CGMMV-contaminated hands. CGMMV contamination analyzed serologically 3 weeks post the infection procedure spread down the row, sequentially,

**37**

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

and the infection ratio was 86% [68]. The contribution of agro-technical work of pruning and trellising to CGMMV disease spread was monitored in an experiment conducted in commercial cucumber greenhouses [68]. In the greenhouses, 5–11 scattered CGMMV-infected plants, which constituted 0.4–0.5% of the plants, were identified, and a survey was conducted on the effects of the intensive agro-technical activities on CGMMV disease spread. The percent increase in infected plants due to agro-technical practice for 40 days, in the various greenhouses, was in the range of

Beneficial pollinators do not only constitute a primary source of tobamovirus disease spread, as was observed in ToBRFV spread analysis [5], but could also promote secondary viral spread between tobamovirus-infected and uninfected plants. Hives containing bumblebees (*Bombus terrestris* L.) placed in a greenhouse of TMV-infected tomato plants (*Lycopersicon esculentum* L. cv. Momotaro) spread the TMV infection to adjacent uninfected tomato plants planted in the greenhouse. TMV viral particles attached to the bumblebee body parts were visualized by electron microscopy and tested positive for TMV in a serological assay. TMV viral particles isolated from the hive components were infectious, as analyzed in a biological assay using *Nicotiana glutinosa* seedlings for inoculation [74]. Regarding the Cucurbitaceae-infecting tobamovirus CGMMV, the honeybee *Apis mellifera* promoted disease spread between infected melon seedlings, which constituted a primary viral source, and adjacent uninfected plants [4]. Efficient secondary viral transmission between the plants occurred when the uninfected plants were placed on the path of the honeybee foraging track, between the beehive and the CGMMV-

Volunteer plants such as weeds could have an important role as tobamovirus reservoirs that may constitute a source of infection. Apparently, weed species could constitute asymptomatic reservoirs of the Cucurbitaceae-infecting tobamovirus CGMMV [75, 76]. Among the weed species susceptible to CGMMV infection that did not show any conspicuous symptom development and their susceptibility to CGMMV infection which was confirmed by laboratory mechanical inoculations were *Molucella laevis*, *Amaranthus graecizans*, and the medicinal plant *Withania somnifera* [76]. Overwintering of tobamoviruses in the weed hosts could promote the tobamovirus spread between cultivated crops of consecutive growing seasons.

In order to prevent the occurrence and establishment of tobamovirus primary infectious source introduced by virus-infected seeds, the appropriate detection methods should be applied. Viral RNA extraction (using Viral RNA Extraction Kit; Bioneer) from ToBRFV-infected tomato (*S. lycopersicum*) seeds (Luria and Dombrovsky, unpublished data) and CGMMV-infected cucumber (*C. sativus* Derben) and melon (*C. melo* Raanan) seeds were successfully executed [60]. Nextgeneration sequencing (NGS) platform has been successfully applied for detection of the tobamoviruses ToBRFV [6] and CGMMV [77]. The NGS detection method

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

11–32% [68].

infected plants.

**6. Management strategies**

**6.1 Sensitive tobamovirus detection methods**

*5.2.4 Weeds*

*5.2.3 Beneficial pollinators*

and the infection ratio was 86% [68]. The contribution of agro-technical work of pruning and trellising to CGMMV disease spread was monitored in an experiment conducted in commercial cucumber greenhouses [68]. In the greenhouses, 5–11 scattered CGMMV-infected plants, which constituted 0.4–0.5% of the plants, were identified, and a survey was conducted on the effects of the intensive agro-technical activities on CGMMV disease spread. The percent increase in infected plants due to agro-technical practice for 40 days, in the various greenhouses, was in the range of 11–32% [68].

#### *5.2.3 Beneficial pollinators*

*Plant Diseases-Current Threats and Management Trends*

*5.2.1 Irrigation water*

*5.2.2 Plant manipulations*

tobamovirus disease spread. Low concentrations of tobamovirus contamination could establish the disease spread in a growing area due to mechanical manipulations [1]. For effective tobamovirus disease transmission, leaf or root injury seems imperative. Although the most common way of tobamovirus disease spread is via physical attachment, root-to-root viral transmission ratios in tobamoviruscontaminated soil are low [67]. Similarly, seeds sown in tobamovirus-contaminated soil showed low infection ratios when compared to seedling planting, which could involve plant injury [68]. However, high concentrations of the contaminating tobamoviruses and repeated exposure to the infectious source reduce the impact of

injury as a necessary determinant in tobamovirus disease spread [63, 65].

muskmelon (*Cucumis melo*) and watermelon plants [72].

Humidity preserves tobamovirus particle viability in soil. Infectious tobamovirus particles of TMV and ToMV were found in environmental waters such as ponds and streams. The occurrence of the tobamoviruses was visualized by electron microscopy, and the infectivity of the tobamovirus particles was examined in a biological assay on laboratory test plants [69]. A quantitative and sensitive approach to detect the tobamoviruses in environmental waters was also applied using sensitive reverse-transcription real-time PCR analysis [70]. Apparently, there were environmental water samples that tested positive for ToMV without the usual sample concentration step. Dispersal of the Cucurbitaceae-infecting tobamovirus CGMMV via irrigation water was tested, for example, in laboratory facilities in the Volcani Center, Israel. In the middle of a planting tray of cucumber (*Cucumis sativus*) plants, one plant was sap-inoculated with the virus and was then separated from adjacent plants by an open plastic vessel to prevent any mechanical transmission of the virus via any other way than that of the irrigation dripping system that was applied. The results inspected a month later showed that CGMMV infection ratios ranged between 36 and 91%, while the control plants were CGMMV free (Dombrovsky and Darzi, unpublished data). CGMMV transmission efficiency via dripping and flooding irrigation systems was examined in a glasshouse experiment. The distances of CGMMV infection of watermelon (*Citrullus lanatus* Thunb.) plants by dripping and flooding irrigation systems were 1.9 and 2.3 m, respectively [71]. CGMMV was also detected in a river close to a farm of CGMMV-infected

Contaminated hands, pruning shears, knives, trellising ropes, and grafting procedures are the most common means for effective tobamovirus transmission. Attempts to quantitate the contribution of mechanical contact to tobamovirus disease spread revealed that a high number of repeated contacts between TMVinfected tobacco leaves and uninfected plants were positively correlated with increased tobamovirus transmission efficiency [63]. Interestingly, there was no correlation between the TMV quantity in the source leaves and the efficiency of viral transmission. Several characteristics of the source of infection could also affect transmission efficiency, such as leaf age and the viral source, whether it was the outcome of systemic spread or it was the primary inoculated material. However, the effects of these parameters on TMV disease spread were not conclusive [73]. Quantitating the disease transmission ratios of the Cucurbitaceae-infecting CGMMV in cucumber plants was conducted, for example, by touching the plants with CGMMV-contaminated hands. CGMMV contamination analyzed serologically 3 weeks post the infection procedure spread down the row, sequentially,

**36**

Beneficial pollinators do not only constitute a primary source of tobamovirus disease spread, as was observed in ToBRFV spread analysis [5], but could also promote secondary viral spread between tobamovirus-infected and uninfected plants. Hives containing bumblebees (*Bombus terrestris* L.) placed in a greenhouse of TMV-infected tomato plants (*Lycopersicon esculentum* L. cv. Momotaro) spread the TMV infection to adjacent uninfected tomato plants planted in the greenhouse. TMV viral particles attached to the bumblebee body parts were visualized by electron microscopy and tested positive for TMV in a serological assay. TMV viral particles isolated from the hive components were infectious, as analyzed in a biological assay using *Nicotiana glutinosa* seedlings for inoculation [74]. Regarding the Cucurbitaceae-infecting tobamovirus CGMMV, the honeybee *Apis mellifera* promoted disease spread between infected melon seedlings, which constituted a primary viral source, and adjacent uninfected plants [4]. Efficient secondary viral transmission between the plants occurred when the uninfected plants were placed on the path of the honeybee foraging track, between the beehive and the CGMMVinfected plants.

#### *5.2.4 Weeds*

Volunteer plants such as weeds could have an important role as tobamovirus reservoirs that may constitute a source of infection. Apparently, weed species could constitute asymptomatic reservoirs of the Cucurbitaceae-infecting tobamovirus CGMMV [75, 76]. Among the weed species susceptible to CGMMV infection that did not show any conspicuous symptom development and their susceptibility to CGMMV infection which was confirmed by laboratory mechanical inoculations were *Molucella laevis*, *Amaranthus graecizans*, and the medicinal plant *Withania somnifera* [76]. Overwintering of tobamoviruses in the weed hosts could promote the tobamovirus spread between cultivated crops of consecutive growing seasons.

#### **6. Management strategies**

#### **6.1 Sensitive tobamovirus detection methods**

In order to prevent the occurrence and establishment of tobamovirus primary infectious source introduced by virus-infected seeds, the appropriate detection methods should be applied. Viral RNA extraction (using Viral RNA Extraction Kit; Bioneer) from ToBRFV-infected tomato (*S. lycopersicum*) seeds (Luria and Dombrovsky, unpublished data) and CGMMV-infected cucumber (*C. sativus* Derben) and melon (*C. melo* Raanan) seeds were successfully executed [60]. Nextgeneration sequencing (NGS) platform has been successfully applied for detection of the tobamoviruses ToBRFV [6] and CGMMV [77]. The NGS detection method

is highly sensitive when compared to the most commonly used serological assays (enzyme-linked immunosorbent assay, Western blot) and the genome sequence analysis performed after PCR amplification. ToBRFV-infected tomato seeds mixed with uninfected seeds in a ratio of 1:600 were successfully detected by the NGS method (Luria and Dombrovsky, unpublished data). Viral RNA extractions, which are easy to perform and need a small amount of starting material, were successfully applied in the NGS analysis [78]. The use of the new technology based on a single-molecule sequencing such as the Oxford Nanopore sequencing platform was successfully applied for detection of plant viruses and bacteria and the detection of the tobamovirus ToBRFV in infected tomato seeds. The sensitivity ratio for detection of ToBRFV-infected tomato seeds by applying the Oxford Nanopore sequencing platform was 1:200 virus-infected seeds mixed with uninfected seeds [10]. Application of sensitive methods for tobamovirus detection in seeds is most critical and needs to be developed for CGMMV-infected cucurbitaceous seeds, in which the viral particles accumulate in the PE envelope underlying the seed coat [59, 60].

#### **6.2 Alleviating soil-associated tobamovirus infectivity**

Soil fumigation with pesticides such as methyl bromide, which had an effect on a wide range of plant pathogens, was successfully used in various crop production facilities. However, since the elimination of its use due to its high toxicity and the detrimental effect on the ozone, several other chemicals such as chloropicrin (trichloronitromethane) had been used [79]. Unlike methyl bromide, many newly used chemicals had no effect on weeds or plant debris, and their efficiency in inactivation of tobamoviruses was questioned. A good alternative to those chemicals are products based on strobilurin fungicide, which originally occurred in the mushroom *Strobilurus tenacellus* [80]. Synthetic compounds such as pyraclostrobin (F 500) that protected the tobacco plant *N. tabacum* (cv. Xanthi nc) against TMV infection could be used in soil preplant treatment. Other chemicals that are based on natural products are alkaloids such as phenanthroquinolizidine that can be extracted from several plant families such as the Vitaceae family [81]. Several formulas of the alkaloid exhibited anti-TMV activity [81]. Similarly, quassinoids isolated from *Brucea javanica* showed anti-TMV activity [82]. Recently, the strong antiviral effect of synthesized bioactive tricyclic spirolactones, which are based on natural polycyclic compounds, has been demonstrated on TMV infection of tobacco plants [83]. Interestingly, the antibiotic Ningnanmycin showed anti-TMV activity both by induction of plant resistance against virus and by inhibition of the virus virulence [84].

Soil steaming treatment using low temperatures (50–60°C) for short time periods (several minutes) had been useful for inactivation of most plant pathogens while preserving soil microflora and minerals [85]. However, these steaming conditions could not be applied for disinfection of tobamovirus-contaminated soil [60]. Interestingly, a combination of mild steaming conditions with chemicals such as potassium hydroxide that are exothermal when reacted with water was effectively applied to disinfection of TMV-infected soil. TMV infectivity ratios were reduced to 3.0%. The increase in the persisted heat in the soil and the higher soil pH could have affected TMV stability [86]. Importantly, regarding CGMMV-contaminated soil, application of intermediate medium composed of CGMMV-free compost, which was prepared from cattle feces, into planting pits prior to planting melon (*C. melo*) seedlings, significantly reduced the initiation of primary infectious foci in the growing area. When combining removal of newly identified infected plants at early growth stage, before trellising, with the implementation of intermediate medium, CGMMV infection ratio at 60 days post planting was 0.3% [60]. Recently, growers have

**39**

**Figure 2.**

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

implemented growth bags that separate between plants, to grow various vegetable

Grafting vegetable crops on *Tobamovirus*-resistant rootstocks could also separate the cultivated crops from the contaminated soil. In particular, *Solanum gilo* accessions were found resistant to the tobamoviruses TMV, ToMV, and the *Pepper mild mottle virus* (PMMoV). Unfortunately, CGMMV-resistant rootstocks scarcely occur and are difficult to find. Using CGMMV-tolerant rootstocks for grafting cucumber plants in field experiment in Northern Israel resulted in viral infectivity ratios of 0.4–0.8% (2 rows, 250 plants in each row). Concomitant growth of ungrafted cucumber plants had CGMMV infectivity ratios in the range of 16–44% (5 rows, 320 plants in each row) (Dombrovsky and Koren, unpublished data). Importantly, it is preferable to use rootstocks that do not cause any reduction in crop yield. Rotations in crop cultivation could reduce buildup of primary infectious tobamovirus inoculum from contaminated soil [87]. For example, it is possible to plant tomato plants after pepper plant plantings since PMMoV does not infect tomatoes. However, because ToMV infects pepper plants, planting tomato crops should not be followed by pepper plant plantings [87]. Importantly, alternating rice and watermelon cultivation reduced CGMMV infection ratios of the watermelons by tenfold (amounting to 7.3%) when compared to consecutive watermelon cultivation [88].

The use of sodium hypochlorite solution on planting facilities and even on seeds was recommended for disinfection of tobamoviruses. However, seeds treated with the hypochlorite solution could show low germination ratios. Trisodium phosphate treatment (TSP) of tobamovirus-infected seeds was also implemented. Importantly, these commonly used disinfection procedures could not be applied for disinfection of tobamovirus-infected cucurbitaceous seeds in which the virus penetrated the seed coat and accumulated in the PE envelope underlying the seed coat [59, 60]. TSP (10%) treatment, combined with heating to 72°C for 72 hours, did not disinfect CGMMV-infected melon (*C. melo* cv. Raanan) seeds. The CGMMV detected in the treated seeds using serological assays and PCR analysis was infectious in a biological assay [60]. However, sodium hypochlorite solution and the new stabilized chlorine product, which has the active ingredient C3Cl3N3NaO3, could be useful for general tobamovirus disinfection of planting facilities [8] such as shears, knives, trellising ropes, planting trays, and irrigation pipes. Careful planting procedures should be implemented, avoiding the infliction of any injury to the plants. Dividing tasks between workers during planting could also reduce secondary tobamovirus

*Limited Cucumber green mottle mosaic virus (CGMMV) infection spread in melon plants grown in growth bags. (a) Melon plants grown in growth bags. (b) CGMMV spread through soil and irrigation water in regular* 

*planting of cucumber plants. Arrows mark CGMMV-infected plants.*

crops in order to reduce tobamovirus disease spread via soil (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

**6.3 Hygienic and careful planting procedures**

#### *Aspects in* Tobamovirus *Management in Intensive Agriculture DOI: http://dx.doi.org/10.5772/intechopen.87101*

*Plant Diseases-Current Threats and Management Trends*

**6.2 Alleviating soil-associated tobamovirus infectivity**

is highly sensitive when compared to the most commonly used serological assays (enzyme-linked immunosorbent assay, Western blot) and the genome sequence analysis performed after PCR amplification. ToBRFV-infected tomato seeds mixed with uninfected seeds in a ratio of 1:600 were successfully detected by the NGS method (Luria and Dombrovsky, unpublished data). Viral RNA extractions, which are easy to perform and need a small amount of starting material, were successfully applied in the NGS analysis [78]. The use of the new technology based on a single-molecule sequencing such as the Oxford Nanopore sequencing platform was successfully applied for detection of plant viruses and bacteria and the detection of the tobamovirus ToBRFV in infected tomato seeds. The sensitivity ratio for detection of ToBRFV-infected tomato seeds by applying the Oxford Nanopore sequencing platform was 1:200 virus-infected seeds mixed with uninfected seeds [10]. Application of sensitive methods for tobamovirus detection in seeds is most critical and needs to be developed for CGMMV-infected cucurbitaceous seeds, in which the viral particles accumulate in the PE envelope underlying the seed coat [59, 60].

Soil fumigation with pesticides such as methyl bromide, which had an effect on a wide range of plant pathogens, was successfully used in various crop production facilities. However, since the elimination of its use due to its high toxicity and the detrimental effect on the ozone, several other chemicals such as chloropicrin (trichloronitromethane) had been used [79]. Unlike methyl bromide, many newly used chemicals had no effect on weeds or plant debris, and their efficiency in inactivation of tobamoviruses was questioned. A good alternative to those chemicals are products based on strobilurin fungicide, which originally occurred in the mushroom *Strobilurus tenacellus* [80]. Synthetic compounds such as pyraclostrobin (F 500) that protected the tobacco plant *N. tabacum* (cv. Xanthi nc) against TMV infection could be used in soil preplant treatment. Other chemicals that are based on natural products are alkaloids such as phenanthroquinolizidine that can be extracted from several plant families such as the Vitaceae family [81]. Several formulas of the alkaloid exhibited anti-TMV activity [81]. Similarly, quassinoids isolated from *Brucea javanica* showed anti-TMV activity [82]. Recently, the strong antiviral effect of synthesized bioactive tricyclic spirolactones, which are based on natural polycyclic compounds, has been demonstrated on TMV infection of tobacco plants [83]. Interestingly, the antibiotic Ningnanmycin showed anti-TMV activity both by induction of plant resistance against virus and by inhibition of the virus

Soil steaming treatment using low temperatures (50–60°C) for short time periods (several minutes) had been useful for inactivation of most plant pathogens while preserving soil microflora and minerals [85]. However, these steaming conditions could not be applied for disinfection of tobamovirus-contaminated soil [60]. Interestingly, a combination of mild steaming conditions with chemicals such as potassium hydroxide that are exothermal when reacted with water was effectively applied to disinfection of TMV-infected soil. TMV infectivity ratios were reduced to 3.0%. The increase in the persisted heat in the soil and the higher soil pH could have affected TMV stability [86]. Importantly, regarding CGMMV-contaminated soil, application of intermediate medium composed of CGMMV-free compost, which was prepared from cattle feces, into planting pits prior to planting melon (*C. melo*) seedlings, significantly reduced the initiation of primary infectious foci in the growing area. When combining removal of newly identified infected plants at early growth stage, before trellising, with the implementation of intermediate medium, CGMMV infection ratio at 60 days post planting was 0.3% [60]. Recently, growers have

**38**

virulence [84].

implemented growth bags that separate between plants, to grow various vegetable crops in order to reduce tobamovirus disease spread via soil (**Figure 2**).

Grafting vegetable crops on *Tobamovirus*-resistant rootstocks could also separate the cultivated crops from the contaminated soil. In particular, *Solanum gilo* accessions were found resistant to the tobamoviruses TMV, ToMV, and the *Pepper mild mottle virus* (PMMoV). Unfortunately, CGMMV-resistant rootstocks scarcely occur and are difficult to find. Using CGMMV-tolerant rootstocks for grafting cucumber plants in field experiment in Northern Israel resulted in viral infectivity ratios of 0.4–0.8% (2 rows, 250 plants in each row). Concomitant growth of ungrafted cucumber plants had CGMMV infectivity ratios in the range of 16–44% (5 rows, 320 plants in each row) (Dombrovsky and Koren, unpublished data). Importantly, it is preferable to use rootstocks that do not cause any reduction in crop yield. Rotations in crop cultivation could reduce buildup of primary infectious tobamovirus inoculum from contaminated soil [87]. For example, it is possible to plant tomato plants after pepper plant plantings since PMMoV does not infect tomatoes. However, because ToMV infects pepper plants, planting tomato crops should not be followed by pepper plant plantings [87]. Importantly, alternating rice and watermelon cultivation reduced CGMMV infection ratios of the watermelons by tenfold (amounting to 7.3%) when compared to consecutive watermelon cultivation [88].

#### **6.3 Hygienic and careful planting procedures**

The use of sodium hypochlorite solution on planting facilities and even on seeds was recommended for disinfection of tobamoviruses. However, seeds treated with the hypochlorite solution could show low germination ratios. Trisodium phosphate treatment (TSP) of tobamovirus-infected seeds was also implemented. Importantly, these commonly used disinfection procedures could not be applied for disinfection of tobamovirus-infected cucurbitaceous seeds in which the virus penetrated the seed coat and accumulated in the PE envelope underlying the seed coat [59, 60]. TSP (10%) treatment, combined with heating to 72°C for 72 hours, did not disinfect CGMMV-infected melon (*C. melo* cv. Raanan) seeds. The CGMMV detected in the treated seeds using serological assays and PCR analysis was infectious in a biological assay [60]. However, sodium hypochlorite solution and the new stabilized chlorine product, which has the active ingredient C3Cl3N3NaO3, could be useful for general tobamovirus disinfection of planting facilities [8] such as shears, knives, trellising ropes, planting trays, and irrigation pipes. Careful planting procedures should be implemented, avoiding the infliction of any injury to the plants. Dividing tasks between workers during planting could also reduce secondary tobamovirus

#### **Figure 2.**

*Limited Cucumber green mottle mosaic virus (CGMMV) infection spread in melon plants grown in growth bags. (a) Melon plants grown in growth bags. (b) CGMMV spread through soil and irrigation water in regular planting of cucumber plants. Arrows mark CGMMV-infected plants.*

infection spread. Weeding the weeds that might be reservoirs of tobamoviruses [76] and supplementing new bumblebee hives for tomato plant cultivation [5] could be important in preventing primary inoculum of tobamovirus infection. Identification and removal of tobamovirus-infected plants in crop cultivation facilities at early stages, before trellising, could reduce secondary tobamovirus disease spread.

#### **6.4 Near-future management strategies**

In the face of the genetic tobamovirus resistance occurring in tomato plants for many years, such as that of the durable *Tm*-2<sup>2</sup> resistance allele [14], a search for ToBRFV-resistant tomato plants could be successful. However, CGMMV-resistant genetic sources for introgression into commercial cucurbitaceous vegetable crop cultivation are scarce [19, 20]. Similarly, sources for CGMMV-resistant rootstocks are limited. Engineering transgenic watermelon rootstocks by transformation of the rootstock *Citrullus lanatus* (Twinser) cv. Gongdae with CGMMV CP gene conferred viral resistance to the plants [89], similar to the phenomenon observed in TMV CP-mediated resistance against TMV [90]. Another approach that could confer viral resistance to susceptible host plants while avoiding the production of transgenic crops is the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology [91]. This mutagenesis system is targeted by guide RNAs to a desired site in the plant cell genome, where Cas9 endonuclease causes double-stranded DNA breaks. The system exploits host cellular repair mechanisms to confer heritable high fidelity change in the genome. Host endogenous genes, such as the *Arabidopsis thaliana TOM1* and *TOM3* genes, and their homologues in tomato and melon plants, are necessary for tobamovirus replication [92, 93]. The host proteins translated from these genes comprise a complex with the tobamovirus replication protein [94]. These host proteins could be targeted by the CRISPR/Cas9 system. Importantly, RNA silencing of these host genes conferred tobamovirus resistance in *Nicotiana tabacum* [95] and in tomatoes [96]. Interestingly, RNA silencing, which is systemic [52, 53, 97], could be transmitted from rootstocks to scions. Hence, engineering rootstocks alone for tobamovirus resistance by RNA silencing of these host genes could confer resistance to the nontransgenic grafted tomato or melon plants. When using the biocontrol approach, plant defense mechanisms that specifically target the infecting tobamovirus, such as the RNA silencing, could be initiated by infecting the susceptible plants with a stable attenuated virus clone or a mutagenized variant of the virus. For example, several attenuated strains were successfully applied to protect tomato plants against ToMV infection, pepper plants against PMMoV infection [98], and muskmelon and cucumber [99] plants against CGMMV infection. However, mutagenized clones might not always be stable, and symptoms might develop in the susceptible plants. Therefore, in order to implement the cross-protection approach, for example, against ToBRFV that infects tomato plants harboring the *Tm*-2<sup>2</sup> resistance allele, it might be beneficial to infect the tomato plants with the stable ToMV that does not show symptoms in the resistant tomato plants. For that purpose, ToMV needs to infect systemically the *Tm*-2<sup>2</sup> resistant tomato plants.

#### **7. Conclusions**

In the recent years, tobamovirus disease spread has been one of the core causes for severe damages observed in various vegetable and ornamental crop productions. Concomitantly, there has been an increase in suggested strategies for tobamovirus disease management. The basic approach of implementing hygienic behavior

**41**

**Author details**

Elisheva Smith and Aviv Dombrovsky\*

Institute of Plant Pathology and Weed Research, Agricultural Research

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

Organization, The Volcani Center, Rishon LeZion, Israel

\*Address all correspondence to: aviv@agri.gov.il

provided the original work is properly cited.

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

while planting has been improved by dividing the planting procedures between workers combined with soil disinfection or the use of intermediate tobamovirusfree medium. This new approach reduced tobamovirus infection substantially. Concurrently, new improved soil disinfectants based on naturally occurring products such as the strobilurin fungicide or plant alkaloids have been produced, eliminating possible harmful side effects of the disinfectants on animals and the environment. Improved methods for detection of tobamovirus-infected seeds have been developed as well. In addition, applicable in the near future are methods exploiting new molecular biology techniques, such as genome editing, to develop tobamovirus-resistant plants. Similarly, methods that engage the plant defense system to invoke resistance to tobamoviruses have been developed, such as the use of attenuated viral strains for plant infection or the use of engineered resistant

We thank Amnon Koren, Neta Luria, Elinor Darzi, and Oded Lachman for

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

plants as rootstocks.

**Acknowledgements**

assistance in data collection.

*Aspects in* Tobamovirus *Management in Intensive Agriculture DOI: http://dx.doi.org/10.5772/intechopen.87101*

while planting has been improved by dividing the planting procedures between workers combined with soil disinfection or the use of intermediate tobamovirusfree medium. This new approach reduced tobamovirus infection substantially. Concurrently, new improved soil disinfectants based on naturally occurring products such as the strobilurin fungicide or plant alkaloids have been produced, eliminating possible harmful side effects of the disinfectants on animals and the environment. Improved methods for detection of tobamovirus-infected seeds have been developed as well. In addition, applicable in the near future are methods exploiting new molecular biology techniques, such as genome editing, to develop tobamovirus-resistant plants. Similarly, methods that engage the plant defense system to invoke resistance to tobamoviruses have been developed, such as the use of attenuated viral strains for plant infection or the use of engineered resistant plants as rootstocks.

#### **Acknowledgements**

*Plant Diseases-Current Threats and Management Trends*

**6.4 Near-future management strategies**

many years, such as that of the durable *Tm*-2<sup>2</sup>

infection spread. Weeding the weeds that might be reservoirs of tobamoviruses [76] and supplementing new bumblebee hives for tomato plant cultivation [5] could be important in preventing primary inoculum of tobamovirus infection. Identification and removal of tobamovirus-infected plants in crop cultivation facilities at early stages, before trellising, could reduce secondary tobamovirus disease spread.

In the face of the genetic tobamovirus resistance occurring in tomato plants for

ToBRFV-resistant tomato plants could be successful. However, CGMMV-resistant genetic sources for introgression into commercial cucurbitaceous vegetable crop cultivation are scarce [19, 20]. Similarly, sources for CGMMV-resistant rootstocks are limited. Engineering transgenic watermelon rootstocks by transformation of the rootstock *Citrullus lanatus* (Twinser) cv. Gongdae with CGMMV CP gene conferred viral resistance to the plants [89], similar to the phenomenon observed in TMV CP-mediated resistance against TMV [90]. Another approach that could confer viral resistance to susceptible host plants while avoiding the production of transgenic crops is the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology [91]. This mutagenesis system is targeted by guide RNAs to a desired site in the plant cell genome, where Cas9 endonuclease causes double-stranded DNA breaks. The system exploits host cellular repair mechanisms to confer heritable high fidelity change in the genome. Host endogenous genes, such as the *Arabidopsis thaliana TOM1* and *TOM3* genes, and their homologues in tomato and melon plants, are necessary for tobamovirus replication [92, 93]. The host proteins translated from these genes comprise a complex with the tobamovirus replication protein [94]. These host proteins could be targeted by the CRISPR/Cas9 system. Importantly, RNA silencing of these host genes conferred tobamovirus resistance in *Nicotiana tabacum* [95] and in tomatoes [96]. Interestingly, RNA silencing, which is systemic [52, 53, 97], could be transmitted from rootstocks to scions. Hence, engineering rootstocks alone for tobamovirus resistance by RNA silencing of these host genes could confer resistance to the nontransgenic grafted tomato or melon plants. When using the biocontrol approach, plant defense mechanisms that specifically target the infecting tobamovirus, such as the RNA silencing, could be initiated by infecting the susceptible plants with a stable attenuated virus clone or a mutagenized variant of the virus. For example, several attenuated strains were successfully applied to protect tomato plants against ToMV infection, pepper plants against PMMoV infection [98], and muskmelon and cucumber [99] plants against CGMMV infection. However, mutagenized clones might not always be stable, and symptoms might develop in the susceptible plants. Therefore, in order to implement the cross-protection approach, for example,

against ToBRFV that infects tomato plants harboring the *Tm*-2<sup>2</sup>

infect systemically the *Tm*-2<sup>2</sup>

**7. Conclusions**

might be beneficial to infect the tomato plants with the stable ToMV that does not show symptoms in the resistant tomato plants. For that purpose, ToMV needs to

resistant tomato plants.

In the recent years, tobamovirus disease spread has been one of the core causes for severe damages observed in various vegetable and ornamental crop productions. Concomitantly, there has been an increase in suggested strategies for tobamovirus disease management. The basic approach of implementing hygienic behavior

resistance allele [14], a search for

resistance allele, it

**40**

We thank Amnon Koren, Neta Luria, Elinor Darzi, and Oded Lachman for assistance in data collection.

### **Author details**

Elisheva Smith and Aviv Dombrovsky\* Institute of Plant Pathology and Weed Research, Agricultural Research Organization, The Volcani Center, Rishon LeZion, Israel

\*Address all correspondence to: aviv@agri.gov.il

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

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**42**

*Plant Diseases-Current Threats and Management Trends*

[9] Baker C, Adkins S. Peppers,

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[11] Mayer A. Über die Mosaikkrankheit

Landwirtschaftliche Versuchs-stationen.

characterization of an isolate of Tomato mottle mosaic virus (ToMMV) infecting tomato and other experimental hosts in a greenhouse in Valencia, Spain. bioRxiv 063255. 2016; DOI: http://doi.

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resistance genes

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1886;**32**:451-467

org/10.1101/063255

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[71] Li JX, Liu SS, Gu QS. Transmission efficiency of *Cucumber green mottle mosaic virus* via seeds, soil, pruning and irrigation water. Journal of Phytopathology. 2015;**5**(5):300-309

[72] Vani S, Varma A. Properties of cucumber green mottle mosaic virus isolated from water of river Jamuna. Indian Phytopathology. 1993;**46**(2):118-122

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[74] Okada K, Kusakari S, Kawaratani M, Negoro J, Ohk ST, Osak T. Tobacco mosaic virus is transmissible from tomato to tomato by pollinating bumblebees. Journal of General Plant Pathology. 2000;**66**:71-74

[75] Boubourakas I, Hatziloukas E, Antignus Y, Katis N. Etiology of

leaf chlorosis and deterioration of the fruit interior of watermelon plants. Journal of Phytopathology. 2004;**152**(10):580-588

[76] Shargil D, Smith E, Lachman O, Reingold V, Darzi E, Tam Y, et al. New weed hosts for *Cucumber green mottle mosaic virus* in wild Mediterranean vegetation. European Journal of Plant Pathology. 2016;**148**(2):473-480

[77] Kehoe MA, Jones RA, Coutts BA. First complete genome sequence of Cucumber green mottle mosaic virus isolated from Australia. Genome Announcements. 2017;**5**(12):e00036-e00017

[78] Luria N, Smith E, Sela N, Lachman O, Koren A, Dombrovsky A. Insights into a watermelon virome contribute to monitoring distribution of whitefly-borne viruses. Phytobiomes. 2019;**3**(1):2471-2906

[79] Duniway J. Status of chemical alternatives to methyl bromide for pre-plant fumigation of soil. Phytopathology. 2002;**92**(12):1337-1343

[80] Sauter H, Steglich W, Anke T. Strobilurins: Evolution of a new class of active substances. Angewandte Chemie, International Edition. 1999;**38**(10):1328-1349

[81] Wang Z, Feng A, Cui M, Liu Y, Wang L, Wang Q. First discovery and stucture-activity relationship study of phenanthroquinolizidines as novel antiviral agents against tobacco mosaic virus (TMV). PLoS One. 2012;**7**(12):e52933

[82] Shen JG, Zhang ZK, Wu ZJ, Ouyang MA, Xie LH, Lin QY. Antiphytoviral activity of bruceine-D from *Brucea javanica* seeds. Pest Management Science: Formerly Pesticide Science. 2008;**64**(2):191-196

[83] Zhu YJ, Wu QF, Fan ZJ, Huo JQ, Zhang JL, Zhao B, et al. Synthesis,

**47**

*Aspects in* Tobamovirus *Management in Intensive Agriculture*

[91] Zaidi SS-e-A, Tashkandi M, Mansoor S, Mahfouz MM. Engineering plant immunity: Using CRISPR/Cas9 to generate virus resistance. Frontiers in

[92] Yamanaka T, Ohta T, Takahashi M, Meshi T, Schmidt R, Dean C, et al. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proceedings of the National Academy of Sciences.

[93] Ishikawa M, Okada Y. Replication of tobamovirus RNA. Proceedings of the Japan Academy, Series B.

[94] Ishibashi K, Ishikawa M. The resistance protein *Tm*-1 inhibits formation of a tomato mosaic virus replication protein-host membrane protein complex. Journal of Virology.

[95] Asano M, Satoh R, Mochizuki A, Tsuda S, Yamanaka T, Nishiguchi M, et al. Tobamovirus-resistant tobacco generated by RNA interference directed against host genes. FEBS Letters.

Plant Science. 2016;**7**

2000;**97**(18):10107-10112

2004;**80**(5):215-224

2013;**87**(14):7933-7939

2005;**579**(20):4479-4484

[96] Ali ME, Ishii Y, Taniguchi J-i, Waliullah S, Kobayashi K, Yaeno T, et al. Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis

TOM1. Archives of Virology. 2018;**163**(5):1357-1362

An endogenous, systemic RNAi pathway in plants. The EMBO Journal.

2010;**29**(10):1699-1712

[97] Dunoyer P, Brosnan CA, Schott G, Wang Y, Jay F, Alioua A, et al. Retracted:

[98] Ichiki T, Nagaoka E, Hagiwara K, Uchikawa K, Tsuda S, Omura T. Integration of mutations responsible for the attenuated phenotype of pepper mild mottle virus strains

*DOI: http://dx.doi.org/10.5772/intechopen.87101*

bioactivity and mode of action of 5A5B6C tricyclic spirolactones as novel antiviral lead compounds. Pest Management Science.

[84] Han Y, Luo Y, Qin S, Xi L, Wan B, Du L. Induction of systemic resistance against tobacco mosaic virus by Ningnanmycin in tobacco. Pesticide Biochemistry and Physiology.

[85] van Loenen MC, Turbett Y, Mullins CE, Feilden NE, Wilson MJ, Leifert C, et al. Low temperature–short duration steaming of soil kills soil-borne pathogens, nematode pests and weeds. European Journal of Plant Pathology.

[86] Luvisi A, Panattoni A, Materazzi A. Heat treatments for sustainable control of soil viruses. Agronomy for Sustainable Development.

[87] Albrechtsen SE. Testing methods for seed-transmitted viruses: Principles and

[88] Park J-W, Jang T-H, Song S-H, Choi H-S, Ko S-J. Studies on the soil transmission of CGMMV and its control with crop rotation. The Korean Journal of Pesticide Science.

[89] Park SM, Lee JS, Jegal S, Jeon BY, Jung M, Park YS, et al. Transgenic watermelon rootstock resistant to CGMMV (*Cucumber green mottle mosaic virus*) infection. Plant Cell Reports.

[90] Bendahmane M, Fitchen JH, Zhang G, Beachy RN. Studies of coat proteinmediated resistance to tobacco mosaic tobamovirus: Correlation between assembly of mutant coat proteins and resistance. Journal of Virology.

2019;**75**(1):292-301

2014;**111**:14-18

2003;**109**(9):993-1002

2015;**35**(2):657-666

protocols. CABI. 2006

2010;**14**(4):473-477

2005;**24**(6):350-356

1997;**71**(10):7942-7950

*Aspects in* Tobamovirus *Management in Intensive Agriculture DOI: http://dx.doi.org/10.5772/intechopen.87101*

bioactivity and mode of action of 5A5B6C tricyclic spirolactones as novel antiviral lead compounds. Pest Management Science. 2019;**75**(1):292-301

*Plant Diseases-Current Threats and Management Trends*

leaf chlorosis and deterioration of the fruit interior of watermelon plants. Journal of Phytopathology.

[76] Shargil D, Smith E, Lachman O, Reingold V, Darzi E, Tam Y, et al. New weed hosts for *Cucumber green mottle mosaic virus* in wild Mediterranean vegetation. European Journal of Plant Pathology. 2016;**148**(2):473-480

[77] Kehoe MA, Jones RA, Coutts BA. First complete genome sequence of Cucumber green mottle mosaic virus isolated from Australia. Genome Announcements.

[78] Luria N, Smith E, Sela N, Lachman O, Koren A, Dombrovsky A. Insights into a watermelon virome contribute

whitefly-borne viruses. Phytobiomes.

Phytopathology. 2002;**92**(12):1337-1343

[79] Duniway J. Status of chemical alternatives to methyl bromide for pre-plant fumigation of soil.

[80] Sauter H, Steglich W, Anke T. Strobilurins: Evolution of a new class of active substances. Angewandte

Chemie, International Edition.

[81] Wang Z, Feng A, Cui M, Liu Y, Wang L, Wang Q. First discovery and stucture-activity relationship study of phenanthroquinolizidines as novel antiviral agents against tobacco mosaic virus (TMV). PLoS One.

[82] Shen JG, Zhang ZK, Wu ZJ, Ouyang MA, Xie LH, Lin QY. Antiphytoviral activity of bruceine-D from *Brucea javanica* seeds. Pest Management Science: Formerly Pesticide Science.

[83] Zhu YJ, Wu QF, Fan ZJ, Huo JQ, Zhang JL, Zhao B, et al. Synthesis,

1999;**38**(10):1328-1349

2012;**7**(12):e52933

2008;**64**(2):191-196

2017;**5**(12):e00036-e00017

to monitoring distribution of

2019;**3**(1):2471-2906

2004;**152**(10):580-588

resilience of its virions. Plant Pathology.

[68] Reingold V, Lachman O, Belausov E, Koren A, Mor N, Dombrovsky A. Epidemiological study of *Cucumber green mottle mosaic virus* in greenhouses enables reduction of disease damage in cucurbit production. The Annals of Applied Biology. 2016;**168**(1):29-40

[69] Jeżewska M, Zarzyńska-Nowak A, Trzmiel K. Detection of infectious tobamoviruses in irrigation and drainage canals in Greater Poland. Journal of Plant Protection Research.

[70] Boben J, Kramberger P, Petrovič N, Cankar K, Peterka M, Štrancar A, et al. Detection and quantification of Tomato mosaic virus in irrigation waters. European Journal of Plant Pathology.

[71] Li JX, Liu SS, Gu QS. Transmission efficiency of *Cucumber green mottle mosaic virus* via seeds, soil, pruning and irrigation water. Journal of Phytopathology. 2015;**5**(5):300-309

[72] Vani S, Varma A. Properties of cucumber green mottle mosaic virus isolated from water of river Jamuna. Indian Phytopathology.

[73] Sacristán S, Díaz M, Fraile A, García-Arenal F. Contact transmission of tobacco mosaic virus: A quantitative analysis of parameters relevant for virus evolution. Journal of Virology.

[74] Okada K, Kusakari S, Kawaratani M, Negoro J, Ohk ST, Osak T. Tobacco mosaic virus is transmissible from tomato to tomato by pollinating bumblebees. Journal of General Plant

[75] Boubourakas I, Hatziloukas E, Antignus Y, Katis N. Etiology of

2018;**67**(3):651-659

2018;**58**(2):202-205

2007;**118**(1):59-71

1993;**46**(2):118-122

2011;**85**(10):4974-4981

Pathology. 2000;**66**:71-74

**46**

[84] Han Y, Luo Y, Qin S, Xi L, Wan B, Du L. Induction of systemic resistance against tobacco mosaic virus by Ningnanmycin in tobacco. Pesticide Biochemistry and Physiology. 2014;**111**:14-18

[85] van Loenen MC, Turbett Y, Mullins CE, Feilden NE, Wilson MJ, Leifert C, et al. Low temperature–short duration steaming of soil kills soil-borne pathogens, nematode pests and weeds. European Journal of Plant Pathology. 2003;**109**(9):993-1002

[86] Luvisi A, Panattoni A, Materazzi A. Heat treatments for sustainable control of soil viruses. Agronomy for Sustainable Development. 2015;**35**(2):657-666

[87] Albrechtsen SE. Testing methods for seed-transmitted viruses: Principles and protocols. CABI. 2006

[88] Park J-W, Jang T-H, Song S-H, Choi H-S, Ko S-J. Studies on the soil transmission of CGMMV and its control with crop rotation. The Korean Journal of Pesticide Science. 2010;**14**(4):473-477

[89] Park SM, Lee JS, Jegal S, Jeon BY, Jung M, Park YS, et al. Transgenic watermelon rootstock resistant to CGMMV (*Cucumber green mottle mosaic virus*) infection. Plant Cell Reports. 2005;**24**(6):350-356

[90] Bendahmane M, Fitchen JH, Zhang G, Beachy RN. Studies of coat proteinmediated resistance to tobacco mosaic tobamovirus: Correlation between assembly of mutant coat proteins and resistance. Journal of Virology. 1997;**71**(10):7942-7950

[91] Zaidi SS-e-A, Tashkandi M, Mansoor S, Mahfouz MM. Engineering plant immunity: Using CRISPR/Cas9 to generate virus resistance. Frontiers in Plant Science. 2016;**7**

[92] Yamanaka T, Ohta T, Takahashi M, Meshi T, Schmidt R, Dean C, et al. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein. Proceedings of the National Academy of Sciences. 2000;**97**(18):10107-10112

[93] Ishikawa M, Okada Y. Replication of tobamovirus RNA. Proceedings of the Japan Academy, Series B. 2004;**80**(5):215-224

[94] Ishibashi K, Ishikawa M. The resistance protein *Tm*-1 inhibits formation of a tomato mosaic virus replication protein-host membrane protein complex. Journal of Virology. 2013;**87**(14):7933-7939

[95] Asano M, Satoh R, Mochizuki A, Tsuda S, Yamanaka T, Nishiguchi M, et al. Tobamovirus-resistant tobacco generated by RNA interference directed against host genes. FEBS Letters. 2005;**579**(20):4479-4484

[96] Ali ME, Ishii Y, Taniguchi J-i, Waliullah S, Kobayashi K, Yaeno T, et al. Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1. Archives of Virology. 2018;**163**(5):1357-1362

[97] Dunoyer P, Brosnan CA, Schott G, Wang Y, Jay F, Alioua A, et al. Retracted: An endogenous, systemic RNAi pathway in plants. The EMBO Journal. 2010;**29**(10):1699-1712

[98] Ichiki T, Nagaoka E, Hagiwara K, Uchikawa K, Tsuda S, Omura T. Integration of mutations responsible for the attenuated phenotype of pepper mild mottle virus strains

**Chapter 4**

**Abstract**

Against Pathogens

in crops of agricultural and economic interest.

biotic and abiotic elicitors

**1. Introduction**

**49**

Plant Metabolites in Plant Defense

*Xóchitl S. Ramírez-Gómez, Sandra N. Jiménez-García,*

*Vicente Beltrán Campos and Ma. Lourdes García Campos*

spread use is due in part to the cultural acceptance of traditional medicine in different regions of the world, as well as its effectiveness in treating various diseases. Many of its active substances or secondary metabolites are formed to a response of various situations that generate stress in their habitat, such as sudden changes in environmental temperature, humidity, rain, drought, and infections by phytopathogens (fungi, bacteria, viruses, nematodes, protozoa). The production of these secondary metabolites is a mechanism of defense of plants. In this context, the objective of this chapter is to study the secondary metabolites of medicinal plants that could have a promising application in the control of different phytopathogens

**Keywords:** medicinal plants, phytopathogens, secondary metabolites, pesticides,

Phytopathogens generally attack plants during their growth, causing alterations in their cellular metabolism and/or interfering with the absorption of nutrients [1]. The crops of cereals, vegetables, and fruits are affected by these organisms during harvest and postharvest [2]. However, one of the main control measures to eradicate phytopathogens is the use of pesticides. Although they are effective, easy to access, and easy to use, they have several disadvantages, generate resistance, and are considered toxic substances, not only for bacteria, fungi, viruses, protozoa, and nematodes but also for the humans, animals, and the environment [3, 4]. In this context, the pesticides can induce acute and chronic toxicity, to persist in the environment and pollute soil and water. So, they are easily incorporated into the food chain, bioaccumulation, and biomagnification [5]. Regarding their toxicity mechanisms, it has been described that they can act as endocrine disruptors and as

On the other hand, the study of medicinal plants as possible natural sources of obtaining active compound (secondary metabolites) against phytopathogens has gained increasing interest in recent years, due to several aspects, mainly that they are obtained from a natural source through the production or synthesis of secondary metabolites considered as nontoxic such as phenols, flavonoids, terpenes, alkaloids, etc. [10–13]. Another advantage is that phytopathogens still do not develop resistance to the antifungal, antimicrobial, and nematicide effect of the phytochemical

reactive species that generate oxidative stress in the cell [6–9].

Medicinal plants are widely used worldwide to treat various diseases. Its wide-

results in a symptomless crossprotecting strain. Archives of Virology. 2005;**150**(10):2009-2020

[99] Slavokhotova AA, Istomina EA, Andreeva EN, Korostyleva TV, Pukhalskij VA, Shijan AN, et al. An attenuated strain of *Cucumber green mottle mosaic virus* as a biological control agent against pathogenic viral strains. American Journal of Plant Sciences. 2016;**7**(05):724-732

#### **Chapter 4**

*Plant Diseases-Current Threats and Management Trends*

results in a symptomless cross-

[99] Slavokhotova AA, Istomina EA, Andreeva EN, Korostyleva TV, Pukhalskij VA, Shijan AN, et al. An attenuated strain of *Cucumber green mottle mosaic virus* as a biological control agent against pathogenic viral strains. American Journal of Plant Sciences.

2005;**150**(10):2009-2020

2016;**7**(05):724-732

protecting strain. Archives of Virology.

**48**

## Plant Metabolites in Plant Defense Against Pathogens

*Xóchitl S. Ramírez-Gómez, Sandra N. Jiménez-García, Vicente Beltrán Campos and Ma. Lourdes García Campos*

#### **Abstract**

Medicinal plants are widely used worldwide to treat various diseases. Its widespread use is due in part to the cultural acceptance of traditional medicine in different regions of the world, as well as its effectiveness in treating various diseases. Many of its active substances or secondary metabolites are formed to a response of various situations that generate stress in their habitat, such as sudden changes in environmental temperature, humidity, rain, drought, and infections by phytopathogens (fungi, bacteria, viruses, nematodes, protozoa). The production of these secondary metabolites is a mechanism of defense of plants. In this context, the objective of this chapter is to study the secondary metabolites of medicinal plants that could have a promising application in the control of different phytopathogens in crops of agricultural and economic interest.

**Keywords:** medicinal plants, phytopathogens, secondary metabolites, pesticides, biotic and abiotic elicitors

#### **1. Introduction**

Phytopathogens generally attack plants during their growth, causing alterations in their cellular metabolism and/or interfering with the absorption of nutrients [1]. The crops of cereals, vegetables, and fruits are affected by these organisms during harvest and postharvest [2]. However, one of the main control measures to eradicate phytopathogens is the use of pesticides. Although they are effective, easy to access, and easy to use, they have several disadvantages, generate resistance, and are considered toxic substances, not only for bacteria, fungi, viruses, protozoa, and nematodes but also for the humans, animals, and the environment [3, 4]. In this context, the pesticides can induce acute and chronic toxicity, to persist in the environment and pollute soil and water. So, they are easily incorporated into the food chain, bioaccumulation, and biomagnification [5]. Regarding their toxicity mechanisms, it has been described that they can act as endocrine disruptors and as reactive species that generate oxidative stress in the cell [6–9].

On the other hand, the study of medicinal plants as possible natural sources of obtaining active compound (secondary metabolites) against phytopathogens has gained increasing interest in recent years, due to several aspects, mainly that they are obtained from a natural source through the production or synthesis of secondary metabolites considered as nontoxic such as phenols, flavonoids, terpenes, alkaloids, etc. [10–13]. Another advantage is that phytopathogens still do not develop resistance to the antifungal, antimicrobial, and nematicide effect of the phytochemical

compounds produced by some medicinal plants. When carrying out an exhaustive search in the literature, it was found that the potential use of the secondary metabolites obtained from medicinal plant extracts is fungicide [14–16]. Most of the research in this area focuses on evaluating the effects of these active compounds on fungi such as *Fusarium*, maybe because it is one of the main phytopathogens that cause economic losses mainly in cereal crops and health problems by their aflatoxins [17, 18]. This chapter shows an overview of the recent research on this topic, emphasizing the effect of biotic and abiotic elicitors on the secondary metabolite production, as well as a brief description of the scientific name of the plant, metabolites with antifungal and antibacterial effect, and their limitations and perspectives of its use in the biological control of phytopathogens.

viruses, bacteria, and fungi is one of them [23]. The above makes sense if we analyze the fact that plants have mechanisms to protect themselves from both biotic and abiotic stress agents. That is, if the phytopathogens (biotic agents) are attacking

**3.1 The bioactive potential of secondary metabolites derived from the**

In this context, it is interesting to analyze the secondary metabolism of plants know which phytochemical substances are produced and what biological activity

Plants are formed by a primary metabolism that is responsible for the physiological processes and development of the plant, such as lipids, carbohydrates, and proteins [23]. The secondary metabolism is not essential in the basic processes of plants. However, these bioactive compounds play an important role in the defense of plants, and these secondary metabolites can be classified as phenolic compounds, carotenoids, terpenes, alkaloids, and sulfur compounds, among others, as shown in

Phenolic compounds are aromatic substances formed during the passage of the shikimic acid pathway or mainly the mevalonic pathway. These can be divided into insoluble compounds such as condensed tannins, lignins, and hydroxamic acids bound to the cell walls, and soluble compounds are phenolic acids, flavonoids, and kinases [25]. Carotenoids are lipophilic molecules and are found in plants giving orange tones. The importance of these compounds is the intervention they have in photosynthesis, and they also protect the photosynthetic apparatus from excess

the plants, why not think what the plant does to defend itself?

*Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

they present.

**Table 1** [24].

**Table 1.**

**51**

*Types of plant secondary metabolites.*

**medicinal plant**

#### **2. Pesticides in the control of phytopathogens**

In the market, there are a variety of pesticides that are used alone or in combination to eradicate, control, or prevent pests [4]. Pesticides can be classified according to the chemical group to which they belong, to their selectivity toward a certain phytopathogen, its mechanism of action, and its use or application. However, the most widely used for their effectiveness and a broad spectrum of activity against various pests and diseases in plants are insecticides, herbicides, and fungicides [4, 19].

Pesticides used in agriculture mainly contaminate the soil by direct application and water by leaching, and it is very easy for them to be present either in trace quantities or high in food and to enter the food chain, which facilitates its accumulation and biomagnification [5, 20]. In general pesticides are considered dangerous substances for living beings since they can produce acute or chronic toxicity; however the magnitude of the poisoning depends on several aspects to be considered such as the physicochemical characteristics of the pesticide, the concentration, the exposure time, the route of entry to organisms, their toxicodynamics and toxicokinetics (absorption, distribution, half-life, metabolism, and elimination), as well as the use of mixtures of different pesticides, the components of their formulation, and the general state of health of the individual [21, 22]. All these aspects influence that pesticides represent a risk or danger for those who use them in the fields of cultivation, as well as for those who consume foods that contain substances in trace quantities in prolonged consumption.

Regarding its toxicity, it has been described that pesticides act as endocrine disruptors and generators of free radicals and enzymatic inhibitors [8, 9]. Unfortunately, the cellular targets to which most of these pesticides are directed coincide with cellular targets that are also present in man, such as the case of the mechanisms of action of organophosphorus insecticides, which inhibit the activity of acetylcholinesterase enzyme present in different insects; unfortunately man and other mammals also have acetylcholinesterase, so their toxicity is not selective toward the pests that they wish to control, but they also affect man, and depending on the magnitude of the poisoning, they can cause death [19–22]. However, until today an ideal pesticide does not exist, and the correct use of herbicides, fungicides, insecticides, etc. has many benefits to control plagues and increase the yield of the crops [19].

#### **3. Secondary metabolites of medicinal plants as biological control of phytopathogens**

There are several methods of biological control against phytopathogens. The use of extracts of medicinal plants to eradicate diseases in crops caused mainly by

#### *Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

compounds produced by some medicinal plants. When carrying out an exhaustive search in the literature, it was found that the potential use of the secondary metabolites obtained from medicinal plant extracts is fungicide [14–16]. Most of the research in this area focuses on evaluating the effects of these active compounds on fungi such as *Fusarium*, maybe because it is one of the main phytopathogens that cause economic losses mainly in cereal crops and health problems by their aflatoxins [17, 18]. This chapter shows an overview of the recent research on this topic, emphasizing the effect of biotic and abiotic elicitors on the secondary metabolite production, as well as a brief description of the scientific name of the plant, metabolites with antifungal and antibacterial effect, and their limitations and perspectives

In the market, there are a variety of pesticides that are used alone or in combination to eradicate, control, or prevent pests [4]. Pesticides can be classified according to the chemical group to which they belong, to their selectivity toward a certain phytopathogen, its mechanism of action, and its use or application. However, the most widely used for their effectiveness and a broad spectrum of activity against various pests and diseases in plants are insecticides, herbicides, and fungicides [4, 19]. Pesticides used in agriculture mainly contaminate the soil by direct application and water by leaching, and it is very easy for them to be present either in trace quantities or high in food and to enter the food chain, which facilitates its accumulation and biomagnification [5, 20]. In general pesticides are considered dangerous substances for living beings since they can produce acute or chronic toxicity; however the magnitude of the poisoning depends on several aspects to be considered such as the physicochemical characteristics of the pesticide, the concentration, the

exposure time, the route of entry to organisms, their toxicodynamics and

toxicokinetics (absorption, distribution, half-life, metabolism, and elimination), as well as the use of mixtures of different pesticides, the components of their formulation, and the general state of health of the individual [21, 22]. All these aspects influence that pesticides represent a risk or danger for those who use them in the fields of cultivation, as well as for those who consume foods that contain substances

Regarding its toxicity, it has been described that pesticides act as endocrine disruptors and generators of free radicals and enzymatic inhibitors [8, 9]. Unfortunately, the cellular targets to which most of these pesticides are directed coincide with cellular targets that are also present in man, such as the case of the mechanisms of action of organophosphorus insecticides, which inhibit the activity of acetylcholinesterase enzyme present in different insects; unfortunately man and other mammals also have acetylcholinesterase, so their toxicity is not selective toward the pests that they wish to control, but they also affect man, and depending on the magnitude of the poisoning, they can cause death [19–22]. However, until today an ideal pesticide does not exist, and the correct use of herbicides, fungicides, insecticides, etc. has many benefits to control plagues and increase the yield of the crops [19].

**3. Secondary metabolites of medicinal plants as biological control of**

of extracts of medicinal plants to eradicate diseases in crops caused mainly by

There are several methods of biological control against phytopathogens. The use

of its use in the biological control of phytopathogens.

*Plant Diseases-Current Threats and Management Trends*

**2. Pesticides in the control of phytopathogens**

in trace quantities in prolonged consumption.

**phytopathogens**

**50**

viruses, bacteria, and fungi is one of them [23]. The above makes sense if we analyze the fact that plants have mechanisms to protect themselves from both biotic and abiotic stress agents. That is, if the phytopathogens (biotic agents) are attacking the plants, why not think what the plant does to defend itself?

In this context, it is interesting to analyze the secondary metabolism of plants know which phytochemical substances are produced and what biological activity they present.

#### **3.1 The bioactive potential of secondary metabolites derived from the medicinal plant**

Plants are formed by a primary metabolism that is responsible for the physiological processes and development of the plant, such as lipids, carbohydrates, and proteins [23]. The secondary metabolism is not essential in the basic processes of plants. However, these bioactive compounds play an important role in the defense of plants, and these secondary metabolites can be classified as phenolic compounds, carotenoids, terpenes, alkaloids, and sulfur compounds, among others, as shown in **Table 1** [24].

Phenolic compounds are aromatic substances formed during the passage of the shikimic acid pathway or mainly the mevalonic pathway. These can be divided into insoluble compounds such as condensed tannins, lignins, and hydroxamic acids bound to the cell walls, and soluble compounds are phenolic acids, flavonoids, and kinases [25]. Carotenoids are lipophilic molecules and are found in plants giving orange tones. The importance of these compounds is the intervention they have in photosynthesis, and they also protect the photosynthetic apparatus from excess


**Table 1.** *Types of plant secondary metabolites.*

energy [25]. The carotenoid contents in plants are affected by various factors, such as plant development, stress conditions, postharvest conditions, or cooking treatments, but the interest of these compounds has been increasing due to their potential antioxidant activity [26]. Terpenes are lipid-soluble compounds that include one- or more five-carbon isoprene units, which are synthesized by all organisms through two pathways, mevalonate and deoxy-D-xylulose [27]. Terpenoids are classified according to the number of isoprene units they contain; terpenes and terpenoids are basic constituents of many types of plant essential oils [28]. Alkaloids are bioactive compounds that generally contain nitrogen derived from an amino acid of great importance because it has physiological and medicinal properties, for example, caffeine, nicotine, morphine, atropine, and quinine [29].

but can protect the plant of abiotic elicitors [36]. On the other hand, phytoalexins are induced against the attack of microbes and insects activated by β-glucosidase by the release of biocidal aglycones [37]. In the same way act the benzoxazinoids (BX), these phytochemical compounds are produced and released by tissue damage and

At present, several biotechnological strategies have been used to increase the productivity of secondary metabolites, using different inducers of secondary metabolites such as at the cellular, organic, and plant levels, as well as the most effective methods to improve the synthesis of these secondary metabolites in endemic and medicinal plants [39]. These secondary metabolites accumulate in plants when they are prone to various stress types, inducers, or signal molecules. Thus, there are different modulating factors of secondary metabolites, as well as microbial, physical, or chemical effects such as abiotic or biotic elicitors, inducing the biosynthesis of specific compound that plays an important role in the adaptations of plants to stress conditions, and these phenomena cause a greater synthesis and accumulation of secondary metabolites [40]. In **Table 2** the authors focus on the abiotic elicitors that are substances of biological origin such as proteins and carbohydrates that are initiator compounds or coupling responses at the cellular level activating several enzymes or signaling canals. There are also microorganisms and chemical compounds with elicitor effect that stress the plant and produce the expression of a greater amount of metabolites or new metabolites which cause physiological changes in the plant against pathogens. As shown in **Table 2**,

glycoprotein-type proteins produce phytoalexins that have been used to identify ion channels in cell membranes and thus transfer signals by external stimuli, as demonstrated by Alami [41] where the *Plantanus x acerifolia* cultures were applied to an inducer of *Ceratocystis fimbriata* f. sp. These, in turn, induced the synthesis of phytoalexins (hydroxycoumarin, scopoletin, and umbelliferone), and upon isolating the glycoprotein produced the synthesis of coumarin by 80%. On the other hand, oligogalacturonic acids are found in the cell wall of the plant inducing the biosynthesis of phytoalexins, whereas chitin is found in the cell wall of fungi, generating signaling factors in plants such as *Hypericum perforatum* production stress in the plant and increasing the production of phenolic compounds for their defense against pathogens [39, 42]. Rhizobacteria function as modelers of secondary metabolites with pharmacological activity. Rhizobacteria colonize the rhizospheres of the plants and improve the growth of the plant, being localized in the bark or root nodules acting as inducers of the enzymes that participate in the metabolic pathways of bioactive compounds and jasmonic acid biosynthesis; these act as signal transducers [43, 44]. Other signal inducers are the mycorrhizal fungi that help the plant to absorb more water and show defense against other pathogens such as fungi, bacteria, or parasites that affect the roots of the plant. These mycorrhizal fungi produce secondary metabolites such as phenolic compounds and alkaloids, among others [45–48]. Elicitors such as salicylic acid, jasmonic acid, hydrogen peroxide, chitosan, etc. act as plant hormones in the expression of genes interacting as target signaling causing a physiological response in the plant which increases the production of phenolic compounds, vitamin C, carotenoids, or defense stimuli against pathogens; there are also synergistic effects between salicylic acid and jasmonic acid providing resistance against pathogens by the induction of the

On the other hand, **Table 3** shows some research that has the influence of different abiotic elicitors that are considered substance and that are not of biological origin such as salt, drought, light or heavy metals, and temperature, among others. **Table 3** shows different perspectives of research on medicinal or aromatic plants in hydroponic crops, outdoors, and the application of elicitors in different stages of growth or postharvest. For example, heavy metals such as Al3+, Cr3+, Co2+, Ni2+,

hydrolysis by β-glucosidase and act as insect repellents too [38].

*Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

octadecanoic acid pathway [49–52, 53].

**53**

Now well, all these compounds mentioned above help the plants to develop complex defense systems against different types of stress for the survival or the systematic forces in their metabolism for resistance against pests and diseases. Stress provoked in the plant involves several signaling response pathways for pathogens and insects, and some of these response pathways are induced by the microorganisms themselves. Also, the plants have specific recognition and signaling systems allowing them to detect the pathogens and initiate an effective defense response [30, 31]. The defence system broadest have the plants against pathogens are the phenolic compounds (phenylpropanoids and flavonoids). These substances have different mechanisms of action they can dissociate the ions of the phenolic hydroxyl and forming phenolates, ionic and hydrogen bonds with peptides and proteins causing a high astringency and protein denaturation. In the other hand, they interfere with the pathogen's cell signalling compounds and affect their physiological activities through enzymatic inhibition, DNA alkylation and altering their reproductive system [31]. The compounds with allelopathic effects affect positively or negatively on the ecosystem's structure to remove or eliminate microorganisms from the plants. Some phenolic compounds are allelochemicals that have been shown to have an activity as antibiotics, antifungals, and antipredator [31]. Phenolic acids, such as benzoic, hydroxybenzoic, vanillic, and caffeic, have antimicrobial and antifungal properties produced by the inhibition of enzymes. Caffeic, chlorogenic, sinapic, ferulic, and p-coumaric acids have antioxidant activity by the inhibition of oxidation of lipids and the elimination of reactive oxygen species. These effects are important to the plant defense [32].

#### **3.2 Improving production of plant secondary metabolites through biotic and abiotic stresses**

Classification of secondary metabolites related to the defense of plants is commonly used in the form of synthesis and accumulation of phytochemicals with interaction effect of the pathogenic plant against plant insect, virus, fungi, and antibacterial compounds. For example, phytoalexins are produced very quickly after infection of a pathogen producing toxicity to an ambiguous environment of fungi or bacteria [33, 34].

Phenylpropanoids and flavonoids have hydroxyl groups that contain phenolic compounds, which dissociate into phenolate ions, and the phenolic hydroxyl groups form ionic bonds and hydrogen bonds with peptides and protons, producing a high astringency and denaturation that thus show an antifungal effect acting together with cellular signaling compounds and physiological activities or acting on the parts of the pathogen, reproductive system, enzymatic inhibition, etc. [35]. The properties of the proteins change with any change in protein conformation, for example, by changing the three-dimensional structure forming covalent bonds with SH, OH or free amino groups there is inactivation or protein function loss. When polyphenols of the plants bind to some proteins of phytopathogens are less toxic for them

#### *Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

energy [25]. The carotenoid contents in plants are affected by various factors, such as plant development, stress conditions, postharvest conditions, or cooking treatments, but the interest of these compounds has been increasing due to their potential antioxidant activity [26]. Terpenes are lipid-soluble compounds that include one- or more five-carbon isoprene units, which are synthesized by all organisms through two pathways, mevalonate and deoxy-D-xylulose [27]. Terpenoids are classified according to the number of isoprene units they contain; terpenes and terpenoids are basic constituents of many types of plant essential oils [28]. Alkaloids are bioactive compounds that generally contain nitrogen derived from an amino acid of great importance because it has physiological and medicinal properties, for

Now well, all these compounds mentioned above help the plants to develop complex defense systems against different types of stress for the survival or the systematic forces in their metabolism for resistance against pests and diseases. Stress provoked in the plant involves several signaling response pathways for pathogens and insects, and some of these response pathways are induced by the microorganisms themselves. Also, the plants have specific recognition and signaling systems allowing them to detect the pathogens and initiate an effective defense response [30, 31]. The defence system broadest have the plants against pathogens are the phenolic compounds (phenylpropanoids and flavonoids). These substances have different mechanisms of action they can dissociate the ions of the phenolic hydroxyl and forming phenolates, ionic and hydrogen bonds with peptides and proteins causing a high astringency and protein denaturation. In the other hand, they interfere with the pathogen's cell signalling compounds and affect their physiological activities through enzymatic inhibition, DNA alkylation and altering their reproductive system [31]. The compounds with allelopathic effects affect positively or negatively on the ecosystem's structure to remove or eliminate microorganisms from the plants. Some phenolic compounds are allelochemicals that have been shown to have an activity as antibiotics, antifungals, and antipredator [31]. Phenolic acids, such as benzoic, hydroxybenzoic, vanillic, and caffeic, have antimicrobial and antifungal properties produced by the inhibition of enzymes. Caffeic, chlorogenic, sinapic, ferulic, and p-coumaric acids have antioxidant activity by the inhibition of oxidation of lipids and the elimination of reactive oxygen species. These effects are

**3.2 Improving production of plant secondary metabolites through biotic and**

Classification of secondary metabolites related to the defense of plants is commonly used in the form of synthesis and accumulation of phytochemicals with interaction effect of the pathogenic plant against plant insect, virus, fungi, and antibacterial compounds. For example, phytoalexins are produced very quickly after infection of a pathogen producing toxicity to an ambiguous environment of

Phenylpropanoids and flavonoids have hydroxyl groups that contain phenolic compounds, which dissociate into phenolate ions, and the phenolic hydroxyl groups form ionic bonds and hydrogen bonds with peptides and protons, producing a high astringency and denaturation that thus show an antifungal effect acting together with cellular signaling compounds and physiological activities or acting on the parts of the pathogen, reproductive system, enzymatic inhibition, etc. [35]. The properties of the proteins change with any change in protein conformation, for example, by changing the three-dimensional structure forming covalent bonds with SH, OH or free amino groups there is inactivation or protein function loss. When polyphenols of the plants bind to some proteins of phytopathogens are less toxic for them

example, caffeine, nicotine, morphine, atropine, and quinine [29].

*Plant Diseases-Current Threats and Management Trends*

important to the plant defense [32].

**abiotic stresses**

fungi or bacteria [33, 34].

**52**

but can protect the plant of abiotic elicitors [36]. On the other hand, phytoalexins are induced against the attack of microbes and insects activated by β-glucosidase by the release of biocidal aglycones [37]. In the same way act the benzoxazinoids (BX), these phytochemical compounds are produced and released by tissue damage and hydrolysis by β-glucosidase and act as insect repellents too [38].

At present, several biotechnological strategies have been used to increase the productivity of secondary metabolites, using different inducers of secondary metabolites such as at the cellular, organic, and plant levels, as well as the most effective methods to improve the synthesis of these secondary metabolites in endemic and medicinal plants [39]. These secondary metabolites accumulate in plants when they are prone to various stress types, inducers, or signal molecules. Thus, there are different modulating factors of secondary metabolites, as well as microbial, physical, or chemical effects such as abiotic or biotic elicitors, inducing the biosynthesis of specific compound that plays an important role in the adaptations of plants to stress conditions, and these phenomena cause a greater synthesis and accumulation of secondary metabolites [40]. In **Table 2** the authors focus on the abiotic elicitors that are substances of biological origin such as proteins and carbohydrates that are initiator compounds or coupling responses at the cellular level activating several enzymes or signaling canals. There are also microorganisms and chemical compounds with elicitor effect that stress the plant and produce the expression of a greater amount of metabolites or new metabolites which cause physiological changes in the plant against pathogens. As shown in **Table 2**, glycoprotein-type proteins produce phytoalexins that have been used to identify ion channels in cell membranes and thus transfer signals by external stimuli, as demonstrated by Alami [41] where the *Plantanus x acerifolia* cultures were applied to an inducer of *Ceratocystis fimbriata* f. sp. These, in turn, induced the synthesis of phytoalexins (hydroxycoumarin, scopoletin, and umbelliferone), and upon isolating the glycoprotein produced the synthesis of coumarin by 80%. On the other hand, oligogalacturonic acids are found in the cell wall of the plant inducing the biosynthesis of phytoalexins, whereas chitin is found in the cell wall of fungi, generating signaling factors in plants such as *Hypericum perforatum* production stress in the plant and increasing the production of phenolic compounds for their defense against pathogens [39, 42]. Rhizobacteria function as modelers of secondary metabolites with pharmacological activity. Rhizobacteria colonize the rhizospheres of the plants and improve the growth of the plant, being localized in the bark or root nodules acting as inducers of the enzymes that participate in the metabolic pathways of bioactive compounds and jasmonic acid biosynthesis; these act as signal transducers [43, 44]. Other signal inducers are the mycorrhizal fungi that help the plant to absorb more water and show defense against other pathogens such as fungi, bacteria, or parasites that affect the roots of the plant. These mycorrhizal fungi produce secondary metabolites such as phenolic compounds and alkaloids, among others [45–48]. Elicitors such as salicylic acid, jasmonic acid, hydrogen peroxide, chitosan, etc. act as plant hormones in the expression of genes interacting as target signaling causing a physiological response in the plant which increases the production of phenolic compounds, vitamin C, carotenoids, or defense stimuli against pathogens; there are also synergistic effects between salicylic acid and jasmonic acid providing resistance against pathogens by the induction of the octadecanoic acid pathway [49–52, 53].

On the other hand, **Table 3** shows some research that has the influence of different abiotic elicitors that are considered substance and that are not of biological origin such as salt, drought, light or heavy metals, and temperature, among others. **Table 3** shows different perspectives of research on medicinal or aromatic plants in hydroponic crops, outdoors, and the application of elicitors in different stages of growth or postharvest. For example, heavy metals such as Al3+, Cr3+, Co2+, Ni2+,

#### *Plant Diseases-Current Threats and Management Trends*


Cu2+, Zn2+, and Cd2+, among others, are considered high toxicity compounds depending on the concentrations applied in the sprinkler system or because they are used as biocontrol since they alter the production of metabolites in plants. Similarly, Zobayed [65] demonstrated that the temperature in high concentrations in *Panax quinqufolius* improves the senescence of the leaves and produces a greater quantity of bioactive compounds in the root of the plant. So the investigations using high or low temperatures demonstrate the production of secondary metabolites, but the temperatures that have been investigated the most are the low producing physiological changes in the plant, increasing the lignification by the production of suberin in the cell wall and the metabolites such as sorbitol, raffinose, proline, melatonin, anthocyanins, etc. However, light by means of ultraviolet radiations generates the production of essential oils and phenolic compounds and decreases the production of toxic compounds in some plants [66]. On the other hand, salinity and drought produce death leading to cellular dehydration or osmotic stress and in certain concentrations can reduce the growth or development of plants but alter many physiological and metabolic processes that stimulate the production of polyphenolic compounds, anthocyanins, terpenes, and alkaloids, among others. Salinity can be produced in plants by ionic or osmotic means and drought by environmental or intentional changes due to water deficit which are always accompanied by temperature or solar radiation [67–69]. Then we can say that the biotic and abiotic factors

*Effect of abiotic elicitor on the production of various secondary metabolites in plants [70–81].*

*Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

**Table 3.**

**55**

are modular secondary metabolites influencing the metabolic level and the

**Table 2.** *Effect of biotic elicitor on the production of various secondary metabolites in plants [54–64].*

#### *Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*


#### **Table 3.**

*Effect of abiotic elicitor on the production of various secondary metabolites in plants [70–81].*

Cu2+, Zn2+, and Cd2+, among others, are considered high toxicity compounds depending on the concentrations applied in the sprinkler system or because they are used as biocontrol since they alter the production of metabolites in plants. Similarly, Zobayed [65] demonstrated that the temperature in high concentrations in *Panax quinqufolius* improves the senescence of the leaves and produces a greater quantity of bioactive compounds in the root of the plant. So the investigations using high or low temperatures demonstrate the production of secondary metabolites, but the temperatures that have been investigated the most are the low producing physiological changes in the plant, increasing the lignification by the production of suberin in the cell wall and the metabolites such as sorbitol, raffinose, proline, melatonin, anthocyanins, etc. However, light by means of ultraviolet radiations generates the production of essential oils and phenolic compounds and decreases the production of toxic compounds in some plants [66]. On the other hand, salinity and drought produce death leading to cellular dehydration or osmotic stress and in certain concentrations can reduce the growth or development of plants but alter many physiological and metabolic processes that stimulate the production of polyphenolic compounds, anthocyanins, terpenes, and alkaloids, among others. Salinity can be produced in plants by ionic or osmotic means and drought by environmental or intentional changes due to water deficit which are always accompanied by temperature or solar radiation [67–69]. Then we can say that the biotic and abiotic factors are modular secondary metabolites influencing the metabolic level and the

**Table 2.**

**54**

*Effect of biotic elicitor on the production of various secondary metabolites in plants [54–64].*

*Plant Diseases-Current Threats and Management Trends*

production of secondary metabolites. Therefore, the current research focuses on the use of elicitors, for the regulation of metabolic pathways, and target signaling in genes that influence the overproduction of secondary metabolites using various applications but taking care of the production performance of fruits, vegetables, or different plants.

Recent studies focused on evaluating the secondary metabolites of medicinal plants that are active against phytopathogens show that the potential use that these compounds can have in the future is for the control of phytopathogenic fungi, mainly against different species of *Fusarium* [14–16]. In this regard, the most active compounds have been found mainly in the essential oil obtained from the aerial parts of various medicinal plants, which suggests that the bioactive compounds are liposoluble; this may explain why they are active mainly against fungi, because the cell wall of these specimens are composed mainly of ergosterol, the active liposoluble compounds present in the essential oil to easily cross the cell wall of the fungus and in the interior act on their cell target, or they can alter the permeability of the wall of the fungus [82]. It can cause rupture and lysis of the fungal cell; however, it is necessary to study the toxicodynamics of these substances in order for them to know how to act in the fungi cell. On the other hand, the antifungal activity has been evaluated in *vitro*, by the agar diffusion and microdilution method; in general terms the range of the evaluated IC50 varies in a range that goes from 0.0035 to 8 mg/ml of the extract. It is important to mention that one of the main limitations of these studies is that this activity has only been evaluated at the laboratory level [83–85]. **Table 4** shows different types of extracts made with medicinal plants, and their biological activity reported *in vitro* tests at the laboratory level.

Finally, in the realization of a retrospective of the secondary metabolite modulating factors in our workgroup, Garcia-Mier [95] demonstrated that the use of mixtures of elicitors such as jasmonic acid, hydrogen peroxide, and chitosan in different concentrations applied in various stages of plant development of the sweet bell red pepper and in different stages of ripening of the fruit has a positive effect on the increase of polyphenolic and carotenoid compounds, where the results showed that the maturation stage of 95% produces a greater quantity of bioactive compounds. On the other hand, Vargas-Hernández [96] demonstrated that the foliar application of hydrogen peroxide in *Capsicum chinense* Jacq. has an effect on the antimicrobial activity, where the different concentrations of hydrogen peroxide potentiated the production of secondary metabolites such as flavonoids, capsaicin, and dihydrocapsaicin, where these metabolites had an effect on microorganisms such as *Staphylococcus aureus*, *Escherichia coli*, *Streptococcus mutant*, *Salmonella thompson*, *Listeria monocytogenes*, *Streptococcus faecalis*, and *Candida albicans*, and the results showed that the application of hydrogen peroxide increases the inhibitory effect against pathogenic microorganisms, showing greater activity against *S. aureus*, *S. Thompson*, and *C. albicans* in the jaguar variety, while the variety Chichen-Itza was more potent against *E. faecalis* and *E. coli*. Also, Zunun-Pérez [97] evaluated the effect of modulating factors of secondary metabolites by spray application that is performed in *Capsicum annuum* L. in weekly applications and 1 day before collection with elicitors such as hydrogen peroxide, salicylic acid, and oligosaccharide of xyloglucan on capsiate concentration and the expression of genes such as phenylalanine ammonia-lyase, aminotransferase, capsaicin synthase, and β-keto acyl synthase where the results showed that hydrogen peroxide in weekly applications significantly increases capsiate concentrations and gene expression and the yields of the production of the plant are not affected by the application of these elicitors.

**57**

*Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

#### *Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

production of secondary metabolites. Therefore, the current research focuses on the use of elicitors, for the regulation of metabolic pathways, and target signaling in genes that influence the overproduction of secondary metabolites using various applications but taking care of the production performance of fruits, vegetables, or

Recent studies focused on evaluating the secondary metabolites of medicinal plants that are active against phytopathogens show that the potential use that these compounds can have in the future is for the control of phytopathogenic fungi, mainly against different species of *Fusarium* [14–16]. In this regard, the most active compounds have been found mainly in the essential oil obtained from the aerial parts of

liposoluble; this may explain why they are active mainly against fungi, because the cell wall of these specimens are composed mainly of ergosterol, the active liposoluble compounds present in the essential oil to easily cross the cell wall of the fungus and in the interior act on their cell target, or they can alter the permeability of the wall of the fungus [82]. It can cause rupture and lysis of the fungal cell; however, it is necessary to study the toxicodynamics of these substances in order for them to know how to act in the fungi cell. On the other hand, the antifungal activity has been evaluated in *vitro*, by the agar diffusion and microdilution method; in general terms the range of the evaluated IC50 varies in a range that goes from 0.0035 to 8 mg/ml of the extract. It is important to mention that one of the main limitations of these studies is that this activity has only been evaluated at the laboratory level [83–85]. **Table 4** shows different types of extracts made with medicinal plants, and their biological activity

Finally, in the realization of a retrospective of the secondary metabolite modulating factors in our workgroup, Garcia-Mier [95] demonstrated that the use of mixtures of elicitors such as jasmonic acid, hydrogen peroxide, and chitosan in different concentrations applied in various stages of plant development of the sweet bell red pepper and in different stages of ripening of the fruit has a positive effect on the increase of polyphenolic and carotenoid compounds, where the results showed that the maturation stage of 95% produces a greater quantity of bioactive compounds. On the other hand, Vargas-Hernández [96] demonstrated that the foliar application of hydrogen peroxide in *Capsicum chinense* Jacq. has an effect on the antimicrobial activity, where the different concentrations of hydrogen peroxide potentiated the production of secondary metabolites such as flavonoids, capsaicin, and dihydrocapsaicin, where these metabolites had an effect on microorganisms such as *Staphylococcus aureus*, *Escherichia coli*, *Streptococcus mutant*, *Salmonella thompson*, *Listeria monocytogenes*, *Streptococcus faecalis*, and *Candida albicans*, and the results showed that the application of hydrogen peroxide increases the inhibitory effect against pathogenic microorganisms, showing greater activity against *S. aureus*, *S. Thompson*, and *C. albicans* in the jaguar variety, while the variety Chichen-Itza was more potent against *E. faecalis* and *E. coli*. Also, Zunun-Pérez [97] evaluated the effect of modulating factors of secondary metabolites by spray application that is performed in *Capsicum annuum* L. in weekly applications and 1 day before collection with elicitors such as hydrogen peroxide, salicylic acid, and oligosaccharide of xyloglucan on capsiate concentration and the expression of genes such as phenylalanine ammonia-lyase, aminotransferase, capsaicin synthase, and β-keto acyl synthase where the results showed that hydrogen peroxide in weekly applications significantly increases capsiate concentrations and gene expression and the yields of the production of the plant are not affected by the application of these elicitors.

various medicinal plants, which suggests that the bioactive compounds are

reported *in vitro* tests at the laboratory level.

*Plant Diseases-Current Threats and Management Trends*

different plants.

**56**



interest. There are different challenges in the use of biopesticides obtained from medicinal plants, such as evaluating the costs of obtaining these compounds on a large scale or exploring the possibility of them being obtained through chemical synthesis to increase yield and reduce costs. On the other hand, the various studies that exist on the effectiveness of these compounds are only at the laboratory level, which is why it is still necessary to explore and evaluate their effectiveness at the

The authors would like to acknowledge the University of Guanajuato for the

\*, Sandra N. Jiménez-García<sup>2</sup>

1 Department of Clinical Nursing, Division of Health Sciences and Engineering,

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

2 Department of Nursing and Obstetrics, Division of Health Sciences and Engineering, University of Guanajuato, Celaya, Guanajuato, Mexico

, Vicente Beltrán Campos<sup>1</sup>

greenhouse and field levels.

**Acknowledgements**

grant of this publication.

**Conflict of interest**

**Author details**

**59**

Xóchitl S. Ramírez-Gómez<sup>1</sup>

and Ma. Lourdes García Campos<sup>1</sup>

provided the original work is properly cited.

University of Guanajuato, Celaya, Guanajuato, Mexico

\*Address all correspondence to: xosofira2002@yahoo.com.mx

The authors declare no conflict of interest.

*Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

**Table 4.**

*Secondary metabolites of medicinal plants with biological activity against phytopathogens [86–94].*

#### **4. Conclusions**

The phytochemicals that produce medicinal plants derived from their secondary metabolism represent a safe and effective alternative to control various phytopathogens that affect various crops of agricultural products of economic and nutritional *Plant Metabolites in Plant Defense Against Pathogens DOI: http://dx.doi.org/10.5772/intechopen.87958*

interest. There are different challenges in the use of biopesticides obtained from medicinal plants, such as evaluating the costs of obtaining these compounds on a large scale or exploring the possibility of them being obtained through chemical synthesis to increase yield and reduce costs. On the other hand, the various studies that exist on the effectiveness of these compounds are only at the laboratory level, which is why it is still necessary to explore and evaluate their effectiveness at the greenhouse and field levels.

#### **Acknowledgements**

The authors would like to acknowledge the University of Guanajuato for the grant of this publication.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Xóchitl S. Ramírez-Gómez<sup>1</sup> \*, Sandra N. Jiménez-García<sup>2</sup> , Vicente Beltrán Campos<sup>1</sup> and Ma. Lourdes García Campos<sup>1</sup>

1 Department of Clinical Nursing, Division of Health Sciences and Engineering, University of Guanajuato, Celaya, Guanajuato, Mexico

2 Department of Nursing and Obstetrics, Division of Health Sciences and Engineering, University of Guanajuato, Celaya, Guanajuato, Mexico

\*Address all correspondence to: xosofira2002@yahoo.com.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

**4. Conclusions**

**Table 4.**

**58**

The phytochemicals that produce medicinal plants derived from their secondary metabolism represent a safe and effective alternative to control various phytopathogens that affect various crops of agricultural products of economic and nutritional

*Secondary metabolites of medicinal plants with biological activity against phytopathogens [86–94].*

*Plant Diseases-Current Threats and Management Trends*

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stress on the growth and production of secondary metabolites in *Prunella vulgaris* L. The Journal of Medicinal Plants Research. 2011;**5**:1749-1755

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[88] Mongalo NI, Dikhoba PM, Soyingbe SO, Makhafola TJ. Antifungal, antioxidant activity and cytotoxicity of south African medicinal plants against mycotoxigenic fungi. Heliyon. 2018;**4**: e00973. DOI: 10.1016/j.heliyon.2018.

[89] Mahlo SM, Chauke HR, McGaw L, Eloff J. Antioxidant and antifungal activity of selected medicinal plant extracts against phytopathogenic fungi.

African Journal of Traditional, Complementary, and Alternative Medicines. 2016;**13**:216-222. DOI:

[90] Costa E, Silva J, de Sousa Carlos Mourão D, de Oliveira Lima FS, de Almeida Sarmento R, Sunti Dalcin M, et al. The efficiency of noni (*Morinda citrifolia* L.) essential oil on the control of leaf spot caused by *Exserohilum turcicum* in maize culture. Medicines.

[91] Nkomo MM, Katerere D, Vismer H, Cruz TT, Stephane S, Balayssac SS, et al. Fusarium inhibition by wild populations of the medicinal plant *Salvia africana*lutea L. linked to metabolomic profiling. BMC Complementary and Alternative Medicine. 2014;**14**(99):2-9. DOI:

[92] Elshafie HS, Grul'ová D, Baranová B, Caputo L, De Martino L, Sedlák V, et al. Antimicrobial activity and chemical composition of essential oil extracted from *Solidago canadensis* L. growing wild in Slovakia. Molecules.

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**69**

**Chapter 5**

**Abstract**

Neglected Disease

management, with emphasis on biological control.

management, semiarid regions

**1. Introduction**

*and Jorge Teodoro de Souza*

Sisal Bole Rot: An Important but

*Yasmim Freitas Figueiredo, Phellippe Arthur Santos Marbach* 

Sisal (*Agave sisalana*) is one of the main sources of hard natural fibre and raw materials for the industry, medicine and handicrafts. Sisal yields a coarse and strong fibre that is increasingly being used in composite materials for automobiles, furniture, construction and plastic and paper products. Extracts of sisal contain substances with anti-inflammatory, antimicrobial and anthelmintic activities. Sisal is adapted to warm environments with low rainfall and is an excellent option for cultivation in semiarid conditions, where other crops cannot be grown. The world's largest sisal producers are Brazil, Tanzania, China, Kenya and Madagascar. Sisal is a labour-intensive crop with great socio-economical importance as it is cultivated in poor areas employing familiar labour. Sisal bole rot is the main disease of sisal, responsible for substantial losses in producing countries. The disease is caused by certain species of the genus *Aspergillus*, especially the ones belonging in the section *Nigri*. The main symptoms are yellowing of the aerial parts and the red-coloured rot of the bole, which causes the plant to die. In this review we are going to address the taxonomy of the causal agents, disease diagnosis and epidemiology and disease

**Keywords:** *Aspergillus welwitschiae*, *Agave sisalana*, biological control, disease

nutritional demand when compared to C3 and C4 plants [1, 5–7].

*Agave sisalana* Perr. ex. Engelm is a monocotyledonous, xerophytic, succulent plant that belongs in the *Asparagaceae* family. The genus *Agave* has more than 200 species, and Mexico is their centre of origin and dispersion, where they have high economic importance and several industrial applications [1, 2]. This genus is able to grow in different conditions, as well as to show excellent adaptation to environments with warm climate, high luminosity and prolonged droughts [3, 4]. Tolerance to abiotic stresses is a striking feature of *A. sisalana*, which confers good performances to this species under conditions that limit the development of most plants [4]. This tolerance is related to morphological and physiological characteristics, such as the CAM metabolism (crassulacean acid). This type of metabolism allows for greater efficiency in water use, higher carbon uptake during the night and low

*Valter Cruz-Magalhães, Jackeline Pereira Andrade,* 

#### **Chapter 5**

## Sisal Bole Rot: An Important but Neglected Disease

*Valter Cruz-Magalhães, Jackeline Pereira Andrade, Yasmim Freitas Figueiredo, Phellippe Arthur Santos Marbach and Jorge Teodoro de Souza*

#### **Abstract**

Sisal (*Agave sisalana*) is one of the main sources of hard natural fibre and raw materials for the industry, medicine and handicrafts. Sisal yields a coarse and strong fibre that is increasingly being used in composite materials for automobiles, furniture, construction and plastic and paper products. Extracts of sisal contain substances with anti-inflammatory, antimicrobial and anthelmintic activities. Sisal is adapted to warm environments with low rainfall and is an excellent option for cultivation in semiarid conditions, where other crops cannot be grown. The world's largest sisal producers are Brazil, Tanzania, China, Kenya and Madagascar. Sisal is a labour-intensive crop with great socio-economical importance as it is cultivated in poor areas employing familiar labour. Sisal bole rot is the main disease of sisal, responsible for substantial losses in producing countries. The disease is caused by certain species of the genus *Aspergillus*, especially the ones belonging in the section *Nigri*. The main symptoms are yellowing of the aerial parts and the red-coloured rot of the bole, which causes the plant to die. In this review we are going to address the taxonomy of the causal agents, disease diagnosis and epidemiology and disease management, with emphasis on biological control.

**Keywords:** *Aspergillus welwitschiae*, *Agave sisalana*, biological control, disease management, semiarid regions

#### **1. Introduction**

*Agave sisalana* Perr. ex. Engelm is a monocotyledonous, xerophytic, succulent plant that belongs in the *Asparagaceae* family. The genus *Agave* has more than 200 species, and Mexico is their centre of origin and dispersion, where they have high economic importance and several industrial applications [1, 2]. This genus is able to grow in different conditions, as well as to show excellent adaptation to environments with warm climate, high luminosity and prolonged droughts [3, 4]. Tolerance to abiotic stresses is a striking feature of *A. sisalana*, which confers good performances to this species under conditions that limit the development of most plants [4]. This tolerance is related to morphological and physiological characteristics, such as the CAM metabolism (crassulacean acid). This type of metabolism allows for greater efficiency in water use, higher carbon uptake during the night and low nutritional demand when compared to C3 and C4 plants [1, 5–7].

Sisal is a monocarpic plant, and the emission of an inflorescence characterises the end of its vegetative cycle, which can occur between 8 and 30 years. The plant multiplies vegetatively through bulbils produced on the inflorescence pole or by stolons that emerge from the rhizome (subterraneous stem) of adult plants. The use of bulbils is the most common form of propagation, but stolons can also be used. The production of seeds is rare, and induction techniques are necessary when this is the objective [8–10]. Most species of *Agave* are highly endemic and have high levels of genetic variation within populations and low differentiation between populations [11]. This limited diversity hinders the establishment of germplasm banks and the search for genes that confer desirable characteristics to these plants.

*Agave sisalana* is a good producer of hard natural fibres [1]. The fibre extracted from this plant occupies the sixth position of importance and represents 2% of the world production of plant fibres [12]. This product is extracted from the leaves of the plant and is traditionally used in the manufacture of cords and ropes [9]. In addition, it is widely used in various industrial sectors. Amongst several applications, sisal fibre has been increasingly used in the reinforcement of building materials, furniture, panels and automobile upholstery [1, 12, 13]. In addition to the various applications and industrial uses, sisal fibre has advantages over synthetic fibres for having lower density (lighter) and lower production cost and is biodegradable and recyclable. Therefore, the use of sisal fibre fits in the growing world tendency that favours the use of sustainable natural resources with less environmental impact [14, 15].

There has been a growing interest in the use of waste or by-products from *Agave* species in biotechnological processes [16, 17]. After fibre extraction the residue is usually discarded [18]. This residue accounts for 98% of the total biomass of the plant and has potential to be used as raw material for biofuels, especially because it is not directly used as food [6, 12, 19]. In order to exploit the economic value of this material, a joint initiative between the Common Fund for Commodities, the United Nations Industrial Development Organization (UNIDO) and the Tanzanian sisal industry financed the first commercial plant for the production of biogas [12]. In addition to some medicinal properties reported [20, 21], *A. sisalana* also produces compounds that have different biological properties [18] of great interest in the pharmaceutical industry such as hecogenin [12, 21–23]. All of the above features place sisal as a strategic species to be exploited in tropical semiarid regions and in temperate latitudes with drought resulting from global climate change [16, 19, 24].

The main world producers of sisal fibre are Brazil, Tanzania, China, Kenya and Madagascar [25]. Other countries, such as Mexico, South Africa, Mozambique, Angola, Indonesia, Thailand, Haiti and Cuba, also produce but in smaller quantities. According to FAO reports, in 2011 Brazil alone produced more than 111 thousand tons of sisal fibre [12].

In Brazil, the semiarid region of Bahia province (northeastern Brazil) is responsible for more than 95% of the country's sisal production [26]. Other provinces that produce smaller amounts of sisal in Brazil are Paraiba, Rio Grande do Norte and Ceará [27]. It is estimated that more than 150,000 families are directly linked to the producing chain of this crop, totalling more than 700,000 small farmers, and more than half a million direct and indirect jobs are involved in activities related to the maintenance, harvesting, extraction and processing of fibre [28–30]. In this sense, sisal has an important economic and social role of the semiarid region of Brazil.

Sisal management is simple because this plant exhibits tolerance to various abiotic stresses. Even under minimal management conditions, the plant presents good development and consequently good fibre production, with low nutritional requirements [12]. However, although it presents all these adaptive advantages to stress conditions, the main problem is of phytosanitary origin. Sisal bole rot, the main disease of sisal, has caused considerable damage to the crop [31]. This disease

**71**

**Figure 1.**

*(B) and (C) Plants killed by the pathogen.*

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

causes the death of infected plants, and despite the economic and social importance

In this chapter we introduce the sisal bole rot disease, a neglected disease that represents the main challenge for sisal production in Brazil and other countries of the world. In addition, we discuss some aspects involved in its symptomatology, aetiology, epidemiology and management. The majority of the results that will be shown were obtained in Brazil, where most of the research on sisal bole rot was done.

The disease was first reported in production areas of Tanzania and Brazil [31, 32]. In Brazil, since the 1990s, the commercial production of sisal has been

*Adult sisal plants under field conditions. (A) Healthy adult plant (white arrow) after leaf harvest for fibre extraction next to an adult plant showing the external symptoms of sisal bole rot (red arrow). The diseased plant has wilted and yellowish leaves that cannot be used for fibre extraction and therefore was not harvested.* 

of sisal, there are few government efforts to control the disease.

**2. Bole rot disease: symptoms and epidemiology**

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

*Plant Diseases-Current Threats and Management Trends*

Sisal is a monocarpic plant, and the emission of an inflorescence characterises the end of its vegetative cycle, which can occur between 8 and 30 years. The plant multiplies vegetatively through bulbils produced on the inflorescence pole or by stolons that emerge from the rhizome (subterraneous stem) of adult plants. The use of bulbils is the most common form of propagation, but stolons can also be used. The production of seeds is rare, and induction techniques are necessary when this is the objective [8–10]. Most species of *Agave* are highly endemic and have high levels of genetic variation within populations and low differentiation between populations [11]. This limited diversity hinders the establishment of germplasm banks and

the search for genes that confer desirable characteristics to these plants.

natural resources with less environmental impact [14, 15].

thousand tons of sisal fibre [12].

*Agave sisalana* is a good producer of hard natural fibres [1]. The fibre extracted from this plant occupies the sixth position of importance and represents 2% of the world production of plant fibres [12]. This product is extracted from the leaves of the plant and is traditionally used in the manufacture of cords and ropes [9]. In addition, it is widely used in various industrial sectors. Amongst several applications, sisal fibre has been increasingly used in the reinforcement of building materials, furniture, panels and automobile upholstery [1, 12, 13]. In addition to the various applications and industrial uses, sisal fibre has advantages over synthetic fibres for having lower density (lighter) and lower production cost and is biodegradable and recyclable. Therefore, the use of sisal fibre fits in the growing world tendency that favours the use of sustainable

There has been a growing interest in the use of waste or by-products from *Agave* species in biotechnological processes [16, 17]. After fibre extraction the residue is usually discarded [18]. This residue accounts for 98% of the total biomass of the plant and has potential to be used as raw material for biofuels, especially because it is not directly used as food [6, 12, 19]. In order to exploit the economic value of this material, a joint initiative between the Common Fund for Commodities, the United Nations Industrial Development Organization (UNIDO) and the Tanzanian sisal industry financed the first commercial plant for the production of biogas [12]. In addition to some medicinal properties reported [20, 21], *A. sisalana* also produces compounds that have different biological properties [18] of great interest in the pharmaceutical industry such as hecogenin [12, 21–23]. All of the above features place sisal as a strategic species to be exploited in tropical semiarid regions and in temperate latitudes with drought resulting from global climate change [16, 19, 24]. The main world producers of sisal fibre are Brazil, Tanzania, China, Kenya and Madagascar [25]. Other countries, such as Mexico, South Africa, Mozambique, Angola, Indonesia, Thailand, Haiti and Cuba, also produce but in smaller quantities. According to FAO reports, in 2011 Brazil alone produced more than 111

In Brazil, the semiarid region of Bahia province (northeastern Brazil) is responsible for more than 95% of the country's sisal production [26]. Other provinces that produce smaller amounts of sisal in Brazil are Paraiba, Rio Grande do Norte and Ceará [27]. It is estimated that more than 150,000 families are directly linked to the producing chain of this crop, totalling more than 700,000 small farmers, and more than half a million direct and indirect jobs are involved in activities related to the maintenance, harvesting, extraction and processing of fibre [28–30]. In this sense, sisal has an important economic and social role of the semiarid region of Brazil. Sisal management is simple because this plant exhibits tolerance to various abiotic stresses. Even under minimal management conditions, the plant presents good development and consequently good fibre production, with low nutritional requirements [12]. However, although it presents all these adaptive advantages to stress conditions, the main problem is of phytosanitary origin. Sisal bole rot, the main disease of sisal, has caused considerable damage to the crop [31]. This disease

**70**

causes the death of infected plants, and despite the economic and social importance of sisal, there are few government efforts to control the disease.

In this chapter we introduce the sisal bole rot disease, a neglected disease that represents the main challenge for sisal production in Brazil and other countries of the world. In addition, we discuss some aspects involved in its symptomatology, aetiology, epidemiology and management. The majority of the results that will be shown were obtained in Brazil, where most of the research on sisal bole rot was done.

#### **2. Bole rot disease: symptoms and epidemiology**

The disease was first reported in production areas of Tanzania and Brazil [31, 32]. In Brazil, since the 1990s, the commercial production of sisal has been

#### **Figure 1.**

*Adult sisal plants under field conditions. (A) Healthy adult plant (white arrow) after leaf harvest for fibre extraction next to an adult plant showing the external symptoms of sisal bole rot (red arrow). The diseased plant has wilted and yellowish leaves that cannot be used for fibre extraction and therefore was not harvested. (B) and (C) Plants killed by the pathogen.*

declining due to economical crises and the occurrence of this disease [33]. Diseased plants produce leaves that are not suitable for fibre extraction as they lose their turgescence, and although these diseased plants survive for some time, they die with the progress of the disease (**Figure 1**) [35]. Plants at advanced stages of the disease are easily identified by the symptoms, which include wilting and yellowing of the aerial part (**Figure 1A**). The main internal symptom of the disease is rotting of the stem with reddening of the tissues, a response of the plant to fungal colonisation. It is thought that there is no relationship between the phenological stage of the plant and the establishment of the disease, since the fungus is capable of infecting both plantlets (**Figure 2E**) and adult plants (**Figures 1** and **2**).

It was reported that the pathogen depends on mechanical injuries and natural openings, mainly on physiologically stressed plants, to start the infection process [32]. In this sense, it is possible that wounds made by insects or by tools used in crop management, such as harvest of the leaves and cultural practices, are ways of pathogen penetration [32, 35, 36]. The histopathology of diseased plants showed that the pathogen penetrates the tissues of the host from the outside, that is, from the epidermis to the parenchyma and later to the central cylinder of the plant [37].

Abreu [36] studied the spatiotemporal distribution of sisal bole rot in producing areas of Bahia Province, Brazil, and found that the disease was present in all

#### **Figure 2.**

*Sisal plantlets with symptoms of sisal bole rot under greenhouse conditions. (A) Healthy sisal plantlets and (B) diseased plantlet with symptoms of sisal bole rot. (C) Stem of healthy plant. (D, F and G) Intermediate symptoms of sisal bole rot, characterised by rotting of the stem. (E and H) Dead plants. The white arrow indicates the production of conidia after colonisation of plant tissues.*

**73**

**Figure 3.**

*number of substitutions per site.*

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

down the progress of the disease.

lishment of the pathogen in the area.

**3. Causal agents**

the studied farms (prevalence of 100%) and, on average, 35% of the plants were infected by the pathogen. This study also showed that the distribution of the disease occurs randomly in the cultivated areas [36]. In the case of sisal bole rot, incidence evaluations are more important than severity, as there are no measures that slow

The lack of more studies on epidemiological aspects of sisal bole rot in different areas where the disease occurs directly impacts the establishment of phytosanitary management practices. More information on these aspects could contribute to the development of strategies to reduce the incidence of the disease. For the moment, what is known is that preventive measures should be employed to avoid the estab-

The disease was first observed in areas of sisal production in Tanzania in the 1930s but was only reported in the 1950s [32]. The causal agent was isolated from diseased plant parts and identified as *Aspergillus niger*. In this study, the authors reported fruiting bodies of *A. niger* in exposed plant tissues and also pointed out that the occurrence of the disease was linked to environmental conditions and the nutritional status of the plant [32]. The first report of this disease in Brazil also occurred in the 1950s, when Machado [38] described a rot of the base of sisal

*Phylogenetic tree of the 27 valid species belonging in the Nigri section of Aspergillus. The red circles indicate species shown to cause sisal bole rot in the A. niger complex. The tree was constructed with sequences of the calmodulin gene, with 456 nucleotides aligned using the maximum likelihood (ML) method and the K2 + G + I substitution model. The bootstrap analysis was performed with 1000 resamplings. The scale represents the* 

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

the studied farms (prevalence of 100%) and, on average, 35% of the plants were infected by the pathogen. This study also showed that the distribution of the disease occurs randomly in the cultivated areas [36]. In the case of sisal bole rot, incidence evaluations are more important than severity, as there are no measures that slow down the progress of the disease.

The lack of more studies on epidemiological aspects of sisal bole rot in different areas where the disease occurs directly impacts the establishment of phytosanitary management practices. More information on these aspects could contribute to the development of strategies to reduce the incidence of the disease. For the moment, what is known is that preventive measures should be employed to avoid the establishment of the pathogen in the area.

#### **3. Causal agents**

*Plant Diseases-Current Threats and Management Trends*

plantlets (**Figure 2E**) and adult plants (**Figures 1** and **2**).

declining due to economical crises and the occurrence of this disease [33]. Diseased plants produce leaves that are not suitable for fibre extraction as they lose their turgescence, and although these diseased plants survive for some time, they die with the progress of the disease (**Figure 1**) [35]. Plants at advanced stages of the disease are easily identified by the symptoms, which include wilting and yellowing of the aerial part (**Figure 1A**). The main internal symptom of the disease is rotting of the stem with reddening of the tissues, a response of the plant to fungal colonisation. It is thought that there is no relationship between the phenological stage of the plant and the establishment of the disease, since the fungus is capable of infecting both

It was reported that the pathogen depends on mechanical injuries and natural openings, mainly on physiologically stressed plants, to start the infection process [32]. In this sense, it is possible that wounds made by insects or by tools used in crop management, such as harvest of the leaves and cultural practices, are ways of pathogen penetration [32, 35, 36]. The histopathology of diseased plants showed that the pathogen penetrates the tissues of the host from the outside, that is, from the epidermis to the parenchyma and later to the central cylinder of the plant [37]. Abreu [36] studied the spatiotemporal distribution of sisal bole rot in producing areas of Bahia Province, Brazil, and found that the disease was present in all

**72**

**Figure 2.**

*Sisal plantlets with symptoms of sisal bole rot under greenhouse conditions. (A) Healthy sisal plantlets and (B) diseased plantlet with symptoms of sisal bole rot. (C) Stem of healthy plant. (D, F and G) Intermediate symptoms of sisal bole rot, characterised by rotting of the stem. (E and H) Dead plants. The white arrow* 

*indicates the production of conidia after colonisation of plant tissues.*

The disease was first observed in areas of sisal production in Tanzania in the 1930s but was only reported in the 1950s [32]. The causal agent was isolated from diseased plant parts and identified as *Aspergillus niger*. In this study, the authors reported fruiting bodies of *A. niger* in exposed plant tissues and also pointed out that the occurrence of the disease was linked to environmental conditions and the nutritional status of the plant [32]. The first report of this disease in Brazil also occurred in the 1950s, when Machado [38] described a rot of the base of sisal

#### **Figure 3.**

*Phylogenetic tree of the 27 valid species belonging in the Nigri section of Aspergillus. The red circles indicate species shown to cause sisal bole rot in the A. niger complex. The tree was constructed with sequences of the calmodulin gene, with 456 nucleotides aligned using the maximum likelihood (ML) method and the K2 + G + I substitution model. The bootstrap analysis was performed with 1000 resamplings. The scale represents the number of substitutions per site.*

stem in the province of Paraíba, Brazil [39]. In Bahia, the largest sisal-producing province in Brazil, the disease was first noticed in a commercial plantation by researchers from the Agency for Agricultural Development of Bahia (EBDA) and Embrapa Semiárido (Brazilian Agricultural Research Institute) in the municipality of Santaluz [33].

In Tanzania and in Brazil, the disease was initially associated with the species *A. niger*. The aetiology of the disease was determined by Koch's postulates from tissue fragments of diseased sisal plants [40]. Species of the genus *Aspergillus* are filamentous fungi belonging in the phylum *Ascomycota* [41]. *Aspergillus niger* and other closely related species form a cluster of morphologically similar species, collectively known as the section *Nigri* (**Figure 3**). The *Nigri* section is comprised of 27 valid species that contain the *A. niger* complex (**Figure 3**). All these species have as main characteristic the formation of black-coloured conidia, uniseriate or biseriate conidiophores and dark colonies (**Figure 4**) [42]. The taxonomy of the section *Nigri* is very complex because many species of this group are difficult to distinguish morphologically [41]. The morphological criteria were the only ones used to identify these species for a long time, and for this reason, many species were misidentified [43, 44].

#### **Figure 4.**

*Macro- and micromorphology of Aspergillus welwitschiae isolated from diseased sisal plants. (A) Obverse and reverse of a plate containing mycelial growth of colony on Blakeslee's malt extract (MEAbl), growing at 25o C for 7 days. (B) Conidiophores of A. welwitschiae and (C) conidia. Scale bars =10 μm.*

**75**

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

fungal structures [44, 45].

cause the disease, including *A. niger*.

**4. Disease management**

nated in diseased plants.

disease to new areas [52].

the disease in the field.

The polyphasic taxonomy integrates molecular, physiological, metabolite production and morphological data for the identification and description of new species of the section *Nigri* [45–48]. The regions recommended for the identification and description of species in the genus *Aspergillus* are fragments of the ITS region of the ribosomal DNA, calmodulin (*caM*), beta-tubulin (*benA*) and the beta subunit of the RNA polymerase (*rpb2*). However, caM sequences were proposed as the most informative markers for the section *Nigri* [49]. The gene *benA* is very informative for the uniseriate/aculeatus clade; however, care must be taken not to use the wrong set of primers (Bt2a/Bt2b) that can also amplify *tubC*, a paralog of *benA*, resulting in misidentification. The alternative primer pair ben2f/Bt2b should be used instead [50]. The other methods used in the polyphasic approach include growth on different media and temperatures, production of secondary metabolites and measurement of all

The initial studies implicated only *A. niger* as the cause of bole rot disease because the authors only took the morphological features of the pathogen into account [32, 40]. Further studies including sequences of the ITS region of the ribosomal DNA and a fragment of the transcription and elongation factor of the RNA polymerase (tef1-alpha) also identified *A. brasiliensis* and *A. tubingensis* in addition to *A. niger* as agents of the disease [31]. Recently, Duarte et al. [37] identified molecular phylogeny strains of *Aspergillus* sp. of the section *Nigri* obtained from diseased plants using a fragment of the calmodulin gene and proposed that *A. welwitschiae* and not *A. niger* is the causal agent of sisal bole rot disease. However, these authors did not include *A. niger* in their study, and therefore, further investigations are still needed to evaluate the ability of other species in the section *Nigri* to

There are no effective control methods available for bole rot disease [51]. Mechanical lesions are used by the pathogen as penetration sites, and this has direct implications for crop management since leaf harvest causes wounds in the plant [32, 36]. Additionally, the pathogen may be spread through the use of tools contami-

Most farmers use plantlets from stolons to establish new plantations, and infected plant material contributes to the spread of the disease to new areas. Therefore, the establishment of new areas using healthy plant material is thought to be one of the most effective ways to prevent the introduction of the pathogen. Removal and destruction of diseased plants from the plantations, balanced fertilisation to prevent stresses and disinfestation of the tools used in diseased plants are other measures recommended to decrease the incidence and avoid the spread of the

Another method investigated to manage the disease is the use of antagonistic microorganisms [53, 34]. Chemical control was never investigated probably because the causal agents are soilborne fungi and farmers have little financial resources. Biological control is an environmentally friendly and viable method to control plant pathogens [54, 45]. Antagonistic bacteria were shown to have potential to control the bole rot disease [53, 34]. Several strains of an undescribed species of *Burkholderia* and strains of *Bacillus* decreased the incidence and severity of the disease under field conditions (**Figures 5** and **6**) [53, 34]. Therefore, it is possible to establish programmes aimed at the development of biological products to manage

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

*Plant Diseases-Current Threats and Management Trends*

of Santaluz [33].

misidentified [43, 44].

stem in the province of Paraíba, Brazil [39]. In Bahia, the largest sisal-producing province in Brazil, the disease was first noticed in a commercial plantation by researchers from the Agency for Agricultural Development of Bahia (EBDA) and Embrapa Semiárido (Brazilian Agricultural Research Institute) in the municipality

In Tanzania and in Brazil, the disease was initially associated with the species *A. niger*. The aetiology of the disease was determined by Koch's postulates from tissue fragments of diseased sisal plants [40]. Species of the genus *Aspergillus* are filamentous fungi belonging in the phylum *Ascomycota* [41]. *Aspergillus niger* and other closely related species form a cluster of morphologically similar species, collectively known as the section *Nigri* (**Figure 3**). The *Nigri* section is comprised of 27 valid species that contain the *A. niger* complex (**Figure 3**). All these species have as main characteristic the formation of black-coloured conidia, uniseriate or biseriate conidiophores and dark colonies (**Figure 4**) [42]. The taxonomy of the section *Nigri* is very complex because many species of this group are difficult to distinguish morphologically [41]. The morphological criteria were the only ones used to identify these species for a long time, and for this reason, many species were

*Macro- and micromorphology of Aspergillus welwitschiae isolated from diseased sisal plants. (A) Obverse and reverse of a plate containing mycelial growth of colony on Blakeslee's malt extract (MEAbl), growing at 25o*

*for 7 days. (B) Conidiophores of A. welwitschiae and (C) conidia. Scale bars =10 μm.*

*C* 

**74**

**Figure 4.**

The polyphasic taxonomy integrates molecular, physiological, metabolite production and morphological data for the identification and description of new species of the section *Nigri* [45–48]. The regions recommended for the identification and description of species in the genus *Aspergillus* are fragments of the ITS region of the ribosomal DNA, calmodulin (*caM*), beta-tubulin (*benA*) and the beta subunit of the RNA polymerase (*rpb2*). However, caM sequences were proposed as the most informative markers for the section *Nigri* [49]. The gene *benA* is very informative for the uniseriate/aculeatus clade; however, care must be taken not to use the wrong set of primers (Bt2a/Bt2b) that can also amplify *tubC*, a paralog of *benA*, resulting in misidentification. The alternative primer pair ben2f/Bt2b should be used instead [50]. The other methods used in the polyphasic approach include growth on different media and temperatures, production of secondary metabolites and measurement of all fungal structures [44, 45].

The initial studies implicated only *A. niger* as the cause of bole rot disease because the authors only took the morphological features of the pathogen into account [32, 40]. Further studies including sequences of the ITS region of the ribosomal DNA and a fragment of the transcription and elongation factor of the RNA polymerase (tef1-alpha) also identified *A. brasiliensis* and *A. tubingensis* in addition to *A. niger* as agents of the disease [31]. Recently, Duarte et al. [37] identified molecular phylogeny strains of *Aspergillus* sp. of the section *Nigri* obtained from diseased plants using a fragment of the calmodulin gene and proposed that *A. welwitschiae* and not *A. niger* is the causal agent of sisal bole rot disease. However, these authors did not include *A. niger* in their study, and therefore, further investigations are still needed to evaluate the ability of other species in the section *Nigri* to cause the disease, including *A. niger*.

#### **4. Disease management**

There are no effective control methods available for bole rot disease [51]. Mechanical lesions are used by the pathogen as penetration sites, and this has direct implications for crop management since leaf harvest causes wounds in the plant [32, 36]. Additionally, the pathogen may be spread through the use of tools contaminated in diseased plants.

Most farmers use plantlets from stolons to establish new plantations, and infected plant material contributes to the spread of the disease to new areas. Therefore, the establishment of new areas using healthy plant material is thought to be one of the most effective ways to prevent the introduction of the pathogen. Removal and destruction of diseased plants from the plantations, balanced fertilisation to prevent stresses and disinfestation of the tools used in diseased plants are other measures recommended to decrease the incidence and avoid the spread of the disease to new areas [52].

Another method investigated to manage the disease is the use of antagonistic microorganisms [53, 34]. Chemical control was never investigated probably because the causal agents are soilborne fungi and farmers have little financial resources. Biological control is an environmentally friendly and viable method to control plant pathogens [54, 45]. Antagonistic bacteria were shown to have potential to control the bole rot disease [53, 34]. Several strains of an undescribed species of *Burkholderia* and strains of *Bacillus* decreased the incidence and severity of the disease under field conditions (**Figures 5** and **6**) [53, 34]. Therefore, it is possible to establish programmes aimed at the development of biological products to manage the disease in the field.

#### **Figure 5.**

*Management of sisal bole rot disease with antagonistic bacteria. (A) and (B) Plantlets treated with Burkholderia sp. and inoculated with the pathogen A. welwitschiae in the field. (C) and (D) Sisal plants inoculated with A. welwitschiae only under field conditions (positive control).*

#### **Figure 6.**

*Incidence of sisal bole rot disease by the application of Burkholderia and Bacillus strains under field conditions. The means represent 25 replicates per treatment. The negative control was treated with water only (CT-) and positive control with A. welwitschiae (CT+). Error bars represent the standard error of the means.*

#### **5. Outlook**

Little is known about the mechanisms used by the pathogen to infect the plant, although *Aspergillus* shows a typical necrotrophic behaviour [37]. More information on the pathogenicity mechanisms could be obtained by the use of omics tools, such as RNAseq, to identify genes expressed by the pathogen during infection. Other microorganisms can influence the establishment and progress of the disease, and in this sense it will be interesting to study the comparative microbiome of diseased

**77**

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

natural populations out of Mexico.

**Acknowledgements**

support.

**Author details**

Valter Cruz-Magalhães1

Phellippe Arthur Santos Marbach3

1 Federal University of Lavras, Lavras, MG, Brazil

3 Reconcavo da Bahia Federal University, BA, Brazil

provided the original work is properly cited.

\*Address all correspondence to: jorge.souza@dfp.ufla.br

and healthy plants. This information may be used to engineer the microbiome to keep the plants healthy, as it has been attempted for other agricultural crops [55]. Sisal bole rot cannot be controlled by any single method, and therefore, the integration of control measures must be adopted. Resistant cultivars are not available for this crop, and unfortunately there are no breeding programmes focusing on sisal bole rot [9]. Breeding programmes are limited by the low genetic diversity of

Preventive measures are thought to be the most effective ways to control bole rot, and these include (i) the use of healthy planting material, (ii) balanced fertilisation to avoid nutritional stresses and (iii) maintaining adequate soil humidity levels to avoid physiological imbalances [52]. When these measures are not able to contain the pathogen, removal of diseased plants is recommended to decrease the source of inoculum of the pathogen [52]. One challenge in this regard is the development strategies to identify diseased plants before the dispersal of pathogen propagules. Sisal residues are commonly used to fertilise plants in the field [52], but only the fermented residue is suitable for this purpose as fresh residues stimulate the spread of the pathogen [56]. Information such as these could be disseminated to farmers to contribute to the management of the disease. Sisal farmers in many parts of the world do not have access to information on the technical aspects of sisal, depend on familiar labour and have little financial resources to invest in the crop. The information generated so far on the management of the disease through the use of antagonistic bacteria are promising, but it is still necessary to develop it into products that can be used by the farmers. New studies aiming at formulating and distributing biological products should be encouraged to contribute to the sustainability of this crop in the long run. The general lack of research on bole rot classifies it as a neglected disease that deserves more attention from research institutes and the government.

The authors thank the Brazilian agencies CNPq and CAPES for the financial

and Jorge Teodoro de Souza1

, Yasmim Freitas Figueiredo1

\*

,

, Jackeline Pereira Andrade2

2 Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

#### *Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

*Plant Diseases-Current Threats and Management Trends*

**76**

**5. Outlook**

**Figure 6.**

**Figure 5.**

Little is known about the mechanisms used by the pathogen to infect the plant, although *Aspergillus* shows a typical necrotrophic behaviour [37]. More information on the pathogenicity mechanisms could be obtained by the use of omics tools, such as RNAseq, to identify genes expressed by the pathogen during infection. Other microorganisms can influence the establishment and progress of the disease, and in this sense it will be interesting to study the comparative microbiome of diseased

*Incidence of sisal bole rot disease by the application of Burkholderia and Bacillus strains under field conditions. The means represent 25 replicates per treatment. The negative control was treated with water only (CT-) and* 

*positive control with A. welwitschiae (CT+). Error bars represent the standard error of the means.*

*Management of sisal bole rot disease with antagonistic bacteria. (A) and (B) Plantlets treated with Burkholderia sp. and inoculated with the pathogen A. welwitschiae in the field. (C) and (D) Sisal plants* 

*inoculated with A. welwitschiae only under field conditions (positive control).*

and healthy plants. This information may be used to engineer the microbiome to keep the plants healthy, as it has been attempted for other agricultural crops [55].

Sisal bole rot cannot be controlled by any single method, and therefore, the integration of control measures must be adopted. Resistant cultivars are not available for this crop, and unfortunately there are no breeding programmes focusing on sisal bole rot [9]. Breeding programmes are limited by the low genetic diversity of natural populations out of Mexico.

Preventive measures are thought to be the most effective ways to control bole rot, and these include (i) the use of healthy planting material, (ii) balanced fertilisation to avoid nutritional stresses and (iii) maintaining adequate soil humidity levels to avoid physiological imbalances [52]. When these measures are not able to contain the pathogen, removal of diseased plants is recommended to decrease the source of inoculum of the pathogen [52]. One challenge in this regard is the development strategies to identify diseased plants before the dispersal of pathogen propagules.

Sisal residues are commonly used to fertilise plants in the field [52], but only the fermented residue is suitable for this purpose as fresh residues stimulate the spread of the pathogen [56]. Information such as these could be disseminated to farmers to contribute to the management of the disease. Sisal farmers in many parts of the world do not have access to information on the technical aspects of sisal, depend on familiar labour and have little financial resources to invest in the crop. The information generated so far on the management of the disease through the use of antagonistic bacteria are promising, but it is still necessary to develop it into products that can be used by the farmers. New studies aiming at formulating and distributing biological products should be encouraged to contribute to the sustainability of this crop in the long run. The general lack of research on bole rot classifies it as a neglected disease that deserves more attention from research institutes and the government.

#### **Acknowledgements**

The authors thank the Brazilian agencies CNPq and CAPES for the financial support.

#### **Author details**

Valter Cruz-Magalhães1 , Jackeline Pereira Andrade2 , Yasmim Freitas Figueiredo1 , Phellippe Arthur Santos Marbach3 and Jorge Teodoro de Souza1 \*

1 Federal University of Lavras, Lavras, MG, Brazil

2 Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil

3 Reconcavo da Bahia Federal University, BA, Brazil

\*Address all correspondence to: jorge.souza@dfp.ufla.br

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

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[16] Yang X, Cushman JC, Borland AM, Edwards EJ, Wullschleger SD, Tuskan GA, et al. A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world. The New Phytologist. 2015;**207**(3):491-504. DOI: 10.1111/ nph.13393

[17] Goldbeck R, Ramos M, Pereira G, Maugeri-Filho F. Cellulase production from a new strain *Acremonium strictum* isolated from the Brazilian biome using different substrates. Bioresource Technology. 2013;**128**:797-803. DOI: 10.1016/j.biortech.2012.10.034

[18] Viel AM, Pereira AR, Neres WE, Dos Santos L, Oliva Neto P, Souza EB, et al. Effect of *Agave sisalana* Perrine extract on the ovarian and uterine tissues and fetal parameters: Comparative interventional study. International Multispeciality Journal of Health. 2017;**3**:129-138

[19] Rodríguez-Garay B. Somatic embryogenesis in *Agave* spp. In: Loyola-Vargas V, Ochoa-Alejo N, editors. Somatic Embryogenesis: Fundamental Aspects and Applications. Cham: Springer; 2016. DOI: 10.1007/978-3-319-33705-0\_16

[20] Chen PY, Kuo YC, Chen CH, Kuo YH, Lee CK. Isolation and immunomodulatory effect of homoisoflavones and flavones from *Agave sisalana* Perrine ex Engelm. Molecules. 2009;**14**:1789-1795. DOI: 10.3390/molecules14051789

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[22] Carneiro F d S, de Oliveira Domingos Queiroz SR, Rodrigues Passos A, Neves do Nascimento M, Souza dos Santos K. Embriogênese somática em *Agave sisalana* Perrine: indução, caracterização anatômica e regeneração. Pesquisa Agropecuária Tropical. 2014;**44**:3

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[24] Rajaud A, de Noblet-Ducoudré N. Tropical semi-arid regions expanding over temperate latitudes under climate change. Climatic ChangeClimatic Change. 2017;**144**(4):703-719. DOI: 10.1007/s10584-017-2052-7

[25] Sharma S, Varshney VK. Chemical analysis of *Agave sisalana* juice for its possible utilization. Acta Chimica and Pharmaceutica Indica. 2012;**2**:60-66

[26] IBGE. Instituto Brasileiro de Geografia e Estatística. Sistema IBGE de Recuperação Automática [Internet]. 2015. Available from: http://www.sidra. ibge.gov.br/ [Accessed: 27 February 2015]

[27] Instituto Brasileiro de Geografia e Estatística (IBGE). Levantamento Sistemático da Produção Agrícola [Internet]. 2011. Available from: http:// www.sidra.ibge.gov.br/ [Accessed: 2011-08-01]

[28] Mattoso LHC, Ferreira FC, Curvelo AAS. In: Leão AL, Carvalho FX, Frollini E, editors. Lignocellulose-Plastic Composites. São Paulo, Brazil: USP and UNESP; 1997

[29] Silva ORR, Beltrão NEM. O agronegócio do sisal no Brasil. Campina Grande, Brasil: Embrapa-CNPA; 1999

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**78**

2010

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BMC Genomics. 2013;**14**:563. DOI:

[8] Das T. Micropropagation of *Agave sisalana*. Plant Cell, Tissue and Organ Culture. 1992;**31**:253-255. DOI: 10.1007/

[9] Nikam TD. High frequency shoot regeneration in *Agave sisalana*. Plant Cell, Tissue and Organ Culture. 1997;**51**(3):225-228. DOI:

[10] Nikam TD, Bansude GM, Kumar KA. Somatic embryogenesis in sisal (*Agave sisalana* Perr. ex. Engelm). Plant Cell Reports. 2003;**22**(3):188-194. DOI:

[11] Eguiarte LE, Aguirre-Planter E, Aguirre X, Colín R, González A, Rocha M, et al. From isozymes to genomics: Population genetics and conservation of Agave in México. The Botanical Review. 2013;**79**(4):483-506. DOI: 10.1007/

[12] Food and Agriculture Organization (FAO). Future fibres [Internet]. 2019. Available from: http://www.fao.org/ economic/futurefibres/fibres/sisal/en/ online [Accessed: 01 February 2019]

[13] Bessadok A, Langevin D, Gouanvé F, Chappey C, Roudesli S, Marais S. Study of water sorption on modified *Agave* fibres. Carbohydrate Polymers.

2009;**76**:74-85. DOI: 10.1016/j.

Part A: Applied Science and

[14] Flores-Sahagun TH, Dos Santos LP, Dos Santos J, Mazzaro I, Mikowski A. Characterization of blue *Agave* bagasse fibers of Mexico. Composites

Manufacturing. 2013;**45**:153-161. DOI: 10.1016/j.compositesa.2012.09.001

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carbpol.2008.09.033

10.1186/1471-2164-14-563

10.1023/A:1005976304198

10.1007/s00299-003-0675-9

s12229-013-9123-x

BF00036233

[1] Nava-Cruz NY, Medina-Morales MA, Martinez JL, Rodriguez R, Aguilar CN. Agave biotechnology: An overview. Critical Reviews in Biotechnology. 2015, 2015;**35**:546-559. DOI: 10.3109/07388551.2014.923813

Christenhusz MJ. The Yucatan Peninsula is the place of origin of sisal (*Agave sisalana*, *Asparagaceae*): Historical accounts, phytogeography and current populations. Botanical Sciences. 2018;**96**:366-379. DOI: 10.17129/

[3] Pinos-Rodríguez JM, Zamudio M, González SS, Mendoza GD, Bárcena R, Ortega ME, et al. Effects of maturity and ensiling of *Agave salmiana* on nutritional quality for lambs. Animal Feed Science and Technology. 2009;**152**:298-306. DOI: 10.1016/j.

[4] Sarwar MB, Ahmad Z, Rashid B, Hassan S, Gregersen PL, Leyva MDLO, et al. De novo assembly of *Agave sisalana* transcriptome in response to drought stress provides insight into the tolerance mechanisms. Scientific Reports. 2019;**9**:1-14. DOI: 10.1038/

[5] Kant P. Could Agave be the species of choice for climate change mitigation? In: Working Paper IGREC-11. New Delhi: Institute of Green Economy, IGREC;

[6] Escamilla-Treviño LL. Potential of plants from the genus *Agave* as bioenergy crops. Bio Energy Research. 2012;**5**:1-9. DOI: 10.1007/

[7] Gross SM, Martin JA, Simpson J, Abraham-Juarez MJ, Wang Z, Visel A. De novo transcriptome assembly of drought tolerant CAM plants, *Agave deserti* and *Agave tequilana*.

anifeedsci.2009.05.002

s41598-018-35891-6

s12155-011-9159-x

[2] Trejo-Torres JC, Gann GD,

botsci.1928

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[31] Santos POD, Silva ACMD, Corrêa ÉB, Magalhães VC, Souza JTD. Additional species of *Aspergillus* causing bole rot disease in *Agave sisalana*. Tropical Plant Pathology. 2014;**39**(4):331-334. DOI: 10.1590/ S1982-56762014000400008

[32] Wallace GB, Dieckmahns EC. Bole rot in sisal. East African Agricultural. 1952;**18**:24-29. DOI: 10.1080/03670074.1952.11664819

[33] Lima EF, Moreira JDAN, Batista FAS, Silva ORRF, Farias FJC, Araújo AE. Podridão vermelha do tronco do sisal (*Agave sisalana* Perrine.) causada por *Botryodiplodia theobromae* pat. Revista de Oleaginosas e Fibrosas. 1998, 1998;**2**:109-112

[34] Barbosa LO, Lima JS, Magalhães VC, Gava CAT, Soares ACF, Marbach PAS, et al. Compatibility and combination of selected bacterial antagonists in the biocontrol of sisal bole rot disease. Biological Control. 2018;**63**(4):595-605. DOI: 10.1007/s10526-018-9872-x

[35] SÁ JO. Controle biológico da podridão vermelha do sisal (*Agave sisalana* Perrine) com *Trichoderma* spp. e Actinobactérias [MSc thesis]. Cruz das Almas: Universidade Federal do Recôncavo da Bahia; 2019

[36] Abreu KCLDM. Epidemiologia da podridão Vermelha do Sisal no Estado da Bahia [MSc thesis]. Cruz das Almas: Universidade Federal do Recôncavo da Bahia; 2010

[37] Duarte EAA, Damasceno CL, Oliveira TASD, Barbosa LDO, Martins FM, Silva JRDQ, et al. Putting the mess in order: A*spergillus welwitschiae* (and not *A. niger*) is the etiological agent of sisal bole rot disease in Brazil. Frontiers in Microbiology. 2018;**9**:1-21. DOI: 10.3389/fmicb.2018.01227

[38] Machado AA. Sobre a Ocorrência de uma Nova Moléstia do Agave na Paraíba. Technical report. Relatório de uma viagem realizada no município de Campina Grande; 1951

[39] Medina JC. O sisal. Secretaria da Agricultura do Estado de São Paulo. São Paulo, Brazil; 1954

[40] Coutinho WM, Suassuna ND, Luz CM, Suinaga FA, Silva ORRF. Bole rot of sisal caused by *Aspergillus niger* in Brazil. Fitopatologia Brasileira. 2006;**31**:605-605. DOI: 10.1590 S0100-41582006000600014

[41] Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, et al. New and revisited species in *Aspergillus* section *Nigri*. Studies in Mycology. 2011;**69**:1-17. DOI: 10.3114/ sim.2011.69.01

[42] Ismail MA. Incidence and significance of black aspergilli in agricultural commodities: A review, with a key to all species accepted to-date. European Journal of Biological Research. 2017;**7**:207-222. DOI: 10.5281/ zenodo.834504

[43] Pitt JL, Hocking AD. Fungi and Food Spoilage. Cambridge: Chapman & Hall; 1997. DOI: 10.1007/978-0-387-92207-2

[44] Samson RA, Houbraken JAMP, Kuijpers AFA, Frank MJ, Frisvad JC. New ochratoxin A or sclerotium producing species in *Aspergillus* section *Nigri*. Studies in Mycology. 2004;**50**:45-61

[45] Varga J, Kocsubé S, Tóth B, Frisvad JC, Perrone G, Susca A, et al. *Aspergillus brasiliensis* sp. nov., a biseriate black *Aspergillus* species with world-wide distribuition. International Journal of Systematic and Evolutionary

**81**

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

> Semi-árida do Nordeste Brasileiro. Campina Grande, Brazil; 2006. p. 44

[53] Magalhães VC, Barbosa LO, Andrade JP, Soares ACF, de Souza JT, Marbach PAS. *Burkholderia* isolates from a sand dune leaf litter display biocontrol activity against the bole rot disease of *Agave sisalana*. Biological Control. 2017;**112**:41-48. DOI: 10.1016/j.

[54] Baker KF. Evolving concepts of biological control of plant pathogens. Annual Review of Phytopathology.

[55] Mueller UG, Sachs JL. Engineering microbiomes to improve plant and animal health. Trends in Microbiology. 2015;**23**:606-617. DOI: 10.1016/j.

[56] do Carmo CO, Tavares PF, da Silva RM, Damasceno CL, Sá JO, Soares ACF. Fatores que afetam a sobrevivência de *Aspergillus niger* e sua relação com a podridão vermelha do caule do sisal.

biocontrol.2017.06.005

1987;**25**:67-85

tim.2015.07.009

Magistra. 2018;**29**:144-153

Microbiology. 2007;**57**:1925-1932. DOI:

[46] Noonim P, Mahakarnchanakul W, Varga J, Frisvad JC, Samson RA. Two novel species of *Aspergillus* section *Nigri* from Thai coffee beans. International Journal of Systematic and Evolutionary Microbiology. 2008;**58**:1727-1734. DOI:

[47] Oliveri C, Torta L, Catara VA. Polyphasic approach to the identification of ochratoxin

Journal of Food Microbiology. 2008;**127**:147-154. DOI: 10.1016/j.

of Systematic and Evolutionary

[49] Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CHW, et al. Phylogeny, identification and nomenclature of the genus *Aspergillus*. Studies in Mycology. 2014;**78**:141-173. DOI: 10.1016/j.

[50] Hubka V, Kolarik M. Betatubulin paralogue tubC is frequently misidentified as the benA gene in *Aspergillus* section *Nigri* taxonomy: Primer specificity testing and taxonomic consequences. Persoonia. 2012;**29**:1-10. DOI: 10.3767/003158512X658123

[51] Embrapa. Cultivo do sisal

WM. Cultivo de sisal na região

[Internet]. 2010. Available from http:// sistemasdeproducao.cnptia.embrapa. br/FontesHTML/Sisal/CultivodoSisal/ doencas.html [Accessed: 25 May 2010]

[52] Suinaga FA, Silva ORRF, Coutinho

10.1099/ijs.0.65463-0

simyco.2014.07.004

ijfoodmicro.2008.06.021

A-producing black *Aspergillus* isolates from vineyards in Sicily. International

[48] Perrone G, Varga J, Susca A, Frisvad JC, Stea G, Kocsubé S, et al. *Aspergillus uvarum* sp. nov., an uniseriate black *Aspergillus* species isolated from grapes in Europe. International Journal

Microbiology. 2008;**58**:1032-1039. DOI:

10.1099/ijs.0.65021-0

10.1099/ijs.0.65694-0

*Sisal Bole Rot: An Important but Neglected Disease DOI: http://dx.doi.org/10.5772/intechopen.86983*

Microbiology. 2007;**57**:1925-1932. DOI: 10.1099/ijs.0.65021-0

*Plant Diseases-Current Threats and Management Trends*

in Microbiology. 2018;**9**:1-21. DOI:

[38] Machado AA. Sobre a Ocorrência de uma Nova Moléstia do Agave na Paraíba. Technical report. Relatório de uma viagem realizada no município de

[39] Medina JC. O sisal. Secretaria da Agricultura do Estado de São Paulo. São

[40] Coutinho WM, Suassuna ND, Luz CM, Suinaga FA, Silva ORRF. Bole rot of sisal caused by *Aspergillus niger* in Brazil. Fitopatologia Brasileira. 2006;**31**:605-605. DOI: 10.1590 S0100-41582006000600014

[41] Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, et al. New and revisited species in *Aspergillus* section *Nigri*. Studies in Mycology. 2011;**69**:1-17. DOI: 10.3114/

[42] Ismail MA. Incidence and significance of black aspergilli in agricultural commodities: A review, with a key to all species accepted to-date. European Journal of Biological Research. 2017;**7**:207-222. DOI: 10.5281/

[43] Pitt JL, Hocking AD. Fungi and Food Spoilage. Cambridge: Chapman & Hall; 1997. DOI: 10.1007/978-0-387-92207-2

[44] Samson RA, Houbraken JAMP, Kuijpers AFA, Frank MJ, Frisvad JC. New ochratoxin A or sclerotium producing species in *Aspergillus* section *Nigri*. Studies in Mycology.

[45] Varga J, Kocsubé S, Tóth B, Frisvad JC, Perrone G, Susca A, et al. *Aspergillus brasiliensis* sp. nov., a biseriate black *Aspergillus* species with world-wide distribuition. International Journal of Systematic and Evolutionary

10.3389/fmicb.2018.01227

Campina Grande; 1951

Paulo, Brazil; 1954

sim.2011.69.01

zenodo.834504

2004;**50**:45-61

Costa LD. Cultivo do Sisal no Nordeste Brasileiro. Ministério da Agricultura, Pecuária e Abastecimento, Circular

[31] Santos POD, Silva ACMD, Corrêa ÉB, Magalhães VC, Souza JTD. Additional species of *Aspergillus* causing bole rot disease in *Agave sisalana*. Tropical Plant Pathology. 2014;**39**(4):331-334. DOI: 10.1590/ S1982-56762014000400008

[32] Wallace GB, Dieckmahns EC. Bole rot in sisal. East African Agricultural. 1952;**18**:24-29. DOI: 10.1080/03670074.1952.11664819

[33] Lima EF, Moreira JDAN, Batista FAS, Silva ORRF, Farias FJC, Araújo AE. Podridão vermelha do tronco do sisal (*Agave sisalana* Perrine.) causada por *Botryodiplodia theobromae* pat. Revista de Oleaginosas e Fibrosas. 1998,

[34] Barbosa LO, Lima JS, Magalhães VC, Gava CAT, Soares ACF, Marbach PAS, et al. Compatibility and combination of selected bacterial antagonists in the biocontrol of sisal bole rot disease. Biological Control. 2018;**63**(4):595-605. DOI: 10.1007/s10526-018-9872-x

[35] SÁ JO. Controle biológico da podridão vermelha do sisal (*Agave sisalana* Perrine) com *Trichoderma* spp. e Actinobactérias [MSc thesis]. Cruz das Almas: Universidade Federal do

[36] Abreu KCLDM. Epidemiologia da podridão Vermelha do Sisal no Estado da Bahia [MSc thesis]. Cruz das Almas: Universidade Federal do Recôncavo da

[37] Duarte EAA, Damasceno CL, Oliveira TASD, Barbosa LDO, Martins FM, Silva JRDQ, et al. Putting the mess in order: A*spergillus welwitschiae* (and not *A. niger*) is the etiological agent of sisal bole rot disease in Brazil. Frontiers

Recôncavo da Bahia; 2019

técnica. 2008; 123

1998;**2**:109-112

**80**

Bahia; 2010

[46] Noonim P, Mahakarnchanakul W, Varga J, Frisvad JC, Samson RA. Two novel species of *Aspergillus* section *Nigri* from Thai coffee beans. International Journal of Systematic and Evolutionary Microbiology. 2008;**58**:1727-1734. DOI: 10.1099/ijs.0.65694-0

[47] Oliveri C, Torta L, Catara VA. Polyphasic approach to the identification of ochratoxin A-producing black *Aspergillus* isolates from vineyards in Sicily. International Journal of Food Microbiology. 2008;**127**:147-154. DOI: 10.1016/j. ijfoodmicro.2008.06.021

[48] Perrone G, Varga J, Susca A, Frisvad JC, Stea G, Kocsubé S, et al. *Aspergillus uvarum* sp. nov., an uniseriate black *Aspergillus* species isolated from grapes in Europe. International Journal of Systematic and Evolutionary Microbiology. 2008;**58**:1032-1039. DOI: 10.1099/ijs.0.65463-0

[49] Samson RA, Visagie CM, Houbraken J, Hong SB, Hubka V, Klaassen CHW, et al. Phylogeny, identification and nomenclature of the genus *Aspergillus*. Studies in Mycology. 2014;**78**:141-173. DOI: 10.1016/j. simyco.2014.07.004

[50] Hubka V, Kolarik M. Betatubulin paralogue tubC is frequently misidentified as the benA gene in *Aspergillus* section *Nigri* taxonomy: Primer specificity testing and taxonomic consequences. Persoonia. 2012;**29**:1-10. DOI: 10.3767/003158512X658123

[51] Embrapa. Cultivo do sisal [Internet]. 2010. Available from http:// sistemasdeproducao.cnptia.embrapa. br/FontesHTML/Sisal/CultivodoSisal/ doencas.html [Accessed: 25 May 2010]

[52] Suinaga FA, Silva ORRF, Coutinho WM. Cultivo de sisal na região

Semi-árida do Nordeste Brasileiro. Campina Grande, Brazil; 2006. p. 44

[53] Magalhães VC, Barbosa LO, Andrade JP, Soares ACF, de Souza JT, Marbach PAS. *Burkholderia* isolates from a sand dune leaf litter display biocontrol activity against the bole rot disease of *Agave sisalana*. Biological Control. 2017;**112**:41-48. DOI: 10.1016/j. biocontrol.2017.06.005

[54] Baker KF. Evolving concepts of biological control of plant pathogens. Annual Review of Phytopathology. 1987;**25**:67-85

[55] Mueller UG, Sachs JL. Engineering microbiomes to improve plant and animal health. Trends in Microbiology. 2015;**23**:606-617. DOI: 10.1016/j. tim.2015.07.009

[56] do Carmo CO, Tavares PF, da Silva RM, Damasceno CL, Sá JO, Soares ACF. Fatores que afetam a sobrevivência de *Aspergillus niger* e sua relação com a podridão vermelha do caule do sisal. Magistra. 2018;**29**:144-153

**83**

**Chapter 6**

**Abstract**

conditions.

**1. Introduction**

*Sahar Abdou Zayan*

Impact of Climate Change on Plant

There has been a remarkable scientific output on the topic of how climate change is likely to affect plant diseases. Climate change influences the occurrence, prevalence, and severity of plant diseases. Projected atmospheric and climate change will thus affect the interaction between crops and pathogens in multiple ways. This will also affect disease management with regard to timing, preference, and efficacy of chemical, physical, and biological measures of control and their utilization within integrated pest management (IPM) strategies. Prediction of future requirements in disease management is of great interest for agro-industries, extension services, and practical farmers. A comprehensive analysis of potential climate change effects on disease control is difficult because current knowledge is limited and fragmented and due to the complexity of future risks for plant disease management, particularly if new crops are introduced in an area. Uncertainty in models of plant disease development under climate change calls for a diversity of management strategies, from more participatory approaches to interdisciplinary science. Involvement of stakeholders and scientists from outside plant pathology shows the importance of trade-offs. All these efforts and integrations will produce effective crop protection strategies using novel technologies as appropriate tools to adapt to altered climatic

Diseases and IPM Strategies

**Keywords:** climate, change, plant, pathology, agriculture

these changes and to prevent a decline in productivity.

however, an activity which is extremely vulnerable to climate change.

Climate change is a major concern for agricultural communities worldwide [1, 2]. The agricultural process consists of three main parts, pathogen, host, and environmental conditions, where the relation between them is the main key for the occurrence of infection from its absence (**Figure 1**), where climate change has great effect on all these factors. Changes in climatic parameters greatly affect crop production and susceptibility to pests as well as insect pest longevity. Climate change affects crop pests and disease susceptibility which in turn affects crop health, and these changes cause deviations in farming practices as to cope with the effects of

Agriculture is an economic activity which is highly reliant on climate and weather in order to produce the food and fiber necessary to sustain human life. Agriculture is,

The effects of climate change on agriculture are characterized by various forms of uncertainty. Firstly, there are uncertainties concerning the rate and magnitude

#### **Chapter 6**

## Impact of Climate Change on Plant Diseases and IPM Strategies

*Sahar Abdou Zayan*

#### **Abstract**

There has been a remarkable scientific output on the topic of how climate change is likely to affect plant diseases. Climate change influences the occurrence, prevalence, and severity of plant diseases. Projected atmospheric and climate change will thus affect the interaction between crops and pathogens in multiple ways. This will also affect disease management with regard to timing, preference, and efficacy of chemical, physical, and biological measures of control and their utilization within integrated pest management (IPM) strategies. Prediction of future requirements in disease management is of great interest for agro-industries, extension services, and practical farmers. A comprehensive analysis of potential climate change effects on disease control is difficult because current knowledge is limited and fragmented and due to the complexity of future risks for plant disease management, particularly if new crops are introduced in an area. Uncertainty in models of plant disease development under climate change calls for a diversity of management strategies, from more participatory approaches to interdisciplinary science. Involvement of stakeholders and scientists from outside plant pathology shows the importance of trade-offs. All these efforts and integrations will produce effective crop protection strategies using novel technologies as appropriate tools to adapt to altered climatic conditions.

**Keywords:** climate, change, plant, pathology, agriculture

#### **1. Introduction**

Climate change is a major concern for agricultural communities worldwide [1, 2]. The agricultural process consists of three main parts, pathogen, host, and environmental conditions, where the relation between them is the main key for the occurrence of infection from its absence (**Figure 1**), where climate change has great effect on all these factors. Changes in climatic parameters greatly affect crop production and susceptibility to pests as well as insect pest longevity. Climate change affects crop pests and disease susceptibility which in turn affects crop health, and these changes cause deviations in farming practices as to cope with the effects of these changes and to prevent a decline in productivity.

Agriculture is an economic activity which is highly reliant on climate and weather in order to produce the food and fiber necessary to sustain human life. Agriculture is, however, an activity which is extremely vulnerable to climate change.

The effects of climate change on agriculture are characterized by various forms of uncertainty. Firstly, there are uncertainties concerning the rate and magnitude

#### **Figure 1.**

*The relation between the pathogen, host, and environmental conditions which are the main factors required for infections to occur.*

of climate change itself. Secondly, there are uncertainties around the response of agriculture-based outputs, for example, with CO2 fertilization. Thirdly, there are uncertainties as to how society responds or even the aptitude to respond to these expected impacts. Some aspects of climate change research are limited by these uncertainties. Most of these uncertainties cannot be quantified, causing a certain level of ignorance in our understandings of future climate change [3].

It is highly possible that climate change will affect food security at the global, regional, and local levels. Climate change can disturb and reduce food availability as well as lower food quality. For example, increases in temperatures, changes in extreme weather events, changes in precipitation patterns, and reductions in water availability could all result in reduced agricultural productivity. Prevalence of extreme weather conditions can also interrupt food delivery and result in increases in food prices due to low supply after extreme events, which are expected to be more frequent in the future. Moreover, increasing temperatures can contribute to spoilage and contamination.

There are four different future scenarios regarding climate change including A1, A2, B1, and B2. The A1 scenario focuses on rapid increases in global economic development, A2 focuses on rapid regional economic development instead of the global one in A1 scenario, B1 focuses on rapid global environmental development regarding agriculture, and B2 focuses on rapid environmental sustainability on regional and local levels.

#### **2. Effect of climate change on plants and plant diseases**

A major example for the devastating effects of climate change is floods caused by rising of sea level that can cause the disappearance of low-level lands and major crop losses. Another example is drought, where insufficiencies in water levels in the soil cause plants to lose their biological functions and even become more susceptible to diseases and pests. Climatic conditions contribute to the disease triangle, which involves the presence of a susceptible host, a pathogen, and suitable environmental conditions for infection to occur, and climate change affects environmental

**85**

unexposed to these species.

with increasing temperatures.

*Impact of Climate Change on Plant Diseases and IPM Strategies*

conditions whether it be in favor of the host or the pathogen. Examples of these conditions include dew, rain, relative humidity, temperature, aeration (wind), soil

equivalent to \$220 million of cherries in Michigan in 2010 and 2012.

Moreover, drought has developed into a major problem in regions with increased summer temperatures as this causes dryness in soils. Even though higher irrigation may be possible in certain regions, water supplies may be also lowered in other locations, causing a lower availability of water for irrigation when more is needed. Many weeds, pests, and fungi thrive under warmer temperatures, wetter climates, and increased CO2 levels. Currently, US farmers spend more than \$11 billion every year to control weeds, which compete with crops for nutritional resources. The ranges and distribution of weeds and pests are likely to increase with climate change. This could cause new problems for farmers' crops previously

Climate change parameters can have effects on both the host and the pathogen, for example, certain degrees of temperature promote pathogen growth, and certain temperatures can cause the host to have higher resistance to pathogenic infections. An example highlighting these events involves wheat and oats, which become more susceptible to rust diseases with increased temperature, while some forage species become more resistant [2]. Moreover, changes in temperature as limited as CO2 changes could cause certain pests to undergo from 1 to 5 additional lifecycles per season, which increases the ability of the pests to overcome plant resistance.

Certain mycotoxins such as fusarium mycotoxins (produced by *Fusarium* spp.) have increased concentrations at harvest due to high humidity and temperature. Humid conditions also increase proliferation of weeds, and weed biomass increases

For any type of crop, the effect exerted by high temperature is highly dependent on the optimal growth and reproduction temperature of the crop. In certain regions, increased temperature may prove beneficial to the types of crops that usually grow there or permit farmers to switch to planting crops that grow in warmer areas. However, that is not always the case; if the higher temperature exceeds a crop's optimum temperature yields will decline, or worse, appearance and infestation of pathogens might occur. Crop yield can be affected by high levels of CO2. A few laboratory experiments showed that high levels of CO2 could positively affect growth. However, certain variables such as varying temperatures, water, ozone, and low nutrient levels may oppose these possible increases in yield. For instance, if the temperature is higher than a crop's optimal temperature requirement, if there are insufficiencies in water and nutrients, increase in yield may be low. Increased levels of CO2 are linked to lower nitrogen and protein content in soybean and alfalfa plants, which results in a great quality reduction. Reduced forage and grain quality can reduce the ability of rangeland and pasture to support livestock which rely on grazing. Although rising CO2 can stimulate plant growth, it also lowers the nutritional value of the majority of food crops. Rising levels of atmospheric carbon dioxide directly affect the concentrations of protein and essential minerals by reducing their content in a variety of plant species, which include rice, soybeans, and wheat. Therefore, the effect of rising CO2 on the crops' nutritional value is considered a possible and indirect threat to human health as well. Moreover, due to the increased use and lowered efficiency of pesticides due to development of pest resistance, human health is additionally threatened by pesticide use as well as their residual toxicity in humans. More extreme temperatures and precipitation might decrease growth in certain crops. As previously mentioned, extreme events such as droughts and floods can decrease yield and damage crops. For instance, increased evening temperatures affected corn yield throughout the US Corn Belt. Additionally, premature budding due to a warmer winter instigated losses

*DOI: http://dx.doi.org/10.5772/intechopen.87055*

moisture, and sunlight intensity.

#### *Impact of Climate Change on Plant Diseases and IPM Strategies DOI: http://dx.doi.org/10.5772/intechopen.87055*

*Plant Diseases-Current Threats and Management Trends*

of climate change itself. Secondly, there are uncertainties around the response of agriculture-based outputs, for example, with CO2 fertilization. Thirdly, there are uncertainties as to how society responds or even the aptitude to respond to these expected impacts. Some aspects of climate change research are limited by these uncertainties. Most of these uncertainties cannot be quantified, causing a certain

*The relation between the pathogen, host, and environmental conditions which are the main factors required for* 

It is highly possible that climate change will affect food security at the global, regional, and local levels. Climate change can disturb and reduce food availability as well as lower food quality. For example, increases in temperatures, changes in extreme weather events, changes in precipitation patterns, and reductions in water availability could all result in reduced agricultural productivity. Prevalence of extreme weather conditions can also interrupt food delivery and result in increases in food prices due to low supply after extreme events, which are expected to be more frequent in the future. Moreover, increasing temperatures can contribute to spoilage and contamination. There are four different future scenarios regarding climate change including A1, A2, B1, and B2. The A1 scenario focuses on rapid increases in global economic development, A2 focuses on rapid regional economic development instead of the global one in A1 scenario, B1 focuses on rapid global environmental development regarding agriculture, and B2 focuses on rapid environmental sustainability on

A major example for the devastating effects of climate change is floods caused by rising of sea level that can cause the disappearance of low-level lands and major crop losses. Another example is drought, where insufficiencies in water levels in the soil cause plants to lose their biological functions and even become more susceptible to diseases and pests. Climatic conditions contribute to the disease triangle, which involves the presence of a susceptible host, a pathogen, and suitable environmental conditions for infection to occur, and climate change affects environmental

level of ignorance in our understandings of future climate change [3].

**2. Effect of climate change on plants and plant diseases**

**84**

regional and local levels.

**Figure 1.**

*infections to occur.*

conditions whether it be in favor of the host or the pathogen. Examples of these conditions include dew, rain, relative humidity, temperature, aeration (wind), soil moisture, and sunlight intensity.

For any type of crop, the effect exerted by high temperature is highly dependent on the optimal growth and reproduction temperature of the crop. In certain regions, increased temperature may prove beneficial to the types of crops that usually grow there or permit farmers to switch to planting crops that grow in warmer areas. However, that is not always the case; if the higher temperature exceeds a crop's optimum temperature yields will decline, or worse, appearance and infestation of pathogens might occur. Crop yield can be affected by high levels of CO2. A few laboratory experiments showed that high levels of CO2 could positively affect growth. However, certain variables such as varying temperatures, water, ozone, and low nutrient levels may oppose these possible increases in yield. For instance, if the temperature is higher than a crop's optimal temperature requirement, if there are insufficiencies in water and nutrients, increase in yield may be low. Increased levels of CO2 are linked to lower nitrogen and protein content in soybean and alfalfa plants, which results in a great quality reduction. Reduced forage and grain quality can reduce the ability of rangeland and pasture to support livestock which rely on grazing. Although rising CO2 can stimulate plant growth, it also lowers the nutritional value of the majority of food crops. Rising levels of atmospheric carbon dioxide directly affect the concentrations of protein and essential minerals by reducing their content in a variety of plant species, which include rice, soybeans, and wheat. Therefore, the effect of rising CO2 on the crops' nutritional value is considered a possible and indirect threat to human health as well. Moreover, due to the increased use and lowered efficiency of pesticides due to development of pest resistance, human health is additionally threatened by pesticide use as well as their residual toxicity in humans. More extreme temperatures and precipitation might decrease growth in certain crops. As previously mentioned, extreme events such as droughts and floods can decrease yield and damage crops. For instance, increased evening temperatures affected corn yield throughout the US Corn Belt. Additionally, premature budding due to a warmer winter instigated losses equivalent to \$220 million of cherries in Michigan in 2010 and 2012.

Moreover, drought has developed into a major problem in regions with increased summer temperatures as this causes dryness in soils. Even though higher irrigation may be possible in certain regions, water supplies may be also lowered in other locations, causing a lower availability of water for irrigation when more is needed. Many weeds, pests, and fungi thrive under warmer temperatures, wetter climates, and increased CO2 levels. Currently, US farmers spend more than \$11 billion every year to control weeds, which compete with crops for nutritional resources. The ranges and distribution of weeds and pests are likely to increase with climate change. This could cause new problems for farmers' crops previously unexposed to these species.

Climate change parameters can have effects on both the host and the pathogen, for example, certain degrees of temperature promote pathogen growth, and certain temperatures can cause the host to have higher resistance to pathogenic infections. An example highlighting these events involves wheat and oats, which become more susceptible to rust diseases with increased temperature, while some forage species become more resistant [2]. Moreover, changes in temperature as limited as CO2 changes could cause certain pests to undergo from 1 to 5 additional lifecycles per season, which increases the ability of the pests to overcome plant resistance.

Certain mycotoxins such as fusarium mycotoxins (produced by *Fusarium* spp.) have increased concentrations at harvest due to high humidity and temperature. Humid conditions also increase proliferation of weeds, and weed biomass increases with increasing temperatures.

Certain parameters can have different effects depending on plant physiology, for example, increased CO2 levels can cause a decrease in plant decomposition rates, which results in higher fungal inoculum levels, and these concentrations may induce the production of more fungal spores. On the other hand, high CO2 concentrations may cause physiological changes to plants, causing them to acquire higher resistance to certain pathogens.

Other extreme conditions may include low water levels and soil erosion which causes a decline in soil fertility and hence plant health.

Fungicide activity is also a major determinant factor; climate change may highly affect fungicide efficiency. Highly frequent rainfalls greatly impact the efficiency of contact fungicides, as rain has the ability to sweep and eliminate contact fungicides from the hosts' surface, rendering them ineffective. However, plants with high metabolic rates have increased intake of fungicides and aren't highly affected by this parameter.

In 2008, the International Food Policy Research Institute estimated that due to climate changes, by 2050, 25 million additional children will have malnutrition due to increased consumption of food products with little efforts done to adapt to and deal with these changes. In addition, the yearly costs to deal with the issue by reducing its impacts by 2050 will be \$7 billion. It will generally be difficult to deal with international trade of crops due to appearance of unexpected pathogens more frequently (Food and Agriculture Organization of the United Nations, 2008).

#### **3. Integrated pest management and mitigating pest management**

IPM stands for integrated pest management, according to the Food and Agricultural Organizations (FAO) in the United States, IPM is an ecosystem approach involving crop protection which combines different strategies and practices toward growing healthier crops and minimizing the use of pesticides to protect the environment. It is an analytical method used to analyze the agroecosystem and its different elements in order to optimally manage these elements to control and minimize pests while protecting the environment and the economic health.

That is, the available methods of control (biological, cultural, chemical, and physical) should be considered and rationally applied by the farmers. However, IPM is more than just a tool and collection of control choices. It also comprises precaution techniques (which mainly include monitoring, prevention, early diagnosis, and forecasting) which assist in the control of pest populations as where data is collected, and preventive actions is recommended as explained in **Figure 2**. A significant part in IPM techniques is proper decision-making for any interferences. Every decision made must be justifiable both ecologically and economically. Consequently, control programs which involve systematic application of chemicals which may harm the environment are unacceptable in IPM processes. As an alternative, precedence is given to alternative control techniques as well as preventive methods. IPM has been applied in various countries and areas that differ in their natural, social, and economic circumstances in addition to their levels of agricultural expansion. However, advancement in crop yield and safety may be realized in any existing conditions through the implementation of IPM. The practice of IPM is not a strict and simple form of submission to regulations and rules, but it rather involves taking actions with environmentally friendly approaches through principles and approaches that contribute to the reduction of the use of chemicals and increasing food security to achieve agricultural sustainability. In order to make IPM as effective as possible, it should be modified to local conditions.

**87**

will be applied.

*Impact of Climate Change on Plant Diseases and IPM Strategies*

The process does not involve a single pest management step, but a collection of pest management steps where decisions, evaluations, and control steps have to be made in order to successfully apply the chosen strategy. In IPM practice, farmers who understand the potential of pests when it comes to crop infestation follow a

*The correlation between forecasting data input, pathogen studies, and environmental conditions as input and* 

Prior to taking any control decision or action, IPM first sets a threshold, which is a point at which the set of involved variable levels specifies that proper control actions must be made in order to control pest populations. Detection of a pest does not mean a certain control action is required. However, the detection of pests or variables at certain thresholds/levels is the determining factor. Therefore, the level at which pests could develop into threats is extremely important while taking pest

A lot of insects, weeds, and other living organisms are not considered pests which require control. Most organisms are not harmful; on the contrary, some of them are useful. IPM programs are to accurately detect and screen for pests, so that proper management decisions can be made in combination with the action deciding thresholds. This process removes the probability that pesticides will be applied when there is no need for their use or that the incorrect type of pesticide

*DOI: http://dx.doi.org/10.5772/intechopen.87055*

four-step approach. These steps include:

*disease modeling to develop preventive measures.*

**3.2 Monitoring and identification of pests**

**3.1 Setting action thresholds**

**Figure 2.**

management decisions.

*Impact of Climate Change on Plant Diseases and IPM Strategies DOI: http://dx.doi.org/10.5772/intechopen.87055*

**Figure 2.**

*Plant Diseases-Current Threats and Management Trends*

causes a decline in soil fertility and hence plant health.

resistance to certain pathogens.

parameter.

Certain parameters can have different effects depending on plant physiology, for example, increased CO2 levels can cause a decrease in plant decomposition rates, which results in higher fungal inoculum levels, and these concentrations may induce the production of more fungal spores. On the other hand, high CO2 concentrations may cause physiological changes to plants, causing them to acquire higher

Other extreme conditions may include low water levels and soil erosion which

Fungicide activity is also a major determinant factor; climate change may highly affect fungicide efficiency. Highly frequent rainfalls greatly impact the efficiency of contact fungicides, as rain has the ability to sweep and eliminate contact fungicides from the hosts' surface, rendering them ineffective. However, plants with high metabolic rates have increased intake of fungicides and aren't highly affected by this

In 2008, the International Food Policy Research Institute estimated that due to climate changes, by 2050, 25 million additional children will have malnutrition due to increased consumption of food products with little efforts done to adapt to and deal with these changes. In addition, the yearly costs to deal with the issue by reducing its impacts by 2050 will be \$7 billion. It will generally be difficult to deal with international trade of crops due to appearance of unexpected pathogens more frequently (Food and Agriculture Organization of the United Nations, 2008).

**3. Integrated pest management and mitigating pest management**

IPM stands for integrated pest management, according to the Food and Agricultural Organizations (FAO) in the United States, IPM is an ecosystem approach involving crop protection which combines different strategies and practices toward growing healthier crops and minimizing the use of pesticides to protect the environment. It is an analytical method used to analyze the agroecosystem and its different elements in order to optimally manage these elements to control and minimize pests while protecting the environment and the economic health.

That is, the available methods of control (biological, cultural, chemical, and physical) should be considered and rationally applied by the farmers. However, IPM is more than just a tool and collection of control choices. It also comprises precaution techniques (which mainly include monitoring, prevention, early diagnosis, and forecasting) which assist in the control of pest populations as where data is collected, and preventive actions is recommended as explained in **Figure 2**. A significant part in IPM techniques is proper decision-making for any interferences. Every decision made must be justifiable both ecologically and economically. Consequently, control programs which involve systematic application of chemicals which may harm the environment are unacceptable in IPM processes. As an alternative, precedence is given to alternative control techniques as well as preventive methods. IPM has been applied in various countries and areas that differ in their natural, social, and economic circumstances in addition to their levels of agricultural expansion. However, advancement in crop yield and safety may be realized in any existing conditions through the implementation of IPM. The practice of IPM is not a strict and simple form of submission to regulations and rules, but it rather involves taking actions with environmentally friendly approaches through principles and approaches that contribute to the reduction of the use of chemicals and increasing food security to achieve agricultural sustainability. In order to make IPM

as effective as possible, it should be modified to local conditions.

**86**

*The correlation between forecasting data input, pathogen studies, and environmental conditions as input and disease modeling to develop preventive measures.*

The process does not involve a single pest management step, but a collection of pest management steps where decisions, evaluations, and control steps have to be made in order to successfully apply the chosen strategy. In IPM practice, farmers who understand the potential of pests when it comes to crop infestation follow a four-step approach. These steps include:

#### **3.1 Setting action thresholds**

Prior to taking any control decision or action, IPM first sets a threshold, which is a point at which the set of involved variable levels specifies that proper control actions must be made in order to control pest populations. Detection of a pest does not mean a certain control action is required. However, the detection of pests or variables at certain thresholds/levels is the determining factor. Therefore, the level at which pests could develop into threats is extremely important while taking pest management decisions.

#### **3.2 Monitoring and identification of pests**

A lot of insects, weeds, and other living organisms are not considered pests which require control. Most organisms are not harmful; on the contrary, some of them are useful. IPM programs are to accurately detect and screen for pests, so that proper management decisions can be made in combination with the action deciding thresholds. This process removes the probability that pesticides will be applied when there is no need for their use or that the incorrect type of pesticide will be applied.

#### **3.3 Prevention**

In order to achieve proper pest control, IPM programs are designed to manage the crop, lawn, or indoor space to prevent the appearance and development of pests. In the case of agricultural crops, this could mean using proper planting methods, for example, rotating between crops, planting resistant plant varieties, and the use of pest-free rootstock. These control techniques can be very useful and efficient in terms of cost and present lower risk to human health and the environment.

#### **3.4 Control**

Once the previously mentioned variables specify that control actions are needed and that protective approaches are ineffective, IPM programs then assess the appropriate control actions in terms of efficiency and risk. Efficient and low-risk control methods are considered first, including targeted and ecofriendly chemicals, such as pheromones which disrupt pest reproduction, or mechanical control, including trapping and weeding. If data generated by the previously mentioned steps specify that less risky management methods are ineffective, then further pest control attempts should be considered, for example, directed use of pesticides. The use of nontargeted pesticides is a less recommended alternative.

A lot of agricultural growers identify their pests prior to pesticide application. Less risky pesticides such as pheromones are employed by a lower subset of growers. In the end, a lot of these farmers are using IPM techniques. The objective is to make more growers use the proper IPM practices. Mostly, crops produced using IPM techniques are not recognized in the marketplace as organic crops. Growers who use IPM practices have no national certifications in certain countries. Due to the complexity of the IPM pest management techniques, it is not possible to use a single IPM description for all crops and all regions of a country. Many growers of certain crops including strawberries and potatoes are attempting to define what IPM means in their crops case as well as the region of growth. Moreover, certified IPM crops are unavailable in a lot of regions. With definitions, farmers can start to market their crops as IPM-grown, which would give consumers alternative and better options while purchasing their food.

The previously mentioned processes of IPM have been redefined and modified over time, and the IPM pyramid was created to provide an easier understanding of the approach. The IPM pyramid consists of three main processes which include preventive or indirect crop protection, risk assessment or monitoring, and responsive or direct crop protection. The three processes aim to increase the efficiency of each step involving crop breading and maintenance. Preventive crop protection involves the use of certified seeds, cultivars which have high tolerance to pathogens, and enhancement of natural enemies of plant pathogens such as microbiological competitors. Risk assessment and monitoring is the most crucial process in IPM; it involves the use of an early warning forecast system which provides information related to current climate and how it could affect plant health and by using such information and understanding plant physiology and susceptibility to pathogens; one can determine timeframes where plants are most susceptible to pathogens and take countermeasures to prevent or minimize pathogen severity (e.g., through the use of fungicides). Direct crop protection basically involves the countermeasures taken to deal with unfavorable conditions, which include the use of antagonistic microorganisms or application of fungicides.

A simple example for how an integrated system for pest management can be created as illustrated in **Figure 3**; generally in order to create such a system, the main information needed include weather data, crop, and disease information. Through knowing the weather data, which is most commonly obtained from meteorological

**89**

IPM systems.

**Figure 3.**

*Impact of Climate Change on Plant Diseases and IPM Strategies*

stations to obtain micro environment weather data and not that of the macro environment, macro environment data can still be used using certain equations to relate to the micro environment. This would allow the prediction and knowledge of current and upcoming weather conditions, in hand with obtaining information about the crop as in which pests affect the grown crops, optimal growth conditions, and lifetime as well as obtaining specific disease information for diseases which have the capability of affecting said crops. One can create a forecasting tool which

*Example of pathogen epidemiology system in potato late blight and its integrated components.*

After obtaining information about the disease, in order to create a disease model, its efficiency is determined through several tests. The general scopes of these tests would be the difference between disease spread and crop loss before and after the implementation of the model. Did delaying the use of pesticides until the threshold point for disease spread determined by the model actually cause a positive difference or was the threshold inaccurate? Was there a noticeable increase in crop yield after implementation? Was there any human error in pesticide management and spraying timelines? Several questions can be answered, and through these

There are several advantages for the use of IPM in mitigating agricultural

Slower development of resistance to pesticides: Pesticide resistance can be incurred by the repeated use of pesticides; this would occur if a farmer is using traditional farming methods as pests would develop resistance to the pesticide due to repeated exposure to the pesticides through development of resistance by natural selection; then the resistant genes would be transferred to the offsprings, incurring permanent resistance to a given pesticide. However, this would not occur with reduced and efficient application of pesticides which is a main strategy adopted by

helps prevent the spread of certain diseases.

problems which include:

answers, the efficiency of the model can be determined.

*DOI: http://dx.doi.org/10.5772/intechopen.87055*

*Impact of Climate Change on Plant Diseases and IPM Strategies DOI: http://dx.doi.org/10.5772/intechopen.87055*

#### **Figure 3.**

*Plant Diseases-Current Threats and Management Trends*

nontargeted pesticides is a less recommended alternative.

better options while purchasing their food.

In order to achieve proper pest control, IPM programs are designed to manage the crop, lawn, or indoor space to prevent the appearance and development of pests. In the case of agricultural crops, this could mean using proper planting methods, for example, rotating between crops, planting resistant plant varieties, and the use of pest-free rootstock. These control techniques can be very useful and efficient in terms of cost and present lower risk to human health and the environment.

Once the previously mentioned variables specify that control actions are needed and that protective approaches are ineffective, IPM programs then assess the appropriate control actions in terms of efficiency and risk. Efficient and low-risk control methods are considered first, including targeted and ecofriendly chemicals, such as pheromones which disrupt pest reproduction, or mechanical control, including trapping and weeding. If data generated by the previously mentioned steps specify that less risky management methods are ineffective, then further pest control attempts should be considered, for example, directed use of pesticides. The use of

A lot of agricultural growers identify their pests prior to pesticide application. Less risky pesticides such as pheromones are employed by a lower subset of growers. In the end, a lot of these farmers are using IPM techniques. The objective is to make more growers use the proper IPM practices. Mostly, crops produced using IPM techniques are not recognized in the marketplace as organic crops. Growers who use IPM practices have no national certifications in certain countries. Due to the complexity of the IPM pest management techniques, it is not possible to use a single IPM description for all crops and all regions of a country. Many growers of certain crops including strawberries and potatoes are attempting to define what IPM means in their crops case as well as the region of growth. Moreover, certified IPM crops are unavailable in a lot of regions. With definitions, farmers can start to market their crops as IPM-grown, which would give consumers alternative and

The previously mentioned processes of IPM have been redefined and modified over time, and the IPM pyramid was created to provide an easier understanding of the approach. The IPM pyramid consists of three main processes which include preventive or indirect crop protection, risk assessment or monitoring, and responsive or direct crop protection. The three processes aim to increase the efficiency of each step involving crop breading and maintenance. Preventive crop protection involves the use of certified seeds, cultivars which have high tolerance to pathogens, and enhancement of natural enemies of plant pathogens such as microbiological competitors. Risk assessment and monitoring is the most crucial process in IPM; it involves the use of an early warning forecast system which provides information related to current climate and how it could affect plant health and by using such information and understanding plant physiology and susceptibility to pathogens; one can determine timeframes where plants are most susceptible to pathogens and take countermeasures to prevent or minimize pathogen severity (e.g., through the use of fungicides). Direct crop protection basically involves the countermeasures taken to deal with unfavorable conditions, which include the use of antagonistic microorganisms or application of fungicides. A simple example for how an integrated system for pest management can be created as illustrated in **Figure 3**; generally in order to create such a system, the main information needed include weather data, crop, and disease information. Through knowing the weather data, which is most commonly obtained from meteorological

**3.3 Prevention**

**3.4 Control**

**88**

*Example of pathogen epidemiology system in potato late blight and its integrated components.*

stations to obtain micro environment weather data and not that of the macro environment, macro environment data can still be used using certain equations to relate to the micro environment. This would allow the prediction and knowledge of current and upcoming weather conditions, in hand with obtaining information about the crop as in which pests affect the grown crops, optimal growth conditions, and lifetime as well as obtaining specific disease information for diseases which have the capability of affecting said crops. One can create a forecasting tool which helps prevent the spread of certain diseases.

After obtaining information about the disease, in order to create a disease model, its efficiency is determined through several tests. The general scopes of these tests would be the difference between disease spread and crop loss before and after the implementation of the model. Did delaying the use of pesticides until the threshold point for disease spread determined by the model actually cause a positive difference or was the threshold inaccurate? Was there a noticeable increase in crop yield after implementation? Was there any human error in pesticide management and spraying timelines? Several questions can be answered, and through these answers, the efficiency of the model can be determined.

There are several advantages for the use of IPM in mitigating agricultural problems which include:

Slower development of resistance to pesticides: Pesticide resistance can be incurred by the repeated use of pesticides; this would occur if a farmer is using traditional farming methods as pests would develop resistance to the pesticide due to repeated exposure to the pesticides through development of resistance by natural selection; then the resistant genes would be transferred to the offsprings, incurring permanent resistance to a given pesticide. However, this would not occur with reduced and efficient application of pesticides which is a main strategy adopted by IPM systems.

Maintaining a balanced ecosystem: Increased use of pesticides might affect nontarget and beneficial organisms; if these organisms are wiped out, the ecosystem will suffer and in turn results in species loss. IPM eradicates pests while minimizing the damage dealt to nontarget species.

Better cost vs. value: Since pesticides incur the highest cost for a farmer during a growing season. Reducing the use of pesticides proves more cost-efficient on the long run than the price for equipment used to determine thresholds, weather conditions, and application of IPM strategies. This is due to limited and efficient pesticide application.

The disadvantages of using IPM strategies include:

More involvement in the technicalities of the method

All individuals involved have to be educated about the available methods and importance of IPM.

Time and energy consuming

IPM strategies are critical strategies; failure to proceed with certain decisions during the IPM process can prove fatal to the entire process due to the need of different control methods for different pests and the need to monitor the application process.

#### **4. Decision support system (DSS)**

In order to produce an efficient model, understanding the decision support system is essential. The decision support system is an informatics tool which uses mathematical models such as equations and statistics to help the decision-maker take action. The three main phases of a decision-making system include intelligence, design, and choice; two other subsequent phases include implementation of the decision and monitoring of its effect and outcomes. The decision-making process in IPM is highly complex and dynamic; it requires a high level of organization and constant update of operators; it requires the presence of databases and means to collect data and information as well as tools to handle data. The decision-making process generally provides the capability to identify when difficulties may occur and how to deal with these difficulties depending on data provided.

The main properties of a successful DSS include ease of use, presentation format, system restrictiveness, decisional guidance, feedback, and interaction support:

#### **4.1 Ease of use**

A DSS system is only beneficial if users perceive a DSS to be easy to use and that using it enhances their performance and productivity. The system should be easy to operate and interact with and requires minimal cognitive efforts; to sum up it should reduce mental effort and time consumed to analyze data and increase comfort of the user.

#### **4.2 Presentation format**

The way information is presented through the program/system may influence the user's judgment/decision; therefore, the way information is presented through the decision support system should be focused on showing key data in an accurate and favorable format.

**91**

*Impact of Climate Change on Plant Diseases and IPM Strategies*

**4.3 System restrictiveness and decisional guidance**

These refer to how much a DSS limits the options of the user and to which extent

The way the system provides messages and the wording of certain commands is important because they promote positive user experience and enhance the decision-

Interaction support means that users are permitted a particular level of interaction with a DSS. The DSS design is the determining factor on the presence as well as the level of interactivity between the user and the system. Users may have control over the system when a certain level of interaction support is present. The received

control over the use of a system may have a motivational effect on its use. The system involves an integration of certain components and interacting factors with a common objective. In the case of pathogen monitoring, a pathogen monitoring system would basically receive input based on environmental factors and properties, and the output is expressed in the form of maps, information, and graphs. The system would then, based on information provided by threshold charts for pathogen favorable growth conditions, give out options that would determine the best possible course of actions in order to prevent disease outbreaks and crop loss. With this, an integrated model is illustrated. Although the systems are accurate,

the main drawbacks include maintenance needs of meteorological stations, the requirement of different systems with different parameters depending on the type of plant, and disease as well as their growth conditions and application of these processes in farms which could be a difficult task due to old farming traditions.

Forecasting of pests and diseases appearance in plants is an additional application that demands a consistent and dependable stock of weather data. There are huge losses in yields due to pests and diseases prevalence which could have been controlled in several situations if the appropriate forecasting techniques were available. Consequently, the forecasting and early warning system, within the Plant Pathology Research Institute of the Agricultural Research Center, was established in the same timeframe of system and climate change applications in agriculture.

Prediction of pest, disease prevalence, and progression based on weather data is

The idea of forecasting and early warning system has been introduced in 1926; with the appearance of computers and information technology, softwares were developed in order to produce convenient warning systems. Currently, early warning systems are being used to deal with pathogens of certain crops such as faba beans. The main concept involves the use of a mobile telemetry automated weather station system to monitor environmental conditions and softwares to interpret the input; based on the information provided, a decision can be made regarding the

extremely crucial when planning and implementing control measures.

**4.6 Forecasting and early warning system**

protection and maintenance of plant health.

it guides the user toward a certain decision. A good DSS provides a reasonable amount of options within the scope of the topic/field at hand, as well as ample guidance to perform the optimal decision and achieve the most beneficial outcome.

*DOI: http://dx.doi.org/10.5772/intechopen.87055*

**4.4 Feedback**

making process.

**4.5 Interaction support**

#### **4.3 System restrictiveness and decisional guidance**

These refer to how much a DSS limits the options of the user and to which extent it guides the user toward a certain decision. A good DSS provides a reasonable amount of options within the scope of the topic/field at hand, as well as ample guidance to perform the optimal decision and achieve the most beneficial outcome.

#### **4.4 Feedback**

*Plant Diseases-Current Threats and Management Trends*

The disadvantages of using IPM strategies include: More involvement in the technicalities of the method

the damage dealt to nontarget species.

Time and energy consuming

**4. Decision support system (DSS)**

application.

process.

provided.

**4.1 Ease of use**

comfort of the user.

favorable format.

**4.2 Presentation format**

importance of IPM.

Maintaining a balanced ecosystem: Increased use of pesticides might affect nontarget and beneficial organisms; if these organisms are wiped out, the ecosystem will suffer and in turn results in species loss. IPM eradicates pests while minimizing

Better cost vs. value: Since pesticides incur the highest cost for a farmer during a growing season. Reducing the use of pesticides proves more cost-efficient on the long run than the price for equipment used to determine thresholds, weather conditions, and application of IPM strategies. This is due to limited and efficient pesticide

All individuals involved have to be educated about the available methods and

IPM strategies are critical strategies; failure to proceed with certain decisions during the IPM process can prove fatal to the entire process due to the need of different control methods for different pests and the need to monitor the application

In order to produce an efficient model, understanding the decision support system is essential. The decision support system is an informatics tool which uses mathematical models such as equations and statistics to help the decision-maker take action. The three main phases of a decision-making system include intelligence, design, and choice; two other subsequent phases include implementation of the decision and monitoring of its effect and outcomes. The decision-making process in IPM is highly complex and dynamic; it requires a high level of organization and constant update of operators; it requires the presence of databases and means to collect data and information as well as tools to handle data. The decision-making process generally provides the capability to identify when difficulties may occur and how to deal with these difficulties depending on data

The main properties of a successful DSS include ease of use, presentation format, system restrictiveness, decisional guidance, feedback, and interaction support:

A DSS system is only beneficial if users perceive a DSS to be easy to use and that using it enhances their performance and productivity. The system should be easy to operate and interact with and requires minimal cognitive efforts; to sum up it should reduce mental effort and time consumed to analyze data and increase

The way information is presented through the program/system may influence the user's judgment/decision; therefore, the way information is presented through the decision support system should be focused on showing key data in an accurate and

**90**

The way the system provides messages and the wording of certain commands is important because they promote positive user experience and enhance the decisionmaking process.

#### **4.5 Interaction support**

Interaction support means that users are permitted a particular level of interaction with a DSS. The DSS design is the determining factor on the presence as well as the level of interactivity between the user and the system. Users may have control over the system when a certain level of interaction support is present. The received control over the use of a system may have a motivational effect on its use.

The system involves an integration of certain components and interacting factors with a common objective. In the case of pathogen monitoring, a pathogen monitoring system would basically receive input based on environmental factors and properties, and the output is expressed in the form of maps, information, and graphs. The system would then, based on information provided by threshold charts for pathogen favorable growth conditions, give out options that would determine the best possible course of actions in order to prevent disease outbreaks and crop loss.

With this, an integrated model is illustrated. Although the systems are accurate, the main drawbacks include maintenance needs of meteorological stations, the requirement of different systems with different parameters depending on the type of plant, and disease as well as their growth conditions and application of these processes in farms which could be a difficult task due to old farming traditions.

#### **4.6 Forecasting and early warning system**

Forecasting of pests and diseases appearance in plants is an additional application that demands a consistent and dependable stock of weather data. There are huge losses in yields due to pests and diseases prevalence which could have been controlled in several situations if the appropriate forecasting techniques were available. Consequently, the forecasting and early warning system, within the Plant Pathology Research Institute of the Agricultural Research Center, was established in the same timeframe of system and climate change applications in agriculture.

Prediction of pest, disease prevalence, and progression based on weather data is extremely crucial when planning and implementing control measures.

The idea of forecasting and early warning system has been introduced in 1926; with the appearance of computers and information technology, softwares were developed in order to produce convenient warning systems. Currently, early warning systems are being used to deal with pathogens of certain crops such as faba beans. The main concept involves the use of a mobile telemetry automated weather station system to monitor environmental conditions and softwares to interpret the input; based on the information provided, a decision can be made regarding the protection and maintenance of plant health.

An early warning system is shown in **Figure 4** which is used to predict and prevent the development of chocolate spot in faba beans that has been developed by Dr. Sahar Zayan, Head of the Early Warning Unit, Plant Pathology Research Institute [4].

A set of computer programs have been successfully produced by the unit's work team, and some of them were applied on crop databases in different governorates. These programs have proven to be successful early warning systems, as they predicted the appearance of diseases before infection and before they reached the epidemic level as well as the reduction of the amount of fungicides used for disease resistance.

The first computer simulation model for prediction of late blight in potato was being produced in Egypt by Prof. Dr. Mohsen Abd El Razek Afifi and Dr. Sahar Zayan in the forecasting and early warning unit in the plant pathology research institute, Agricultural Research Center. The model was applied in the fields, and it produced results which assisted in the protection of the potato crops from infection and the reduction of pesticide application periods which was equivalent to 75% in certain regions.

After the construction of the first Egyptian computer model, the creator named it EGY-BLIGHTCAST, and its efficiency was verified in all the computer laboratories (Workstations) as well as the field conditions by the potato producing private sector companies [5]; the model was applied in 1998 and 1999, and it preserved the crop from the risks of epidemic infection; pesticide savings reached 50% in a season and 75% in another, and the productivity increased by a ton and 300 kilograms per acre which was denoted in official reports by the applying company. Afterward the model was developed in 2002 and 2003; it was used in different regions (hotspots of late blight on potato crops) in the main governorates for potato production in Egypt throughout 2004–2008; the methodologies for prediction of late blight were linked and modified based on short-term observation.

Throughout several growing seasons of potato and the analysis of the relation between 24-hour meteorological data which were collected in real-time from the forecasting station (AdconTelemetry A733) and its effect on the possibility of daily infection by diseases triggered by late blight, it is possible for the EGY-BLIGHTCAST (DDIP) model to accurately predict the outbreak of the late blight disease and to drastically reduce the cost of necessary fungicide to control the outbreak when compared to routine spraying programs (schedule based programs)

**93**

**Author details**

Giza, Egypt

Sahar Abdou Zayan

*Impact of Climate Change on Plant Diseases and IPM Strategies*

in light of the field conditions. The main roles for the analysis of the model validation system were discussed through a study which was published in the year 2009. Moreover, several computer simulation models were produced on the same basis for a number of important diseases on strategic crops in Egypt, including downy and powdery mildew in grapes—downy mildew in onions and early blight in tomatoes. All of these forecasting models were applied in test fields, and their efficiency in disease prediction was proven as well as the actual savings in application of pesti-

In the year 2015, the system and model production techniques were developed by Dr. Sahar Zayan, and a study was published for a computer model for brown spot on beans which received an Intellectual Property Rights (IPR) license from official

With the appearance of climate change phenomena, farmers and decisionmakers will need more decision support systems especially plant disease forecasting

I would like to thank my team who helped me in developing this work.

Unit of Forecasting and Early Warning, Plant Pathology Research Institute (ARC),

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

\*Address all correspondence to: drsahar.abdo@gmail.com

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.87055*

cides used in disease control.

authorities in Egypt [6].

systems.

**Thanks**

**Figure 4.** *A certified early warning system for faba bean chocolate spot [4].*

*Impact of Climate Change on Plant Diseases and IPM Strategies DOI: http://dx.doi.org/10.5772/intechopen.87055*

in light of the field conditions. The main roles for the analysis of the model validation system were discussed through a study which was published in the year 2009. Moreover, several computer simulation models were produced on the same basis for a number of important diseases on strategic crops in Egypt, including downy and powdery mildew in grapes—downy mildew in onions and early blight in tomatoes. All of these forecasting models were applied in test fields, and their efficiency in disease prediction was proven as well as the actual savings in application of pesticides used in disease control.

In the year 2015, the system and model production techniques were developed by Dr. Sahar Zayan, and a study was published for a computer model for brown spot on beans which received an Intellectual Property Rights (IPR) license from official authorities in Egypt [6].

With the appearance of climate change phenomena, farmers and decisionmakers will need more decision support systems especially plant disease forecasting systems.

#### **Thanks**

*Plant Diseases-Current Threats and Management Trends*

and modified based on short-term observation.

*A certified early warning system for faba bean chocolate spot [4].*

Institute [4].

resistance.

certain regions.

An early warning system is shown in **Figure 4** which is used to predict and prevent the development of chocolate spot in faba beans that has been developed by Dr. Sahar Zayan, Head of the Early Warning Unit, Plant Pathology Research

team, and some of them were applied on crop databases in different governorates. These programs have proven to be successful early warning systems, as they predicted the appearance of diseases before infection and before they reached the epidemic level as well as the reduction of the amount of fungicides used for disease

A set of computer programs have been successfully produced by the unit's work

The first computer simulation model for prediction of late blight in potato was being produced in Egypt by Prof. Dr. Mohsen Abd El Razek Afifi and Dr. Sahar Zayan in the forecasting and early warning unit in the plant pathology research institute, Agricultural Research Center. The model was applied in the fields, and it produced results which assisted in the protection of the potato crops from infection and the reduction of pesticide application periods which was equivalent to 75% in

After the construction of the first Egyptian computer model, the creator named it EGY-BLIGHTCAST, and its efficiency was verified in all the computer laboratories (Workstations) as well as the field conditions by the potato producing private sector companies [5]; the model was applied in 1998 and 1999, and it preserved the crop from the risks of epidemic infection; pesticide savings reached 50% in a season and 75% in another, and the productivity increased by a ton and 300 kilograms per acre which was denoted in official reports by the applying company. Afterward the model was developed in 2002 and 2003; it was used in different regions (hotspots of late blight on potato crops) in the main governorates for potato production in Egypt throughout 2004–2008; the methodologies for prediction of late blight were linked

Throughout several growing seasons of potato and the analysis of the relation between 24-hour meteorological data which were collected in real-time from the forecasting station (AdconTelemetry A733) and its effect on the possibility of daily infection by diseases triggered by late blight, it is possible for the EGY-BLIGHTCAST (DDIP) model to accurately predict the outbreak of the late blight disease and to drastically reduce the cost of necessary fungicide to control the outbreak when compared to routine spraying programs (schedule based programs)

**92**

**Figure 4.**

I would like to thank my team who helped me in developing this work.

#### **Author details**

Sahar Abdou Zayan Unit of Forecasting and Early Warning, Plant Pathology Research Institute (ARC), Giza, Egypt

\*Address all correspondence to: drsahar.abdo@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

#### **References**

[1] FAO. Climate Change, Agriculture and Food Security [Internet]. Agriculture Organization. 2008. Available from: http://www.fao.org/ faostat/en/#data/RF

[2] Coakley S, Scherm H, Chakraborty S. Climate change and plant disease management. Annual Review of Phytopathology. 1999;**37**(1):399-426

[3] Anita W, Dominic M, Neil A. Climate Change and Agriculture Impacts, Adaptation and Mitigation: Impacts, Adaptation and Mitigation. OECD Publishing; 2010

[4] Zayan S, Morsy M. BCS-CAST: An early warning computerized model for Faba bean chocolate spot in Egypt. In: Proceedings of 5th International Conference on Alternative Methods of Crop Protection; 11-13 March 2015; Lille, France. Lille, France: Association Françoise of Plant Protection (AFPP); 2015

[5] Afifi M, Zayan S. Implementation of Egy-Blight Cast the first computer simulation model for potato late blight in Egypt. In: Proceedings of Agriculture: Africa's "Engine for Growth"-Plant Science & Biotechnology Hold the Key at Roth Amsted Research; Harpenden, UK: Association of Applied Biologists; October 2009. 12-14

[6] Medany M, Zayan S, Fiani D. New communication methodologies in an innovative digital extension system in Egypt. In: The Ciheam Watch Letter no 38 in February 2017 on "Rural Innovation and Digital Revolution in Agriculture". 2017

**95**

Section 2

Host-Pathogen

Coevolution

Section 2

Host-Pathogen Coevolution

**94**

*Plant Diseases-Current Threats and Management Trends*

[1] FAO. Climate Change, Agriculture

[2] Coakley S, Scherm H, Chakraborty S. Climate change and plant disease management. Annual Review of Phytopathology. 1999;**37**(1):399-426

[3] Anita W, Dominic M, Neil A. Climate Change and Agriculture Impacts, Adaptation and Mitigation: Impacts, Adaptation and Mitigation. OECD

[4] Zayan S, Morsy M. BCS-CAST: An early warning computerized model for Faba bean chocolate spot in Egypt. In: Proceedings of 5th International Conference on Alternative Methods of Crop Protection; 11-13 March 2015; Lille, France. Lille, France: Association Françoise of Plant Protection (AFPP);

[5] Afifi M, Zayan S. Implementation of Egy-Blight Cast the first computer simulation model for potato late blight in Egypt. In: Proceedings of Agriculture: Africa's "Engine for Growth"-Plant Science & Biotechnology Hold the Key at Roth Amsted Research; Harpenden, UK: Association of Applied Biologists;

[6] Medany M, Zayan S, Fiani D. New communication methodologies in an innovative digital extension system in Egypt. In: The Ciheam Watch Letter no 38 in February 2017 on "Rural Innovation and Digital Revolution in

and Food Security [Internet]. Agriculture Organization. 2008. Available from: http://www.fao.org/

faostat/en/#data/RF

**References**

Publishing; 2010

October 2009. 12-14

Agriculture". 2017

2015

**97**

expressed genes

**1. Introduction**

**Chapter 7**

**Abstract**

Infection

*and Simon L. Elliot*

Asymptomatic Phytoplasma

*Philip Donkersley, Farley W.S. Silva, Murilo S. Alves,* 

*Claudine M. Carvalho, Abdullah M. Al-Sadi*

understanding the evolution of pathogens within perennial hosts.

**Keywords:** *Citrus aurantifolia*, acid lime, silent infection, phytoplasma, differentially

Vector-borne plant pathogens of perennial crop species provide an opportunity to study the impacts of long-term infections, in terms of epidemiology and vector ecology. Crop diseases directly threaten global food security; an estimated 16% of food production globally is lost despite our efforts to control crop diseases [1]. Perennial crops generally have advantages over annuals in terms of energetic efficiency; for example, constant canopy development increases photosynthesis efficiency [2], which results in 30% increases in carbon turnover than those maintained by annual crops [2]. Pathogens must evolve to infect and reproduce

Reveal a Novel and Troublesome

Asymptomatic infections are by their nature challenging to study and even more difficult to monitor across broad geographical ranges, particularly as methods are reliant on expensive molecular techniques. The plant pathogen that causes Witches' Broom disease of lime (*Candidatus* Phytoplasma aurantifolia) is a major limiting factor in lime production across the Middle East and was recently detected in Brazil, but without the typical symptoms from the Middle East. Here, we discuss the difficulty of monitoring asymptomatic infections and highlight the threat posed by highlight future outbreaks. Asymptomatic infections have important implications for understanding the evolution of pathogens within perennial hosts. We use three model systems of asymptomatic infections: (i) a Phytoplasma and (ii) a bacterial infection of lime (*Citrus aurantifolia*) and (iii) an "out-group" Phytoplasma of Cassava (*Manihot esculenta*) to demonstrate consistency across divergent hosts. We found that although all plants in the study were intentionally infected, assays typically did not confirm this diagnosis. Emergent technologies monitoring gene expression could be used to both study novel biology associated with asymptomatic infections and develop monitoring technologies. We highlight the difficulty of monitoring asymptomatic infections in possible future outbreaks and have important implications for

#### **Chapter 7**

## Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection

*Philip Donkersley, Farley W.S. Silva, Murilo S. Alves, Claudine M. Carvalho, Abdullah M. Al-Sadi and Simon L. Elliot*

#### **Abstract**

Asymptomatic infections are by their nature challenging to study and even more difficult to monitor across broad geographical ranges, particularly as methods are reliant on expensive molecular techniques. The plant pathogen that causes Witches' Broom disease of lime (*Candidatus* Phytoplasma aurantifolia) is a major limiting factor in lime production across the Middle East and was recently detected in Brazil, but without the typical symptoms from the Middle East. Here, we discuss the difficulty of monitoring asymptomatic infections and highlight the threat posed by highlight future outbreaks. Asymptomatic infections have important implications for understanding the evolution of pathogens within perennial hosts. We use three model systems of asymptomatic infections: (i) a Phytoplasma and (ii) a bacterial infection of lime (*Citrus aurantifolia*) and (iii) an "out-group" Phytoplasma of Cassava (*Manihot esculenta*) to demonstrate consistency across divergent hosts. We found that although all plants in the study were intentionally infected, assays typically did not confirm this diagnosis. Emergent technologies monitoring gene expression could be used to both study novel biology associated with asymptomatic infections and develop monitoring technologies. We highlight the difficulty of monitoring asymptomatic infections in possible future outbreaks and have important implications for understanding the evolution of pathogens within perennial hosts.

**Keywords:** *Citrus aurantifolia*, acid lime, silent infection, phytoplasma, differentially expressed genes

#### **1. Introduction**

Vector-borne plant pathogens of perennial crop species provide an opportunity to study the impacts of long-term infections, in terms of epidemiology and vector ecology. Crop diseases directly threaten global food security; an estimated 16% of food production globally is lost despite our efforts to control crop diseases [1]. Perennial crops generally have advantages over annuals in terms of energetic efficiency; for example, constant canopy development increases photosynthesis efficiency [2], which results in 30% increases in carbon turnover than those maintained by annual crops [2]. Pathogens must evolve to infect and reproduce

within a single year in annual cropping systems, and thus typically demonstrate more aggressive pathologies [3], which often require multiple hosts, such as potato blight (*Phytophthora infestans*) and wheat rust (*Puccinia graminis*). As the host plant remains in situ after harvest, perennial cropping systems therefore theoretically allow for evolution of slower pathologies, which may be cryptic in nature.

Globally, plant pathogens are spreading faster than ever, due to climate change, increased crop and germplasm trading, failure of border biocontrol and associated spread of vector species. Here, we shall introduce and discuss a complex vectorborne plant pathogens of a perennial tropical cash-crop plant: *Citrus*. *Citrus* is the world's principal fruit crop, with about 60 million megatons grown annually [4]. Limes account for ~5% of global *Citrus* production [4]. Lime is cultivated in tropical, subtropical and temperate regions from 40°N to 40°S [5, 6]. Countries in the Middle East, as well as India, Pakistan, Brazil, Argentina and Mexico grow lime as a key part of their agricultural economies [7, 8].

The production of lime in the Middle East has been markedly impacted by Witches' Broom Disease of Lime (WBDL) [7]. Symptoms of witches' broom disease of lime (WBDL) were first observed in Oman in the 1970s [9]. Infected trees present with "witches' brooms": shoot structures characterized compactness and small, pale green leaves. In the advanced stages of the disease, leaves become dry, brooms become increasingly more prevalent, and fruits become significantly smaller and less marketable. Finally, the tree collapses within 4 or 5 years after infection [10].

Asymptomatic ("silent") infections have recently been detected in lime trees in Brazil [11] and Oman [12]. This silent infection was observed through molecular testing of plant material, yet the host plants themselves show no obvious visible symptoms. These infected trees do however, also collapse within the 5 year post infection period [13], making this asymptomatic variant potentially even more of a threat to global lime production.

Detailed research into this system has been limited, some suggest that the silent infection may be due to ultra-low pathogen titre levels within the host plant [12, 14] or due to different interactions with plant defences [15] or insect vectors [16, 17]. Silent infections are difficult to monitor and pose a significant risk to global food security, given that the limited knowledge we have suggests they may be as destructive as symptomatic [18], but we do not yet know the full extent of their range.

The Phytoplasma "*Candidatus* Phytoplasma aurantifolia" has been identified as the causative agent of WBDL [19]. Phytoplasma are wall-less gram-positive bacteria belonging to the class Mollicutes [20]. They are found in the phloem sieve tubes of plants and in the gut, salivary glands and other organs of Hemipteran insect vectors [21]. Phytoplasma are obligate biotrophic organisms, which lack many essential genes that encode for components of metabolic pathways; and they likely import metabolites such as nucleotides, amino acids, and fatty acids from the host plant [22]. Phytoplasma are the only known organisms that lack ATP-synthase subunits, which are thought to be essential for life [22]. Owing to the inability to culture them *in vitro* and their inaccessibility in host plants [19, 22], the molecular mechanisms that underlie Phytoplasma infections within host plants remain largely unknown [10]. Phytoplasma may be able to overcome plant defences by producing specific proteins: effectors [15]. The effectors (e.g., SAP11 and SAP54) may modulate host plant growth and interactions with the insect vectors [16, 23].

Although studies using proteomics [10, 24] and cDNA-amplified fragment length polymorphism (cDNA-AFLP) [25] have investigated differentially expressed genes (DEGs) in plants infected by "*Ca*. P. aurantifolia," these studies provide only a brief snapshot of gene expression and regulation during infection. Recent developments in high throughput "omics" based approaches now allow a detailed

**99**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

developing our knowledge of these differentially expressed genes.

a poorly understood, emerging area of plant pathology.

came from a previously published study by *Citrus* [27].

harvest, when it can cause 100% crop losses [18].

with "*Ca*. P. aurantifolia," but showing no visible symptoms [17].

**2.1 Sample locations**

**2.2 Plant material**

examination of plant pathogen interactions, and these have been applied to symptomatic infections of "*Ca*. P. aurantifolia" in the Middle East [18, 26]. Our study examined DEGs in symptomatic and asymptomatic infections of the Phytoplasma in acid lime trees. Although asymptomatic infections have been linked to fitness benefits in the vectors of this pathogen [17], our knowledge of understanding of gene expression differences in an asymptomatic infection are extremely limited. One way to understand the effects and biology of asymptomatic infections is by

Within this chapter, we shall discuss two studies on asymptomatic infections of crop plants [1]. Reliable detection of asymptomatic plant pathogens is the greatest limitation on controlling and limiting their global spread. We first discuss and test the potential for currently employed molecular tools to misidentify "healthy" plants. We study three asymptomatic infections (a Phytoplasma of lime, a Phytoplasma of cassava and *Citrus* Huanglongbing) and compare the rate of falsenegatives detecting the disease [2]. Asymptomatic infections in Brazil represent a novel biology by the Phytoplasma infecting lime trees. This novel pathology needs to be explored to better understand the infection process, and also presents us with an opportunity to design superior detection tools. We compare the gene expression of infected symptomatic and asymptomatic plants using qPCR. These findings provide an important and novel examination of the nature of asymptomatic infections,

**2. Pathogen detection in the absence of visible symptoms: study system**

In order to comprehensively study the most ubiquitous methods used globally for asymptomatic infections of crop plants, we used three model systems: the aforementioned Phytoplasma causing Witches' Broom Disease of Lime (WBDL), a closely related Phytoplasma causing Cassava (*Manihot esculenta* L.) Witches' Broom, and an out group pathogen of lime—"*Candidatus Liberibacter* asiaticus," causative organism of Huanglongbing disease of lime. Data for the first two pathosystems was collected for the present study, whereas data from Huanglongbing

Acid lime (*C. aurantifolia* L.) trees were grown on a *Citrus* orchard maintained at Universidade Federal de Viçosa (UFV), Brazil (S20°45′585″; W042°50′908″). The site was chosen as plant material there had previously been found to be infected

Leaf samples of cassava (*M. esculenta*) grown in a glasshouse at UFV and deliberately infected with a cassava witches' broom (Phytoplasma 16SrIII-A) were also taken. For details regarding this pathogen, please see [18]. Although this disease can display typical symptoms of witches' broom (e.g., stunting, leaf chlorosis, deformation, and reduced size), the infections in Brazil do not display symptoms until

*Citrus* leaf samples from Brazil were taken from four 15-year adult trees and 10 1-year saplings; for each adult tree 30 leaves were collected and for saplings 10 samples were collected in a semi-random fashion. Cassava leaf samples were collected from eight 1-year adult plants, 10 leaves were sampled from each cassava plant.

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

#### *Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

*Plant Diseases-Current Threats and Management Trends*

key part of their agricultural economies [7, 8].

threat to global lime production.

their range.

within a single year in annual cropping systems, and thus typically demonstrate more aggressive pathologies [3], which often require multiple hosts, such as potato blight (*Phytophthora infestans*) and wheat rust (*Puccinia graminis*). As the host plant remains in situ after harvest, perennial cropping systems therefore theoretically allow for evolution of slower pathologies, which may be cryptic in nature.

Globally, plant pathogens are spreading faster than ever, due to climate change, increased crop and germplasm trading, failure of border biocontrol and associated spread of vector species. Here, we shall introduce and discuss a complex vectorborne plant pathogens of a perennial tropical cash-crop plant: *Citrus*. *Citrus* is the world's principal fruit crop, with about 60 million megatons grown annually [4]. Limes account for ~5% of global *Citrus* production [4]. Lime is cultivated in tropical, subtropical and temperate regions from 40°N to 40°S [5, 6]. Countries in the Middle East, as well as India, Pakistan, Brazil, Argentina and Mexico grow lime as a

The production of lime in the Middle East has been markedly impacted by Witches' Broom Disease of Lime (WBDL) [7]. Symptoms of witches' broom disease of lime (WBDL) were first observed in Oman in the 1970s [9]. Infected trees present with "witches' brooms": shoot structures characterized compactness and small, pale green leaves. In the advanced stages of the disease, leaves become dry, brooms become increasingly more prevalent, and fruits become significantly smaller and less marketable. Finally, the tree collapses within 4 or 5 years after infection [10]. Asymptomatic ("silent") infections have recently been detected in lime trees in Brazil [11] and Oman [12]. This silent infection was observed through molecular testing of plant material, yet the host plants themselves show no obvious visible symptoms. These infected trees do however, also collapse within the 5 year post infection period [13], making this asymptomatic variant potentially even more of a

Detailed research into this system has been limited, some suggest that the silent infection may be due to ultra-low pathogen titre levels within the host plant [12, 14] or due to different interactions with plant defences [15] or insect vectors [16, 17]. Silent infections are difficult to monitor and pose a significant risk to global food security, given that the limited knowledge we have suggests they may be as destructive as symptomatic [18], but we do not yet know the full extent of

The Phytoplasma "*Candidatus* Phytoplasma aurantifolia" has been identified as the causative agent of WBDL [19]. Phytoplasma are wall-less gram-positive bacteria belonging to the class Mollicutes [20]. They are found in the phloem sieve tubes of plants and in the gut, salivary glands and other organs of Hemipteran insect vectors [21]. Phytoplasma are obligate biotrophic organisms, which lack many essential genes that encode for components of metabolic pathways; and they likely import metabolites such as nucleotides, amino acids, and fatty acids from the host plant [22]. Phytoplasma are the only known organisms that lack ATP-synthase subunits, which are thought to be essential for life [22]. Owing to the inability to culture them *in vitro* and their inaccessibility in host plants [19, 22], the molecular mechanisms that underlie Phytoplasma infections within host plants remain largely unknown [10]. Phytoplasma may be able to overcome plant defences by producing specific proteins: effectors [15]. The effectors (e.g., SAP11 and SAP54) may modulate host

Although studies using proteomics [10, 24] and cDNA-amplified fragment length polymorphism (cDNA-AFLP) [25] have investigated differentially expressed genes (DEGs) in plants infected by "*Ca*. P. aurantifolia," these studies provide only a brief snapshot of gene expression and regulation during infection. Recent developments in high throughput "omics" based approaches now allow a detailed

plant growth and interactions with the insect vectors [16, 23].

**98**

examination of plant pathogen interactions, and these have been applied to symptomatic infections of "*Ca*. P. aurantifolia" in the Middle East [18, 26]. Our study examined DEGs in symptomatic and asymptomatic infections of the Phytoplasma in acid lime trees. Although asymptomatic infections have been linked to fitness benefits in the vectors of this pathogen [17], our knowledge of understanding of gene expression differences in an asymptomatic infection are extremely limited. One way to understand the effects and biology of asymptomatic infections is by developing our knowledge of these differentially expressed genes.

Within this chapter, we shall discuss two studies on asymptomatic infections of crop plants [1]. Reliable detection of asymptomatic plant pathogens is the greatest limitation on controlling and limiting their global spread. We first discuss and test the potential for currently employed molecular tools to misidentify "healthy" plants. We study three asymptomatic infections (a Phytoplasma of lime, a Phytoplasma of cassava and *Citrus* Huanglongbing) and compare the rate of falsenegatives detecting the disease [2]. Asymptomatic infections in Brazil represent a novel biology by the Phytoplasma infecting lime trees. This novel pathology needs to be explored to better understand the infection process, and also presents us with an opportunity to design superior detection tools. We compare the gene expression of infected symptomatic and asymptomatic plants using qPCR. These findings provide an important and novel examination of the nature of asymptomatic infections, a poorly understood, emerging area of plant pathology.

#### **2. Pathogen detection in the absence of visible symptoms: study system**

In order to comprehensively study the most ubiquitous methods used globally for asymptomatic infections of crop plants, we used three model systems: the aforementioned Phytoplasma causing Witches' Broom Disease of Lime (WBDL), a closely related Phytoplasma causing Cassava (*Manihot esculenta* L.) Witches' Broom, and an out group pathogen of lime—"*Candidatus Liberibacter* asiaticus," causative organism of Huanglongbing disease of lime. Data for the first two pathosystems was collected for the present study, whereas data from Huanglongbing came from a previously published study by *Citrus* [27].

#### **2.1 Sample locations**

Acid lime (*C. aurantifolia* L.) trees were grown on a *Citrus* orchard maintained at Universidade Federal de Viçosa (UFV), Brazil (S20°45′585″; W042°50′908″). The site was chosen as plant material there had previously been found to be infected with "*Ca*. P. aurantifolia," but showing no visible symptoms [17].

Leaf samples of cassava (*M. esculenta*) grown in a glasshouse at UFV and deliberately infected with a cassava witches' broom (Phytoplasma 16SrIII-A) were also taken. For details regarding this pathogen, please see [18]. Although this disease can display typical symptoms of witches' broom (e.g., stunting, leaf chlorosis, deformation, and reduced size), the infections in Brazil do not display symptoms until harvest, when it can cause 100% crop losses [18].

#### **2.2 Plant material**

*Citrus* leaf samples from Brazil were taken from four 15-year adult trees and 10 1-year saplings; for each adult tree 30 leaves were collected and for saplings 10 samples were collected in a semi-random fashion. Cassava leaf samples were collected from eight 1-year adult plants, 10 leaves were sampled from each cassava plant.

The sampling strategy for both lime Phytoplasma and cassava Phytoplasma aimed to collect a spatially diverse group of samples (orientated on x, y and z axes relative to the trunk), with the position of each leaf sampled noted with respect to its branches from the main trunk. For all sample types locations, leaf midrib samples (the larger vein along the midline of a leaf) were taken. The midribs were immediately frozen in liquid nitrogen after harvesting and then transported to the laboratory, where they were stored at −80°C until total DNA and RNA isolation.

#### **2.3 Molecular detection of Phytoplasma**

The presence/absence of the Phytoplasma in the leaf samples of both acid lime and cassava was analysed using PCR for Phytoplasma detection. To this end, total DNA was extracted from acid lime leaf samples using the DNeasy Qiagen Plant Mini Prep kit following manufacturer's instructions. Then, total DNA was extracted from the cassava leaf samples following the protocol of [28], with modifications that are detailed in [18].

We then used a nested PCR using universal primers for Phytoplasma detection. Extracted DNA of both *Citrus* and cassava Phytoplasma were amplified using 16S rRNA PCR primers P4 (5′-CAT CAT TTA GTT GGG CAC TT-3′) and 23rev (5′-CGT CCT TCA TCG GCT CTT-3′) in the initial reaction, and the resulting amplicon was diluted (1:10) and used as template DNA for nested PCR amplification using the P3 (5′-GGA TGG ATC ACC TCC TT-3′) and 23rev primers [18, 29, 30].

PCR amplification was carried out using a Loccus Biotechnologia TC9639 Thermal Cycler (LB, São Paulo, Brazil) in 20 μl volumes, such that each reaction contained the following: 2.0 μl (20 pmol) of each primer, 8.0 μl water (DNA-free water; Qiagen, SP, Brazil), 4.0 μl sample extracted DNA and 0.1 μl Invitrogen *Taq* DNA Polymerase (5 U/μl) (ThermoFisher Scientific, Brazil), 1.3 μl MgCl (50 mm) 2.6 μl dNTPs (10 mm), 2.0 μl PCR buffer (200 mm Tris-HCl pH 8.4, 500 mm KCl). For the first round PCR, initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 45 s, 55°C for 45 s and 72°C for 3 min, with a final elongation step at 72°C for 7 min. For the nested reactions, the conditions were 95°C for 3 min, followed by 32 cycles of 95°C for 45 s, 54°C for 45 s and 72°C for 3 min, with a final elongation step at 72°C for 7 min. The resulting amplicon was then visualised on agarose gel electrophoresis using SybrSafe DNA stain to confirm the presence/ absence of both Phytoplasma from each leaf sample of each plant host.

Data on the successful amplification of "*Candidatus* C. liberibacter" were obtained from the Coy et al. [27] study. Briefly, this study compares the efficacy of the current method of detection for C. *liberibacter asiaticus* within plant and insect samples is by a presence/absence PCR assay using a 16S rDNA gene target. Specifically they examined these methods for sensitivity to low bacterial titers or suboptimal PCR conditions that can result in false-negatives. This study concluded that the high incidence of false negatives using this system could contributes to the under-reporting of plant pathogen infections. Hence, the data paralleled our present study, and were used for direct comparison of this pathosystem with our own presented here.

#### **3. Pathogen detection in the absence of visible symptoms: results and discussion**

Detection of "*Ca*. Phytoplasma aurantifolia" by 23S-PCR on asymptomatic acid lime (*C. aurantifolia*) plants showed that all plants sampled in this study were technically infected (**Table 1**), meaning that each plant had at least one sample that positively detected the Phytoplasma. The proportion of samples that failed to detect the pathogen was, on average, in adult trees 38.5% ± 6.62 (n = 3), and in saplings

**101**

**Table 1.**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

46.25% ± 22.6 (n = 10). Within cassava (*Manihot esculenta*), false-negative rate was 48.75% ± 17.3 (n = 8). False-negative molecular tests have also been found in molecular techniques for detecting Huanglongbing infections in *Citrus* plants. Thus particular study found a false-negative rate of 54.9%, using a nested PCR assay [27], and identified that more sensitive molecular tests involving qPCR addressed this issue, albeit not in a manner applicable to growers and germplasm suppliers expect-

*Results of asymptomatic infections of "Ca. Phytoplasma aurantifolia" detection using 23S-PCR from adult* 

**Tree Infected Detection likelihood (%)**

A 21/31 67.74 B 24/44 54.55 C 28/45 62.22 SA 5/10 50.00 SB 6/10 60.00 SC 6/10 60.00 SD 6/10 60.00 SE 4/10 40.00 SF 3/10 30.00 SG 3/10 30.00 SH 10/10 100.00

The evidence for false-negative across multiple plant pathosystems has notable implications across the field. One of the base assumptions of plant pathology is the suitability of a biological sample to represent the entire host plant. These falsenegatives mean that multiple biological samples per plant may be required to correctly identify the presence of a pathogen. A hypothetical plant with α leaves and a false-negative rate of *β* ± SD, to guarantee a correct identification (under P = 1.00)

*n* = (*α* × *β*) + 1 (1)

Due to the nature of additive probabilities (Eq. (2)), the probability of, for example, 38 continuous false-negatives on a tree of 100 leaves would be P = 4.83<sup>−</sup>22. Consequently, a decision support system based on the likelihood of having an infected tree can be developed in order to determine the appropriate number of samples required to avoid a false-negative. For example, for P = 0.05, minimum sample number would be n = 4.19; for P = 0.005, n = 8.94; for P = 0.001, n = 12.25 (**Figure 1a**). For cassava similarly the minimum sample number for the same prob-

Asymptomatic plant pathogens are particularly troublesome within perennial crops as they are not removed at the end of the growing season and act as reservoirs of infectious materials to be dispersed to new hosts by insects (and other vehicles). Persistence of asymptomatic infections in hosts may also cause problems through subtle direct damage or sublethal infections leading to plant-by-plant transmission

(1 − *β*)*<sup>n</sup>*−*<sup>k</sup>* (2)

*k*=*x* α ( \_\_ *α <sup>k</sup>*) *<sup>β</sup><sup>k</sup>*

abilities would be (in order): n = 3.55, n = 7.20 and n = 9.76 (**Figure 1b**).

ing to provide disease-free planting material [31].

*(A–C) and sapling (SA-SH) Citrus acid lime plants.*

the minimum sample number (n) must be:

∑

*DOI: http://dx.doi.org/10.5772/intechopen.86650*


*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

#### **Table 1.**

*Plant Diseases-Current Threats and Management Trends*

**2.3 Molecular detection of Phytoplasma**

The sampling strategy for both lime Phytoplasma and cassava Phytoplasma aimed to collect a spatially diverse group of samples (orientated on x, y and z axes relative to the trunk), with the position of each leaf sampled noted with respect to its branches from the main trunk. For all sample types locations, leaf midrib samples (the larger vein along the midline of a leaf) were taken. The midribs were immediately frozen in liquid nitrogen after harvesting and then transported to the laboratory, where they were stored at −80°C until total DNA and RNA isolation.

The presence/absence of the Phytoplasma in the leaf samples of both acid lime and cassava was analysed using PCR for Phytoplasma detection. To this end, total DNA was extracted from acid lime leaf samples using the DNeasy Qiagen Plant Mini Prep kit following manufacturer's instructions. Then, total DNA was extracted from the cassava leaf samples following the protocol of [28], with modifications that are detailed in [18]. We then used a nested PCR using universal primers for Phytoplasma detection. Extracted DNA of both *Citrus* and cassava Phytoplasma were amplified using 16S rRNA PCR primers P4 (5′-CAT CAT TTA GTT GGG CAC TT-3′) and 23rev (5′-CGT CCT TCA TCG GCT CTT-3′) in the initial reaction, and the resulting amplicon was diluted (1:10) and used as template DNA for nested PCR amplification using the P3

(5′-GGA TGG ATC ACC TCC TT-3′) and 23rev primers [18, 29, 30].

absence of both Phytoplasma from each leaf sample of each plant host.

direct comparison of this pathosystem with our own presented here.

**3. Pathogen detection in the absence of visible symptoms: results and** 

Detection of "*Ca*. Phytoplasma aurantifolia" by 23S-PCR on asymptomatic acid lime (*C. aurantifolia*) plants showed that all plants sampled in this study were technically infected (**Table 1**), meaning that each plant had at least one sample that positively detected the Phytoplasma. The proportion of samples that failed to detect the pathogen was, on average, in adult trees 38.5% ± 6.62 (n = 3), and in saplings

Data on the successful amplification of "*Candidatus* C. liberibacter" were obtained from the Coy et al. [27] study. Briefly, this study compares the efficacy of the current method of detection for C. *liberibacter asiaticus* within plant and insect samples is by a presence/absence PCR assay using a 16S rDNA gene target. Specifically they examined these methods for sensitivity to low bacterial titers or suboptimal PCR conditions that can result in false-negatives. This study concluded that the high incidence of false negatives using this system could contributes to the under-reporting of plant pathogen infections. Hence, the data paralleled our present study, and were used for

PCR amplification was carried out using a Loccus Biotechnologia TC9639 Thermal Cycler (LB, São Paulo, Brazil) in 20 μl volumes, such that each reaction contained the following: 2.0 μl (20 pmol) of each primer, 8.0 μl water (DNA-free water; Qiagen, SP, Brazil), 4.0 μl sample extracted DNA and 0.1 μl Invitrogen *Taq* DNA Polymerase (5 U/μl) (ThermoFisher Scientific, Brazil), 1.3 μl MgCl (50 mm) 2.6 μl dNTPs (10 mm), 2.0 μl PCR buffer (200 mm Tris-HCl pH 8.4, 500 mm KCl). For the first round PCR, initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 45 s, 55°C for 45 s and 72°C for 3 min, with a final elongation step at 72°C for 7 min. For the nested reactions, the conditions were 95°C for 3 min, followed by 32 cycles of 95°C for 45 s, 54°C for 45 s and 72°C for 3 min, with a final elongation step at 72°C for 7 min. The resulting amplicon was then visualised on agarose gel electrophoresis using SybrSafe DNA stain to confirm the presence/

**100**

**discussion**

*Results of asymptomatic infections of "Ca. Phytoplasma aurantifolia" detection using 23S-PCR from adult (A–C) and sapling (SA-SH) Citrus acid lime plants.*

46.25% ± 22.6 (n = 10). Within cassava (*Manihot esculenta*), false-negative rate was 48.75% ± 17.3 (n = 8). False-negative molecular tests have also been found in molecular techniques for detecting Huanglongbing infections in *Citrus* plants. Thus particular study found a false-negative rate of 54.9%, using a nested PCR assay [27], and identified that more sensitive molecular tests involving qPCR addressed this issue, albeit not in a manner applicable to growers and germplasm suppliers expecting to provide disease-free planting material [31].

The evidence for false-negative across multiple plant pathosystems has notable implications across the field. One of the base assumptions of plant pathology is the suitability of a biological sample to represent the entire host plant. These falsenegatives mean that multiple biological samples per plant may be required to correctly identify the presence of a pathogen. A hypothetical plant with α leaves and a false-negative rate of *β* ± SD, to guarantee a correct identification (under P = 1.00) the minimum sample number (n) must be:

$$n = (a \times \beta) \text{ + 1} \tag{1}$$

$$\sum\_{k=\infty}^{\mathfrak{g}} \left(\frac{a}{k}\right) \rho^k \left(1-\rho\right)^{n-k} \tag{2}$$

Due to the nature of additive probabilities (Eq. (2)), the probability of, for example, 38 continuous false-negatives on a tree of 100 leaves would be P = 4.83<sup>−</sup>22. Consequently, a decision support system based on the likelihood of having an infected tree can be developed in order to determine the appropriate number of samples required to avoid a false-negative. For example, for P = 0.05, minimum sample number would be n = 4.19; for P = 0.005, n = 8.94; for P = 0.001, n = 12.25 (**Figure 1a**). For cassava similarly the minimum sample number for the same probabilities would be (in order): n = 3.55, n = 7.20 and n = 9.76 (**Figure 1b**).

Asymptomatic plant pathogens are particularly troublesome within perennial crops as they are not removed at the end of the growing season and act as reservoirs of infectious materials to be dispersed to new hosts by insects (and other vehicles). Persistence of asymptomatic infections in hosts may also cause problems through subtle direct damage or sublethal infections leading to plant-by-plant transmission

**Figure 1.**

*Probability function for false negatives using PCR-based detection for asymptomatic Phytoplasma infections. The additive probability of sequential false negatives as the sample size increases in (a) Citrus Phytoplasma in adult Citrus trees (false negative rate = 38.5%); (b) cassava Phytoplasma (false negative rate = 48.75%)*.

[32]. The use of accurate and timely diagnostic methods is undoubtedly one of the best ways to monitor pathogen ranges in asymptomatic infected plants, and thus avoid dissemination to new hosts and ranges. Generally, traditional methods of identification based on visual symptoms and culturing in laboratories are time-consuming, labourintensive, costly and have "very low sensitivity and specificity" [33, 34].

Molecular methods are the mainstream alternative to symptomology and laboratory culture. The results of this present (and a previous) study [27] have demonstrated a potential flaw in molecular methods: the frequency of falsenegatives. Whereas classical plant pathology can rely on a non-destructive inspection of the entire host plant, culture and molecular methods must only use a small "representative" destructive subsample of the plant. The major limitation to this is the quality of the representation of the host plant within this subsample. We have demonstrated here that a single biological sample from an infected plant may not be representative of the whole plant and therefore multiple samples from within the same host plant can result in different results from molecular testing for pathogens. We found false-negative rates between 38 and 49%, meaning that approximately a minimum of one in three samples would fail to detect a pathogen if taken alone. Although this calls into question the use of single biological samples for identifying pathogens by molecular methods, these methods have to strike a balance between precision and cost [35]. We calculate, based on these false-negative rates, minimum sample numbers (per plant) between 3 and 5 samples, which may make these methods prohibitively expensive for widespread use within agriculture.

By comparison, real-time PCR used to detect and quantify pathogens in symptomless plant tissues is a promising tool to improve our understanding of "silent" infections [36]. Different methods of DNA amplification that rely on conventional and quantitative PCRs have also been developed to detect and identify "*Ca.* Liberibacter" species associated with Huanglongbing (HLB) in *Citrus* [27, 37]. But other simpler methods, such as direct tissue blot immunoassay, have been used to facilitate detection of pathogens in asymptomatic plants of *Citrus* [38]. Molecular tools have been developed for identification of WBDL from field samples [7, 39], but remain prohibitively expensive for widespread implementation by growers. Much research effort and resources have been devoted to development of on-the-spot diagnostics in plant pathology, and have shown success in control and monitoring the spread of some plant diseases (e.g., *Potato Virus Y*), but do not exist for Phytoplasma

**103**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

developed; but have not been adopted for widespread use yet [34, 41].

**4. Novel asymptomatic infection biology: study system**

of the "silent" symptoms in this newly emerged pathosystem.

yellowing and necrosis) and uninfected (healthy tissue).

**4.1 Sample locations**

**4.2 Plant material**

**4.3 RNA extraction**

**4.4 Gene expression**

yet [40]. *In-situ* kits for testing Phytoplasma using immunofluorescence have been

Successful identification of asymptomatic infections by the Phytoplasma causing Witches' Broom Disease of Lime (WBDL) provide a unique opportunity to compare the pathology with its' symptomatic counterpart. A recent study by Mardi et al. [26] using a high-throughput genomics approach identified 2805 differentially expressed genes in symptomatically infected *Citrus* plants. This study revealed the key potential molecular pathways through which the Phytoplasma infects and parasitizes its host. Correspondingly, here we studied 25 of these that were differentially expressed by more than 128-fold and 4 further genes identified as significantly differentially expressed in recent infections found in Brazil (Alves et al. unpublished data). These genes allowed us to design a targeted study to understand how the symptomatic and asymptomatic infections differ, and potentially identify some

Acid lime (*C. aurantifolia*) trees were grown at the same *Citrus* orchard at UFV mentioned previously. Lime leaves were also collected from cultivated areas in Muscat, Oman (N23°58′591″, E58°40′590″). Omani samples were collected from a farm with symptomatic infected trees (drastic reduction in growth, generalized leaf

Six *Citrus* plants were sampled each in Brazil and Oman (three symptomatic and three healthy plants), for three biological replicates. Samples from Brazil were confirmed for Phytoplasma by PCR (see Section 2), samples from Oman were confirmed by symptoms (drastic reduction in growth, generalized leaf yellowing and necrosis).

Total RNA was extracted from the three biological replicates of limes infected with "*Ca*. P aurantifolia" and three healthy acid lime leaves (from both Brazil and Oman) using the RNeasy Plant Mini Kit (Qiagen, SP, Brazil). RNA quantity and quality were determined using a Nano-Drop ND 1000 spectrophotometer (Thermo Scientific, MA, USA). five hundred nanogram of total RNA from each replicate was reverse-transcribed in a 20 μl reaction using 1 μl of Invitrogen SuperScript® III Reverse Transcriptase (Thermo Scientific), 1 μl oligo(dT)18 (100 nm), 1 μl dTT (100 mm), 2 μl dNTP (10 mm), 4 μl 5× first-strand buffer (250 mm Tris-HCl

Gene specific primers were designed for 15 genes belonging to key pathways with possible implication in disease progression and resistance identified by Mardi et al. [26] and four by Alves et al. (unpublished). The sequence of primers, amplicon length, optimal primer and enzymatic efficiency for each primer pair is presented in **Table 2**. Mardi genes were amplified only for Brazilian samples,

(pH 8.3), 375 mm KCl, 15 mm MgCl2) and RNAse free water (Qiagen).

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

yet [40]. *In-situ* kits for testing Phytoplasma using immunofluorescence have been developed; but have not been adopted for widespread use yet [34, 41].

### **4. Novel asymptomatic infection biology: study system**

Successful identification of asymptomatic infections by the Phytoplasma causing Witches' Broom Disease of Lime (WBDL) provide a unique opportunity to compare the pathology with its' symptomatic counterpart. A recent study by Mardi et al. [26] using a high-throughput genomics approach identified 2805 differentially expressed genes in symptomatically infected *Citrus* plants. This study revealed the key potential molecular pathways through which the Phytoplasma infects and parasitizes its host. Correspondingly, here we studied 25 of these that were differentially expressed by more than 128-fold and 4 further genes identified as significantly differentially expressed in recent infections found in Brazil (Alves et al. unpublished data). These genes allowed us to design a targeted study to understand how the symptomatic and asymptomatic infections differ, and potentially identify some of the "silent" symptoms in this newly emerged pathosystem.

#### **4.1 Sample locations**

*Plant Diseases-Current Threats and Management Trends*

[32]. The use of accurate and timely diagnostic methods is undoubtedly one of the best ways to monitor pathogen ranges in asymptomatic infected plants, and thus avoid dissemination to new hosts and ranges. Generally, traditional methods of identification based on visual symptoms and culturing in laboratories are time-consuming, labour-

*Probability function for false negatives using PCR-based detection for asymptomatic Phytoplasma infections. The additive probability of sequential false negatives as the sample size increases in (a) Citrus Phytoplasma in adult Citrus trees (false negative rate = 38.5%); (b) cassava Phytoplasma (false negative rate = 48.75%)*.

Molecular methods are the mainstream alternative to symptomology and laboratory culture. The results of this present (and a previous) study [27] have demonstrated a potential flaw in molecular methods: the frequency of falsenegatives. Whereas classical plant pathology can rely on a non-destructive inspection of the entire host plant, culture and molecular methods must only use a small "representative" destructive subsample of the plant. The major limitation to this is the quality of the representation of the host plant within this subsample. We have demonstrated here that a single biological sample from an infected plant may not be representative of the whole plant and therefore multiple samples from within the same host plant can result in different results from molecular testing for pathogens. We found false-negative rates between 38 and 49%, meaning that approximately a minimum of one in three samples would fail to detect a pathogen if taken alone. Although this calls into question the use of single biological samples for identifying pathogens by molecular methods, these methods have to strike a balance between precision and cost [35]. We calculate, based on these false-negative rates, minimum sample numbers (per plant) between 3 and 5 samples, which may make these methods prohibitively expensive for widespread use within agriculture.

By comparison, real-time PCR used to detect and quantify pathogens in symptomless plant tissues is a promising tool to improve our understanding of "silent" infections [36]. Different methods of DNA amplification that rely on conventional

and quantitative PCRs have also been developed to detect and identify "*Ca.* Liberibacter" species associated with Huanglongbing (HLB) in *Citrus* [27, 37]. But other simpler methods, such as direct tissue blot immunoassay, have been used to facilitate detection of pathogens in asymptomatic plants of *Citrus* [38]. Molecular tools have been developed for identification of WBDL from field samples [7, 39], but remain prohibitively expensive for widespread implementation by growers. Much research effort and resources have been devoted to development of on-the-spot diagnostics in plant pathology, and have shown success in control and monitoring the spread of some plant diseases (e.g., *Potato Virus Y*), but do not exist for Phytoplasma

intensive, costly and have "very low sensitivity and specificity" [33, 34].

**102**

**Figure 1.**

Acid lime (*C. aurantifolia*) trees were grown at the same *Citrus* orchard at UFV mentioned previously. Lime leaves were also collected from cultivated areas in Muscat, Oman (N23°58′591″, E58°40′590″). Omani samples were collected from a farm with symptomatic infected trees (drastic reduction in growth, generalized leaf yellowing and necrosis) and uninfected (healthy tissue).

#### **4.2 Plant material**

Six *Citrus* plants were sampled each in Brazil and Oman (three symptomatic and three healthy plants), for three biological replicates. Samples from Brazil were confirmed for Phytoplasma by PCR (see Section 2), samples from Oman were confirmed by symptoms (drastic reduction in growth, generalized leaf yellowing and necrosis).

#### **4.3 RNA extraction**

Total RNA was extracted from the three biological replicates of limes infected with "*Ca*. P aurantifolia" and three healthy acid lime leaves (from both Brazil and Oman) using the RNeasy Plant Mini Kit (Qiagen, SP, Brazil). RNA quantity and quality were determined using a Nano-Drop ND 1000 spectrophotometer (Thermo Scientific, MA, USA). five hundred nanogram of total RNA from each replicate was reverse-transcribed in a 20 μl reaction using 1 μl of Invitrogen SuperScript® III Reverse Transcriptase (Thermo Scientific), 1 μl oligo(dT)18 (100 nm), 1 μl dTT (100 mm), 2 μl dNTP (10 mm), 4 μl 5× first-strand buffer (250 mm Tris-HCl (pH 8.3), 375 mm KCl, 15 mm MgCl2) and RNAse free water (Qiagen).

#### **4.4 Gene expression**

Gene specific primers were designed for 15 genes belonging to key pathways with possible implication in disease progression and resistance identified by Mardi et al. [26] and four by Alves et al. (unpublished). The sequence of primers, amplicon length, optimal primer and enzymatic efficiency for each primer pair is presented in **Table 2**. Mardi genes were amplified only for Brazilian samples,


*Plant Diseases-Current Threats and Management Trends*

**Table 2.**

**105**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

whereas we were able to study the smaller number Alves genes were amplified for

The selected genes were quantified using the Applied Biosystems StepOne™ Real Time PCR system (Thermo Scientific). qRT-PCR was performed in a 10-

μl of SYBR Green PCR Master Mix, 4

(**Table 1**), 50 ng of template cDNA. The thermal cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s, and a final

gene was used to estimate gene expression in RNA concentration values of ng

′-TTT CTT CCT CAA CTT CAC TTG TAT CC-3

Standard curves for each gene were examined in the amplification plot and the standard curve plot was prepared in ABI 7500 software v.2.0.6. Reaction efficiency, R square and slope values were calculated by the ABI 7500 software v.2.0.6 program (**Table 3**) and were used to determine the copy number of infection-related RNA in

′),

The detected expression of selected transcripts was measured using the absolute quantification method. We prepared standard curves for each target gene (0.01–

Ubiquitin 1 and Tubulin alpha were used as internal reference genes, with primer

α-tub-F (5

′-GAT AGG CGT TCC AGT AAC AAC GA-3

**−1/slope**

−4.69701 0.212901 1.159017 115.9

−4.99484 0.200206 1.148863 114.9

−4.17641 0.23944 1.180534 118.1

−1.01071 0.989403 1.985363 198.5

−1.69178 0.591093 1.506388 150.6

−4.35679 0.229527 1.17245 117.2

−4.19476 0.238393 1.179678 117.9

−4.67463 0.213921 1.159836 115.9

−1.68595 0.593137 1.508523 150.8

−1.32334 0.755662 1.688406 168.8

−4.3699 0.228838 1.171891 117.2

−2.41499 0.41408 1.332449 133.2

−4.77939 0.209232 1.156072 115.6

−3.94001 0.253806 1.192349 119.2

−5.796 0.172533 1.127035 112.7

—

—

—

—

) in order to quantify each genes expression relative to a standard internal

T) value of each gene relative to the internal control

μl

μ l − 1

′-TGG

′), respectively.

**E E (%)**

— 112.8

— 132.5

— 119.1

— 102.7

μl of each primer mix

′), UBi-IR (5

′-CTG CAA GGG TTC TTG GTG

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

both Brazilian and Oman samples.

extension step at 72°C for 5 min.

control gene. Cycle threshold (C

TCA TAG GCT GTT CGA TCA C-3

**Unigene Slope**

α-tub-R (5

[42]. Triplicate reactions were used for each sample.

—

—

—

—

*qPCR efficiency values for Phytoplasma related genes in C. aurantifolia.*

reaction containing 5

10 ng μ l − 1

TTC-3

sets UBi-IF (5

each sample.

U352

U2265

U27316

U75775

U26576

U72184

U59125

U68165

U68593

U77887

U3869

U17275

U41653

U17606

U24969

WRKY33

WRKY70

MYBR1

JAZ6

**Table 3.**

′) and

 *Primer sequences for potential infection related differentially expressed genes in Citrus aurantifolia used in qPCR.*

#### *Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

*Plant Diseases-Current Threats and Management Trends*

**104**

**Unigene**

U352 U2265 U27316 U75775 U26576 U72184 U59125 U68165 U68593 U77887

U3869 U17275 U41653 U17606 U24969 WRKY33 WRKY70

MYBR1

JAZ6

**Table 2.**

**Forward primer**

TGGCTCTGGATGGCATTG

TGCTGCATTGGTTCTGTC

ATGCGATACACAACCCAATCT

GAAGGAGCTGACGTTTTC

GATTGTCCGCCCAGTAGTG

CAAAGAGATGGGCAAAGAG

TATGGGGATAAGGGGTGT

CTGCTGAGATTACATGGTT

GACTCTCTTTCAATGCCA

CATGCCATCCTCTTCACT

CTCCTCCTCCTCCTCCAAAG

AACACCCATTTGCATTCTC

GAGAGTAGCAAGACCTCAAG

CTCACCGCAGATTTTGAACCAC

GCCTCCGTTTCCAATTCTC

GATGATGAAAATGAACCTGATGCT

AGACCGGAGAGGATGCTACAAG

AATGGATCCAACTTGGTTTTGAA

ACAATGATGCAACCCCACTTC

*Primer sequences for potential infection related differentially expressed genes in Citrus aurantifolia used in qPCR.*

**Reverse primer**

GTGCTTCTGGGATAGTGA

GACTGCAAAGGACTCCAAG

CGGCCATGAGACCAAAACT

CTTCTGCCTCTTCCCTCTC

CACGCGATCAGCCAAACTC

GCCAAATTACAAACCAAACGA

TGCCACAACTAACCTCCTC

CTCTTCAGGGAATTGCAC

TTGAAAGCACAGGTTCCGA

GGGTTGGGTTGAGTATCT

GCGAACCCATCACACTACAT

GGTTTGTATGCCTTCGATG

TATCACCAGCCTCACTTCAC

ACATCCGTCTTCTCATCCACA

GATACCGAGGATTTCATGGC

CAATTCTTGGCTCCCTCACAGT

CCCATATTTCCTCCATGCAAA

ATCCAAACTCGCCCTGGTT

TGCTGCAGCCCTTTCTTTTC

**Amplicon length (bp)**

133

130

126

160

174

121

182

147

119

123

117

130

114

158

131

144

152

110

120

whereas we were able to study the smaller number Alves genes were amplified for both Brazilian and Oman samples.

The selected genes were quantified using the Applied Biosystems StepOne™ Real Time PCR system (Thermo Scientific). qRT-PCR was performed in a 10-μl reaction containing 5 μl of SYBR Green PCR Master Mix, 4 μl of each primer mix (**Table 1**), 50 ng of template cDNA. The thermal cycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s, and a final extension step at 72°C for 5 min.

The detected expression of selected transcripts was measured using the absolute quantification method. We prepared standard curves for each target gene (0.01– 10 ng μl −1 ) in order to quantify each genes expression relative to a standard internal control gene. Cycle threshold (CT) value of each gene relative to the internal control gene was used to estimate gene expression in RNA concentration values of ng μl −1 [42]. Triplicate reactions were used for each sample.

Ubiquitin 1 and Tubulin alpha were used as internal reference genes, with primer sets UBi-IF (5′-TTT CTT CCT CAA CTT CAC TTG TAT CC-3′), UBi-IR (5′-TGG TCA TAG GCT GTT CGA TCA C-3′), α-tub-F (5′-CTG CAA GGG TTC TTG GTG TTC-3′) and α-tub-R (5′-GAT AGG CGT TCC AGT AAC AAC GA-3′), respectively. Standard curves for each gene were examined in the amplification plot and the standard curve plot was prepared in ABI 7500 software v.2.0.6. Reaction efficiency, R square and slope values were calculated by the ABI 7500 software v.2.0.6 program (**Table 3**) and were used to determine the copy number of infection-related RNA in each sample.


#### **Table 3.**

*qPCR efficiency values for Phytoplasma related genes in C. aurantifolia.*

#### **4.5 Statistical analysis**

Analyses of differential gene expression in asymptomatic Phytoplasma infections of acid lime were performed using the *R* statistical software v3.3.2 [43]. Non-metric multidimensional scaling (NMDS) was used to analyse differential gene expression and partition variation between symptomatic/asymptomatic and infected/uninfected groups across all genes [44]. Here, we analysed normalised gene copy number by NMDS using the "*metaMDS*" function [45]. NMDS was performed using the Bray-Curtis dissimilarity index on two ordinal scales for optimal NMDS stress values. Interactions between these and infection type were tested and assigned significance using the "*envfit*" function. Significant differences in DEGs between asymptomatic infected and healthy *Citrus* plants were tested using Student's *t*-tests. Matched gene expression data between Brazil and Oman were further analysed using *post-hoc* Tukey HSD tests to test differential expression based on sample location and symptom type.

#### **5. Novel asymptomatic infection biology: results and discussion**

Gene expression profiles were determined by qPCR for 15-disease related genes identified previously for infections of "*Ca*. Phytoplasma aurantifolia" in *Citrus aurantifolia* adult trees by Mardi et al. [26]. NMDS showed that a two-dimensional solution was sufficient to achieve low stress values to enable us to interpret disease-related gene expression (stress = 0.049). Infection status (asymptomatic/ uninfected) of leaf samples was significantly correlated with the NMDS analysis of gene expression (**Figure 2**, R2 = 0.533, P < 0.001), demonstrating clear differences in

#### **Figure 2.**

*Surface NMDS ordinations of differential gene expression from samples of "Ca. Phytoplasma aurantifolia" asymptomatic infected and uninfected (healthy) acid lime trees from Brazil denoted by open circles; their position is determined by where they fall on ordinal axes 1 and 2. Red names are species centroids for each Unigene. Polygons indicate clustering of each infection type, which are interpreted as how each gene (and the overall gene expression composition) correlates with the infection properties.*

**107**

**Table 4.**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

**Transcript Uninfected** 

**expression (ng μl −1 )**

U352 Beta-galactosidase 3 9.74 8.47 ↓ (p < 0.001) Energy

U77887 Gibberellin 2-oxidase 10.24 8.20 NS (p = 0.195) Growth

U75775 Nitrite reductase 17.27 12.58 ↓ (p = 0.001) Protein

*Functional characterisation of DEGs expressed in response to infection by "Ca. P. aurantifolia" and mean* 

*differential expression between asymptomatic infected and healthy C. aurantifolia plants.*

host plant gene expression in response to infection by this asymptomatic infection. When examining the direction and significance of differential expression of each of these 15-disease related genes, several significant decreases were found (**Table 4**). Expression of four genes related to stress tolerance, cell replication, energy production and protein production (CRT/DRE binding factor; NAC domain-containing protein 71; beta-galactosidase 3; nitrite reductase) were significantly decreased in asymptomatic infected plants. Genes related to immune response (mitogen-activated protein kinase 1; cyclic nucleotide-gated ion channel 1; brassinosteroid insensitive-1-associated receptor kinase) were not significantly differentially expressed however.

> **Asymptomatic expression (ng μl −1 )**

**Differential expression**

11.22 13.67 NS (p = 0.321) ABA-signalling

8.15 7.82 NS (p = 0.537) Abiotic stress

12.32 7.76 ↓ (p = 0.050) Abiotic stress

32.74 15.30 ↓ (p = 0.011) Cell Replication

7.54 7.38 NS (p = 0.664) Growth

21.27 18.40 NS (p = 0.140) Growth

27.04 22.42 NS (p = 0.118) Immune response

17.12 16.97 NS (p = 0.918) Immune response

20.04 21.12 NS (p = 0.453) Immune response

15.42 15.73 NS (p = 0.659) JA-signalling

6.42 6.52 NS (p = 0.623) Light response

7.85 8.38 NS (p = 0.051) Protein

**Functional characterisation**

tolerance

tolerance

production

regulation

regulation

regulation

production

production

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

**Unigene ID**

U24969 Probable LRR

U72184 Zinc finger A20

U59125 CRT/DRE binding

U27316 NAC domain-

U68165 Ent-copalyl

U41653 LRR receptor-like

U26576 Mitogen-activated

U68593 Cyclic nucleotide-

U17606 Brassinosteroid

U3869 Jasmonate ZIM

U17275 Phytochrome-

U2265 Amino acid

receptor-like serine/ threonine-protein kinase

and AN1 domaincontaining stressassociated protein 3

factor

containing protein 71

diphosphate synthase

serine/threonineprotein kinase GSO1

protein kinase 1

gated ion channel 1

insensitive-1 associated receptor kinase

domain-containing protein 6

interacting factor 3

transporter

*Significant differences were tested by students T test.*

#### *Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

*Plant Diseases-Current Threats and Management Trends*

Analyses of differential gene expression in asymptomatic Phytoplasma infections of acid lime were performed using the *R* statistical software v3.3.2 [43]. Non-metric multidimensional scaling (NMDS) was used to analyse differential gene expression and partition variation between symptomatic/asymptomatic and infected/uninfected groups across all genes [44]. Here, we analysed normalised gene copy number by NMDS using the "*metaMDS*" function [45]. NMDS was performed using the Bray-Curtis dissimilarity index on two ordinal scales for optimal NMDS stress values. Interactions between these and infection type were tested and assigned significance using the "*envfit*" function. Significant differences in DEGs between asymptomatic infected and healthy *Citrus* plants were tested using Student's *t*-tests. Matched gene expression data between Brazil and Oman were further analysed using *post-hoc* Tukey HSD tests to test differential expression based on sample location and symptom type.

**5. Novel asymptomatic infection biology: results and discussion**

solution was sufficient to achieve low stress values to enable us to interpret disease-related gene expression (stress = 0.049). Infection status (asymptomatic/ uninfected) of leaf samples was significantly correlated with the NMDS analysis of

Gene expression profiles were determined by qPCR for 15-disease related genes identified previously for infections of "*Ca*. Phytoplasma aurantifolia" in *Citrus aurantifolia* adult trees by Mardi et al. [26]. NMDS showed that a two-dimensional

= 0.533, P < 0.001), demonstrating clear differences in

**4.5 Statistical analysis**

gene expression (**Figure 2**, R2

**106**

**Figure 2.**

*Surface NMDS ordinations of differential gene expression from samples of "Ca. Phytoplasma aurantifolia" asymptomatic infected and uninfected (healthy) acid lime trees from Brazil denoted by open circles; their position is determined by where they fall on ordinal axes 1 and 2. Red names are species centroids for each Unigene. Polygons indicate clustering of each infection type, which are interpreted as how each gene (and the* 

*overall gene expression composition) correlates with the infection properties.*

host plant gene expression in response to infection by this asymptomatic infection. When examining the direction and significance of differential expression of each of these 15-disease related genes, several significant decreases were found (**Table 4**). Expression of four genes related to stress tolerance, cell replication, energy production and protein production (CRT/DRE binding factor; NAC domain-containing protein 71; beta-galactosidase 3; nitrite reductase) were significantly decreased in asymptomatic infected plants. Genes related to immune response (mitogen-activated protein kinase 1; cyclic nucleotide-gated ion channel 1; brassinosteroid insensitive-1-associated receptor kinase) were not significantly differentially expressed however.


#### **Table 4.**

*Functional characterisation of DEGs expressed in response to infection by "Ca. P. aurantifolia" and mean differential expression between asymptomatic infected and healthy C. aurantifolia plants.*

The genes MYBR, JAZ6, WRKY37 and WRKY70 were targeted for amplification from samples from both Oman and Brazil (Alves et al. unpublished). MYBR gene expression was not significantly different between Brazil and Oman (F = 3.725, P = 0.067 **Figure 3a**); *posthoc* tests showed no significant difference between infected/uninfected in Oman (P = 0.998) or Brazil (P = 0.354). JAZ6 expression was significantly different between Brazil and Oman (F = 24.016, P < 0.001 **Figure 3b**); *posthoc* tests showed a significant difference between infected/uninfected in Oman (P = 0.043), but not Brazil (P = 0.588). WRKY70 expression was significantly different between Brazil and Oman (F = 50.002, P < 0.001 **Figure 3c**); *posthoc* tests showed a significant difference between infected/uninfected both in Oman (P < 0.001), and Brazil (P = 0.004). WRKY37 expression was significantly different between Brazil and Oman (F = 9.617, P = 0.004 **Figure 3d**); *posthoc* tests showed a significant difference between infected/uninfected both in Oman (P < 0.001), and Brazil (P < 0.001).

Disease symptoms are, taken at their most literal, an observable change in host homeostasis in response to the presence of a pathogen. The mechanism underlying symptoms (or lack thereof) within the host plant is broad, but mostly resides in genetic changes (host immune response, genomic mutations, RNA silencing) in either the host or pathogen. The nature of asymptomatic infections is complex and poorly understood. Some may express pathogenesis genes at a lower level and be kept in the host without causing overt symptoms [46].

**Figure 3.**

*Differential gene expression of disease-related genes amplified in Brazilian (asymptomatic) and Omani (symptomatic) acid lime trees infected with the Phytoplasma "Ca*. *Phytoplasma aurantifolia."*

**109**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

expected given the asymptomatic nature of the infections (**Table 4**).

infections, and perhaps opens up new routes for detecting these pathogens.

Previous research into symptomatic infections of "*Ca*. P. aurantifolia" infecting acid lime has indicated production of several metabolites significantly altered during infection. In Iran, infections are associated with catechin and epicatechin production in leaves [47, 48]. Amino and organic acid concentrations (such as proline, arginine, glutamate, citrate and salicylate) are also significantly increased immediately after inoculation [47]. Studies have shown that "*Ca*. P aurantifolia" also alters the concentration of limonene, ocimene and trans-caryophyllene [7]. Much like the DEGs we have identified in the present study, each of these chemicals could act as measurable indicators to diagnose the infected lime at the early stages of the WBDL progression. A distinct host plant genomic response to infection by this asymptomatic infection has significant implications for the diseases' insect vectors. Management strategies for insect-vectored pathogens specifically target the vector-plant interactions, relying on monitoring and suppressing these vectors in order to reduce the frequency and severity of disease outbreaks [49]. Many vector-borne plant diseases alter host plant phenotypes in ways that can influence their vectors biology and behaviour [50–52], with significant implications for disease transmission.

Infected plants are often better for their vectors than uninfected in terms of vector growth rates, reproduction and longevity [17, 53]; although the opposite is certainly true in some pathosystems [53] and some vectors actively avoid infected hosts that represent inferior hosts [54]. We have previously demonstrated that an asymptomatic infection results in significant increases in vector life history traits (reproduction and growth rates) than with a symptomatic infection [17]. In future studies, the distinct expression profile detected within the plant host here could be usefully explored in relation to differential gene expression in the insect host, in

We also specifically consider differences between two agricultural loci—the Middle East and South America—by examining a gene set directly related to the plant-pathogen (Phytoplasma) interaction. Four genes (JAZ6, MYBR, WRKY70 and WRKY33)

order to fully understand this vector-host-pathogen complex [16, 23].

We examined a group of host plant (*C. aurantifolia*) infection related genes identified by [26] in the context of an asymptomatic infection. This previous study established by next generation sequencing of host plant RNA expression (RNAseq) that 2805 genes are differentially expressed in symptomatic infected compared with healthy uninfected plants. Of these, 71 genes were significantly deregulated; of them, 52 were upregulated and 19 down-regulated in response to Phytoplasma infection [26]. Here, using quantitative PCR methods, we studied a subset of these genes that were expressed by more than 128-fold and their differential expression in relation to healthy vs. asymptomatic infected lime plants in Brazil (**Table 4**). We demonstrate that the asymptomatic infection does result in detectable changes in host plant gene expression (**Figure 2**). Specifically, however, no significant change in expression of *Citrus* immune response genes was found here, which would be

Certain genes related to stress tolerance, cell replication and energy production had their expression significantly reduced in infected plants (**Table 4**). The latter may be the best candidate for a "symptom" of these "silent" infections: Phytoplasma are obligate biotrophic organisms and their parasitism may be through host ATP-synthase subunits [22]. When comparing these results with those of [26], the stress tolerance gene also shows a significant reduction in expression in symptomatic infected lime plants. However, cell replication and energy production genes were significantly deregulated in the asymptomatic infections, which was distinct to the symptomatic infection in [26]. This may be one of the first accounts of a significant alteration of gene expression by a host plant infected by an asymptomatic plant pathogen. The demonstrated response by the plant clearly indicates that these are not truly "silent"

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

#### *Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

*Plant Diseases-Current Threats and Management Trends*

kept in the host without causing overt symptoms [46].

Brazil (P < 0.001).

The genes MYBR, JAZ6, WRKY37 and WRKY70 were targeted for amplification from samples from both Oman and Brazil (Alves et al. unpublished). MYBR gene expression was not significantly different between Brazil and Oman (F = 3.725, P = 0.067 **Figure 3a**); *posthoc* tests showed no significant difference between

infected/uninfected in Oman (P = 0.998) or Brazil (P = 0.354). JAZ6 expression was significantly different between Brazil and Oman (F = 24.016, P < 0.001 **Figure 3b**); *posthoc* tests showed a significant difference between infected/uninfected in Oman (P = 0.043), but not Brazil (P = 0.588). WRKY70 expression was significantly different between Brazil and Oman (F = 50.002, P < 0.001 **Figure 3c**); *posthoc* tests showed a significant difference between infected/uninfected both in Oman (P < 0.001), and Brazil (P = 0.004). WRKY37 expression was significantly different between Brazil and Oman (F = 9.617, P = 0.004 **Figure 3d**); *posthoc* tests showed a significant difference between infected/uninfected both in Oman (P < 0.001), and

Disease symptoms are, taken at their most literal, an observable change in host homeostasis in response to the presence of a pathogen. The mechanism underlying symptoms (or lack thereof) within the host plant is broad, but mostly resides in genetic changes (host immune response, genomic mutations, RNA silencing) in either the host or pathogen. The nature of asymptomatic infections is complex and poorly understood. Some may express pathogenesis genes at a lower level and be

**108**

**Figure 3.**

*Differential gene expression of disease-related genes amplified in Brazilian (asymptomatic) and Omani (symptomatic) acid lime trees infected with the Phytoplasma "Ca*. *Phytoplasma aurantifolia."*

We examined a group of host plant (*C. aurantifolia*) infection related genes identified by [26] in the context of an asymptomatic infection. This previous study established by next generation sequencing of host plant RNA expression (RNAseq) that 2805 genes are differentially expressed in symptomatic infected compared with healthy uninfected plants. Of these, 71 genes were significantly deregulated; of them, 52 were upregulated and 19 down-regulated in response to Phytoplasma infection [26]. Here, using quantitative PCR methods, we studied a subset of these genes that were expressed by more than 128-fold and their differential expression in relation to healthy vs. asymptomatic infected lime plants in Brazil (**Table 4**). We demonstrate that the asymptomatic infection does result in detectable changes in host plant gene expression (**Figure 2**). Specifically, however, no significant change in expression of *Citrus* immune response genes was found here, which would be expected given the asymptomatic nature of the infections (**Table 4**).

Certain genes related to stress tolerance, cell replication and energy production had their expression significantly reduced in infected plants (**Table 4**). The latter may be the best candidate for a "symptom" of these "silent" infections: Phytoplasma are obligate biotrophic organisms and their parasitism may be through host ATP-synthase subunits [22]. When comparing these results with those of [26], the stress tolerance gene also shows a significant reduction in expression in symptomatic infected lime plants. However, cell replication and energy production genes were significantly deregulated in the asymptomatic infections, which was distinct to the symptomatic infection in [26]. This may be one of the first accounts of a significant alteration of gene expression by a host plant infected by an asymptomatic plant pathogen. The demonstrated response by the plant clearly indicates that these are not truly "silent" infections, and perhaps opens up new routes for detecting these pathogens.

Previous research into symptomatic infections of "*Ca*. P. aurantifolia" infecting acid lime has indicated production of several metabolites significantly altered during infection. In Iran, infections are associated with catechin and epicatechin production in leaves [47, 48]. Amino and organic acid concentrations (such as proline, arginine, glutamate, citrate and salicylate) are also significantly increased immediately after inoculation [47]. Studies have shown that "*Ca*. P aurantifolia" also alters the concentration of limonene, ocimene and trans-caryophyllene [7]. Much like the DEGs we have identified in the present study, each of these chemicals could act as measurable indicators to diagnose the infected lime at the early stages of the WBDL progression.

A distinct host plant genomic response to infection by this asymptomatic infection has significant implications for the diseases' insect vectors. Management strategies for insect-vectored pathogens specifically target the vector-plant interactions, relying on monitoring and suppressing these vectors in order to reduce the frequency and severity of disease outbreaks [49]. Many vector-borne plant diseases alter host plant phenotypes in ways that can influence their vectors biology and behaviour [50–52], with significant implications for disease transmission.

Infected plants are often better for their vectors than uninfected in terms of vector growth rates, reproduction and longevity [17, 53]; although the opposite is certainly true in some pathosystems [53] and some vectors actively avoid infected hosts that represent inferior hosts [54]. We have previously demonstrated that an asymptomatic infection results in significant increases in vector life history traits (reproduction and growth rates) than with a symptomatic infection [17]. In future studies, the distinct expression profile detected within the plant host here could be usefully explored in relation to differential gene expression in the insect host, in order to fully understand this vector-host-pathogen complex [16, 23].

We also specifically consider differences between two agricultural loci—the Middle East and South America—by examining a gene set directly related to the plant-pathogen (Phytoplasma) interaction. Four genes (JAZ6, MYBR, WRKY70 and WRKY33)

are modulated during Phytoplasma infection in lime trees (**Figure 3**). Interestingly, an inverse expression profile for this gene set could be verified by comparing infected lime trees from Brazil and Oman (**Figure 3**). While JAZ6 and WRKY33 are up-regulated in infected (symptomatic) Omani samples, the same genes present lower gene expression in infected (asymptomatic) Brazilian samples (**Figure 3**). The same inverse relation can be verified for WRKY70, which is down-regulated in infected Omani samples, but presents a significantly higher expression in Brazilian samples (**Figure 3**). Such expression profiles of this gene set represent a signature of symptomatic and asymptomatic Phytoplasma infected plants, which can be used to distinguish earlier Phytoplasma infections. This specific expression profile can be associated to the distinct "*Ca*. P. aurantifolia"-related strains responsible for different infections (symptomatic and asymptomatic) in lime trees. The differential expression of plant transcriptional regulation-related genes reflects the possible action of strain-specific Phytoplasma effectors, as verified for other plant-Phytoplasma interactions [16].

Finally, we should also address the previously reported benefits of asymptomatic infections for their host plants. Asymptomatic infections may result in induced systemic resistance (ISR) [55]: pathogens acquired at low titres elicit a set of systemic plant defences (i.e., oxidative burst, phytoalexins and pathogenesis-related proteins) which prepare hosts to more successfully resist later, more severe infections [56–58]. The use of ISR to induce resistance in plants by application of exogenous (chemical or organic) inducers, has been used in integrated programs of disease management. Preinoculation of sour orange (*Citrus aurantium*) seedlings with a hypovirulent isolate of *Phytophthora Citrus* root rot protected them from later infections [59]. Li et al. [60] have also demonstrated the effects chemical inducers on resistance of *Citrus* groves to HLB disease of *Citrus*. Over-expression of an *Arabidopsis* gene (a positive regulator of ISR) in transgenic "Duncan" grapefruit and "Hamlin" sweet orange increased their resistance to *Citrus* canker [61]. Although ISR may be a useful alternative for disease control, it has to be cautiously assessed. In some cases the use of ISR compounds may not provide the expected protection against disease: for example, spraying ISRs onto sweet orange plants did not reduced incidence of *Citrus* canker [59].

#### **6. Conclusions**

This study has addressed two key questions regarding the nature of asymptomatic infections: [1] that being invisible or "silent" infections (and the consequent reliance on molecular tools for detection) makes them inherently challenging to monitor; and [2] that this organism interacts with its plant host in a distinct manner that we have observed in the present study. The key findings are that asymptomatic infections from three case studies all demonstrate high rates of false-negative discovery; meaning that repeated testing of the same plant can give both negative and positive results and that a single positive result is taken as meaning the plant is infected. We also demonstrate that infection by the Phytoplasma "*Ca*. P aurantifolia" is associated with significantly different genetic expression by its acid lime host, giving a first unique insight into the biology of a "silent" infection.

The Phytoplasma "*Ca*. P aurantifolia" is the aetiological agent of Witches' Broom Disease of Lime (WBDL). Although in the Middle East this disease causes high economic impact on lime production, in Brazil emerging infections are notably symptomless [17]. Asymptomatic infections are not particularly rare in plant pathology. *Colletotrichum* fungi, for example, are symbionts that interact with a range of plants as either symptomatic pathogens or asymptomatic endophytes [62]. Yet we do not understand whether the symbiont can use both strategies or if certain strains display the pathogenic or endophytic strategy. Asymptomatic infections also exists for plant

**111**

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection*

viruses: Pelargonium line pattern virus (PLPV; family Tombusviridae) can be asymptomatic when infecting geranium, which may be due to plant defences such as RNA silencing [63]. In some cases, a resource allocation trade-off mechanism between replication and virulence factor production may explain the emergence of asymptomatic modes of a pathogen, for example, in *Ralstonia solanacearum* populations [64]. As "*Ca.* P. aurantifolia" and other asymptomatic plant pathogens like it spread to novel sites of infection globally [31], and as these infections become more difficult to detect [11], new rapid detection methods will be required in order to effectively detect sources of pathogen and monitor its evolution. This study has presented both the difficulties in monitoring "silent" infections using PCR based methods, but has also identified target genes that behave consistently and distinctly during infection

This study was financed in part by the Coordenação de Aperfeiçoamento de

, Murilo S. Alves3

1 Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

, Claudine M. Carvalho3

,

SLE has been the beneficiary of CNPq productivity grants (grant nos.

The authors would like to thanks Vale S.A. for financing of the project. Thanks to SQU for partial support of the work through the grant EG/AGR/

Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

*DOI: http://dx.doi.org/10.5772/intechopen.86650*

by this Phytoplasma.

**Acknowledgements**

CROP/16/01.

**Conflict of interest**

**Author details**

Philip Donkersley1

Abdullah M. Al-Sadi4

Minas Gerais, Brazil

Minas Gerais, Brazil

309221/2013-7 & 309845/2016-5).

The authors declare no conflict of interest.

4 Crop Sciences, Sultan Qaabos University, Oman

provided the original work is properly cited.

\*Address all correspondence to: donkersleyp@gmail.com

\*, Farley W.S. Silva<sup>2</sup>

and Simon L. Elliot2

2 Department of Entomology, Universidade Federal de Viçosa, Viçosa,

3 Department of Phytopathology, Universidade Federal de Viçosa, Viçosa,

*Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection DOI: http://dx.doi.org/10.5772/intechopen.86650*

viruses: Pelargonium line pattern virus (PLPV; family Tombusviridae) can be asymptomatic when infecting geranium, which may be due to plant defences such as RNA silencing [63]. In some cases, a resource allocation trade-off mechanism between replication and virulence factor production may explain the emergence of asymptomatic modes of a pathogen, for example, in *Ralstonia solanacearum* populations [64].

As "*Ca.* P. aurantifolia" and other asymptomatic plant pathogens like it spread to novel sites of infection globally [31], and as these infections become more difficult to detect [11], new rapid detection methods will be required in order to effectively detect sources of pathogen and monitor its evolution. This study has presented both the difficulties in monitoring "silent" infections using PCR based methods, but has also identified target genes that behave consistently and distinctly during infection by this Phytoplasma.

#### **Acknowledgements**

*Plant Diseases-Current Threats and Management Trends*

are modulated during Phytoplasma infection in lime trees (**Figure 3**). Interestingly, an inverse expression profile for this gene set could be verified by comparing infected lime trees from Brazil and Oman (**Figure 3**). While JAZ6 and WRKY33 are up-regulated in infected (symptomatic) Omani samples, the same genes present lower gene expression in infected (asymptomatic) Brazilian samples (**Figure 3**). The same inverse relation can be verified for WRKY70, which is down-regulated in infected Omani samples, but presents a significantly higher expression in Brazilian samples (**Figure 3**). Such expression profiles of this gene set represent a signature of symptomatic and asymptomatic Phytoplasma infected plants, which can be used to distinguish earlier Phytoplasma infections. This specific expression profile can be associated to the distinct "*Ca*. P. aurantifolia"-related strains responsible for different infections (symptomatic and asymptomatic) in lime trees. The differential expression of plant transcriptional regulation-related genes reflects the possible action of strain-specific Phytoplasma

Finally, we should also address the previously reported benefits of asymptomatic

This study has addressed two key questions regarding the nature of asymptomatic infections: [1] that being invisible or "silent" infections (and the consequent reliance on molecular tools for detection) makes them inherently challenging to monitor; and [2] that this organism interacts with its plant host in a distinct manner that we have observed in the present study. The key findings are that asymptomatic infections from three case studies all demonstrate high rates of false-negative discovery; meaning that repeated testing of the same plant can give both negative and positive results and that a single positive result is taken as meaning the plant is infected. We also demonstrate that infection by the Phytoplasma "*Ca*. P aurantifolia" is associated with significantly different genetic expression by its acid lime host,

The Phytoplasma "*Ca*. P aurantifolia" is the aetiological agent of Witches' Broom

Disease of Lime (WBDL). Although in the Middle East this disease causes high economic impact on lime production, in Brazil emerging infections are notably symptomless [17]. Asymptomatic infections are not particularly rare in plant pathology. *Colletotrichum* fungi, for example, are symbionts that interact with a range of plants as either symptomatic pathogens or asymptomatic endophytes [62]. Yet we do not understand whether the symbiont can use both strategies or if certain strains display the pathogenic or endophytic strategy. Asymptomatic infections also exists for plant

infections for their host plants. Asymptomatic infections may result in induced systemic resistance (ISR) [55]: pathogens acquired at low titres elicit a set of systemic plant defences (i.e., oxidative burst, phytoalexins and pathogenesis-related proteins) which prepare hosts to more successfully resist later, more severe infections [56–58]. The use of ISR to induce resistance in plants by application of exogenous (chemical or organic) inducers, has been used in integrated programs of disease management. Preinoculation of sour orange (*Citrus aurantium*) seedlings with a hypovirulent isolate of *Phytophthora Citrus* root rot protected them from later infections [59]. Li et al. [60] have also demonstrated the effects chemical inducers on resistance of *Citrus* groves to HLB disease of *Citrus*. Over-expression of an *Arabidopsis* gene (a positive regulator of ISR) in transgenic "Duncan" grapefruit and "Hamlin" sweet orange increased their resistance to *Citrus* canker [61]. Although ISR may be a useful alternative for disease control, it has to be cautiously assessed. In some cases the use of ISR compounds may not provide the expected protection against disease: for example, spraying ISRs onto

effectors, as verified for other plant-Phytoplasma interactions [16].

sweet orange plants did not reduced incidence of *Citrus* canker [59].

giving a first unique insight into the biology of a "silent" infection.

**110**

**6. Conclusions**

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

SLE has been the beneficiary of CNPq productivity grants (grant nos. 309221/2013-7 & 309845/2016-5).

The authors would like to thanks Vale S.A. for financing of the project.

Thanks to SQU for partial support of the work through the grant EG/AGR/ CROP/16/01.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Philip Donkersley1 \*, Farley W.S. Silva<sup>2</sup> , Murilo S. Alves3 , Claudine M. Carvalho3 , Abdullah M. Al-Sadi4 and Simon L. Elliot2

1 Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom

2 Department of Entomology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

3 Department of Phytopathology, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

4 Crop Sciences, Sultan Qaabos University, Oman

\*Address all correspondence to: donkersleyp@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

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*Candidatus* Liberibacter asiaticus in the Asian *Citrus* psyllid, *Diaphorina citri* Kuwayama. Journal of Microbiological

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**117**

**Chapter 8**

**Abstract**

management

**1. Introduction**

Emerging Bacterial Disease

and Management

manage the leaf scald disease in sugarcane.

(Leaf Scald) of Sugarcane in

China: Pathogenesis, Diagnosis,

*Muralidharan Govindaraju, Yisha Li and Muqing Zhang*

Sugarcane is the major industrial crop grown in tropical and sub-tropical regions in China. More than 100 sugarcane diseases are identified around the globe; half have been reported in China. Many varieties of sugarcane were replaced due to the infection of new pathogenic disease. Recently, leaf scald was found in China, which is one of the major sugarcane diseases also seriously affecting growth of sugarcane. Several isolates were recovered and identified using ELISA and PCR assays from the symptomatic leaf samples in Guangxi, China. The genomes of our isolates from *X. albilineans* were re-sequenced and revealed that rpf gene encoded regulation of pathogenicity factors mainly involved in the pathogenesis of sugarcane. The disease is mainly transferred through seed cane. In the past, hot water treatment was used to manage the disease. Healthy seed cane from resistant cultivars could effectively

**Keywords:** sugarcane, leaf scald, *X. albilineans*, pathogenesis, bacterial disease,

Sugarcane (*Saccharum* spp.) is an industrially important crop of tropical and sub-tropical regions cultivated mainly for production of sucrose, biofuel, and ethanol [1]. China is one of the third largest sugarcane (*Saccharum officinarum* L.)-producing countries, followed by Brazil and India [2]. In the 1980s, Fujian and Guangdong were the two major sugarcane-producing provinces in China. Due to social, economic and environmental factors, major sugarcane-producing areas were moved to Guangxi and Yunnan provinces which accounts for 64 and 24% of total sugarcane-growing regions during 2015/2016. Since then, approximately 60% of the total cane yield is produced in Guangxi [2]. Sugarcane is the host of many serious plant pathogens that can mainly affect cane production and yield. In sugarcane, more than 100 pathogens have been reported to cause disease, Including fungi, bacteria, phytoplasma, and virus [3]. Several of these diseases are considered to be the most severe threat to sugarcane production in Guangxi, especially leaf scald disease. Sugarcane leaf scald was first reported in the 1980s in Taiwan, China [4, 5], and recently found in 2015 in Guangxi, China. The pathogen from the recovered isolates was identified to be *Xanthomonas albilineans*.

#### **Chapter 8**

*Plant Diseases-Current Threats and Management Trends*

[62] Rojas EI, Rehner SA, Samuels GJ, Van Bael SA, Herre EA, Cannon P, et al. *Colletotrichum gloeosporioides* s.l. associated with *Theobroma cacao* and other plants in Panamá: Multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes. Mycologia.

2010;**102**(6):1318-1338. DOI:

DOI: 10.1016/j.virol.2016.11.018

in the plant pathogen *Ralstonia solanacearum*. PLoS Pathogens. 2016;**12**(10):e1005939. DOI: 10.1371/

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[64] Peyraud R, Cottret L, Marmiesse L, Gouzy J, Genin S. A resource allocation trade-off between virulence and

proliferation drives metabolic versatility

[63] Pérez-Cañamás M, Blanco-Pérez M, Forment J, Hernández C. *Nicotiana benthamiana* plants asymptomatically infected by *Pelargonium* line pattern virus show unusually high accumulation of viral small RNAs that is neither associated with DCL induction nor RDR6 activity. Virology. 2017;**501**:136-146.

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[55] Heil M, Bostock RM. Induced systemic resistance (ISR) against pathogens in the context of induced plant defences. Annals of Botany. 2002;**89**(5):503-512. DOI: 10.1093/aob/

[56] Phytoalexins KJ, Metabolism S. Stress metabolism, and disease resistance in plants. Annual Review of Phytopathology. 2003;**33**:275-297

[57] Lamb C, Dixon RA. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology. 2002;**48**:251-275. DOI: 10.1146/annurev.arplant.48.1.251

[58] van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defenserelated proteins in infected plants. Annual Review of Phytopathology. 2006;**44**:135-162. DOI: 10.1146/annurev.

[59] Graham JH, Colburn GC, Chung KR, Cubero J. Protection of *Citrus* roots against infection by *Phytophthora* spp. by hypovirulent *P. nicotianae* is not related to induction of systemic acquired resistance. Plant and Soil. 2012;**358**: 39-49. DOI: 10.1007/s11104-011-1119-x

[60] Li J, Trivedi P, Wang N. Field evaluation of plant defense inducers for the control of *Citrus* Huanglongbing. Phytopathology. 2015;**106**:37-47. DOI:

[61] Zhang X, Francis MI, Dawson WO, Graham JH, Orbović V, Triplett EW, et al. Over-expression of the *Arabidopsis* NPR1 gene in *Citrus* increases resistance to *Citrus* canker. European Journal of Plant Pathology. 2010;**128**(1):91-100. DOI: 10.1007/s10658-010-9633-x

10.1094/phyto-08-15-0196-r

phyto.44.070505.143425

10.1093/ee/21.3.578

mcf076

**116**

## Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis, and Management

*Muralidharan Govindaraju, Yisha Li and Muqing Zhang*

#### **Abstract**

Sugarcane is the major industrial crop grown in tropical and sub-tropical regions in China. More than 100 sugarcane diseases are identified around the globe; half have been reported in China. Many varieties of sugarcane were replaced due to the infection of new pathogenic disease. Recently, leaf scald was found in China, which is one of the major sugarcane diseases also seriously affecting growth of sugarcane. Several isolates were recovered and identified using ELISA and PCR assays from the symptomatic leaf samples in Guangxi, China. The genomes of our isolates from *X. albilineans* were re-sequenced and revealed that rpf gene encoded regulation of pathogenicity factors mainly involved in the pathogenesis of sugarcane. The disease is mainly transferred through seed cane. In the past, hot water treatment was used to manage the disease. Healthy seed cane from resistant cultivars could effectively manage the leaf scald disease in sugarcane.

**Keywords:** sugarcane, leaf scald, *X. albilineans*, pathogenesis, bacterial disease, management

#### **1. Introduction**

Sugarcane (*Saccharum* spp.) is an industrially important crop of tropical and sub-tropical regions cultivated mainly for production of sucrose, biofuel, and ethanol [1]. China is one of the third largest sugarcane (*Saccharum officinarum* L.)-producing countries, followed by Brazil and India [2]. In the 1980s, Fujian and Guangdong were the two major sugarcane-producing provinces in China. Due to social, economic and environmental factors, major sugarcane-producing areas were moved to Guangxi and Yunnan provinces which accounts for 64 and 24% of total sugarcane-growing regions during 2015/2016. Since then, approximately 60% of the total cane yield is produced in Guangxi [2]. Sugarcane is the host of many serious plant pathogens that can mainly affect cane production and yield. In sugarcane, more than 100 pathogens have been reported to cause disease, Including fungi, bacteria, phytoplasma, and virus [3]. Several of these diseases are considered to be the most severe threat to sugarcane production in Guangxi, especially leaf scald disease. Sugarcane leaf scald was first reported in the 1980s in Taiwan, China [4, 5], and recently found in 2015 in Guangxi, China. The pathogen from the recovered isolates was identified to be *Xanthomonas albilineans*.

#### **1.1 Economic influence of leaf scald disease in sugarcane industry**

Leaf scald disease (**Figure 1**) is also known as leaf burning disease, and it is caused by *X. albilineans* (Ashby) Dowson [3]. The major host plants mainly include *S. officinarum*, *Zea mays*, *Panicum antidotale*, *Bambusa vulgaris*, *Pennisetum purpureum*, and *Paspalum conjugatum*. The disease is mainly distributed in Australia, the USA, the Philippines, Myanmar, Thailand, Java, Laos, and Vietnam [6, 7]. Now, it is the most important quarantine disease in Taiwan, Guangxi, Guangdong, Yunnan, Fujian, Jiangxi, and Hainan in China [8]. Leaf scald was the reason for major losses in sugarcane at the beginning of the century when noble canes, *Saccharum officinarum*, was cultivated [9]. The major impact of the disease was reduced by the cultivation of interspecific hybrids. The susceptible cultivars were rapidly destroyed. Sugarcane cultivars resistant to leaf scald disease include F156, F160, F170, F173, and NCO310 in Taiwan, China. Other varieties resistant to this disease include Q42, Q50, Q98, Q813, P0J36, POJ2725, CP807, CP29–CP116, Co290, Co301, Co331, Co421, and B34104. The varieties susceptible to leaf scald disease are CP29–CP291, Co281, Co419, Co7301, Q44, Q63, Q66, B34104, B37161, B070, GT 46, GT 06–2081, GT 08–1589, LC 03–1137, and ROC1 [7, 10].

#### **1.2 Symptomology**

The major characteristic symptoms of leaf scald disease are divided into three phases: latent, chronic, and acute phases.

#### *1.2.1 Latent phase*

During this period, infected plants do not show any symptoms that occur in tolerant varieties and under favorable conditions for growth of plant. Stress can activate the infected plant to pass from the phase of latent into chronic or acute phase.

**119**

**Figure 2.**

*Chronic phase of infection with pencil line stripe.*

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis…*

lessons may be noticed in severely affected stalks of plants (**Figure 2**).

show the chronic streaks on re-tillering of diseased stalks [9].

The chronic phase is characterized by "white pencil line" stripe 1–2 mm wide and patches of chlorotic tissue on leaves, side shooting and burning of leaf tips. At a later stage, the margins' stripe may become diffuse, and a red pencil line may be formed in the middle of the stripe. In dry weather, the leaf stripes dry out from the leaf apex to the margin, and finally the entire leaf become wilt. Infected plants exhibit shorter internodes, and the node of the stalk produces small tillerings, and a leaf of the tillerings shows white streaks. The disease-infected sections of stalks show reddening, and discolored vessels can pass through the internodes. Necrotic

In the acute phase of the disease, plant dies without showing any major symp-

LSD symptoms were recorded every month in the field. Disease severity was rated according to the procedure described [11]. However, all inoculated sugarcane stalks were rated individually, symptom severity ranging from 0 to 5. The ratings were used to calculate mean disease severity (DS): DS = [(1 × FL + 2 × ML + 3 × CB + 4 × N + 5 × D)/5 × T] 100. However, FL = number of stalks with one or two pencil-line streaks (rating 1), ML = number of stalks with more than two pencil-line streaks (rating 2),

toms. The infected stalks section does not show reddening of the vessels and tillerings joined into main stalk (**Figure 3**). It occurs mainly in drought condition of sugarcane growing period. In physiological water shortage condition, the leaves

*DOI: http://dx.doi.org/10.5772/intechopen.88333*

*1.2.2 Chronic phase*

*1.2.3 Acute phase*

**1.3 Field assessment**

**Figure 1.** *Major sugarcane areas affected by leaf scald in China.*

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88333*

#### *1.2.2 Chronic phase*

*Plant Diseases-Current Threats and Management Trends*

and ROC1 [7, 10].

*1.2.1 Latent phase*

**1.2 Symptomology**

phases: latent, chronic, and acute phases.

*Major sugarcane areas affected by leaf scald in China.*

**1.1 Economic influence of leaf scald disease in sugarcane industry**

Leaf scald disease (**Figure 1**) is also known as leaf burning disease, and it is caused by *X. albilineans* (Ashby) Dowson [3]. The major host plants mainly include *S. officinarum*, *Zea mays*, *Panicum antidotale*, *Bambusa vulgaris*, *Pennisetum purpureum*, and *Paspalum conjugatum*. The disease is mainly distributed in Australia, the USA, the Philippines, Myanmar, Thailand, Java, Laos, and Vietnam [6, 7]. Now, it is the most important quarantine disease in Taiwan, Guangxi, Guangdong, Yunnan, Fujian, Jiangxi, and Hainan in China [8]. Leaf scald was the reason for major losses in sugarcane at the beginning of the century when noble canes, *Saccharum officinarum*, was cultivated [9]. The major impact of the disease was reduced by the cultivation of interspecific hybrids. The susceptible cultivars were rapidly destroyed. Sugarcane cultivars resistant to leaf scald disease include F156, F160, F170, F173, and NCO310 in Taiwan, China. Other varieties resistant to this disease include Q42, Q50, Q98, Q813, P0J36, POJ2725, CP807, CP29–CP116, Co290, Co301, Co331, Co421, and B34104. The varieties susceptible to leaf scald disease are CP29–CP291, Co281, Co419, Co7301, Q44, Q63, Q66, B34104, B37161, B070, GT 46, GT 06–2081, GT 08–1589, LC 03–1137,

The major characteristic symptoms of leaf scald disease are divided into three

During this period, infected plants do not show any symptoms that occur in tolerant varieties and under favorable conditions for growth of plant. Stress can activate the infected plant to pass from the phase of latent into chronic or acute phase.

**118**

**Figure 1.**

The chronic phase is characterized by "white pencil line" stripe 1–2 mm wide and patches of chlorotic tissue on leaves, side shooting and burning of leaf tips. At a later stage, the margins' stripe may become diffuse, and a red pencil line may be formed in the middle of the stripe. In dry weather, the leaf stripes dry out from the leaf apex to the margin, and finally the entire leaf become wilt. Infected plants exhibit shorter internodes, and the node of the stalk produces small tillerings, and a leaf of the tillerings shows white streaks. The disease-infected sections of stalks show reddening, and discolored vessels can pass through the internodes. Necrotic lessons may be noticed in severely affected stalks of plants (**Figure 2**).

#### *1.2.3 Acute phase*

In the acute phase of the disease, plant dies without showing any major symptoms. The infected stalks section does not show reddening of the vessels and tillerings joined into main stalk (**Figure 3**). It occurs mainly in drought condition of sugarcane growing period. In physiological water shortage condition, the leaves show the chronic streaks on re-tillering of diseased stalks [9].

#### **1.3 Field assessment**

LSD symptoms were recorded every month in the field. Disease severity was rated according to the procedure described [11]. However, all inoculated sugarcane stalks were rated individually, symptom severity ranging from 0 to 5. The ratings were used to calculate mean disease severity (DS): DS = [(1 × FL + 2 × ML + 3 × CB + 4 × N + 5 × D)/5 × T] 100. However, FL = number of stalks with one or two pencil-line streaks (rating 1), ML = number of stalks with more than two pencil-line streaks (rating 2),

**Figure 2.** *Chronic phase of infection with pencil line stripe.*

**Figure 3.** *Acute phase of infected plants leads to death.*

CB = number of stalks with leaf chlorosis or bleaching (rating 3), N = number of stalks with leaf necrosis (rating 4), D = number of dead stalks or stalks with side shooting (rating 5), and T = total number of stalks. The rating of 5 was attributed to stalks with dead inoculated leaves 1 month after inoculation [11].

#### **1.4 Phylogenetic**

The multilocus sequence analysis (MLSA) of 119 strains of *Xanthomonas* genus is distributed into two uneven groups, with group 2 containing all but five species, namely, *X. albilineans, X. theicola, X. sacchari, X. translucens*, and *X. hyacinthi*, which were clustered into group 1 [12].

Three serovars associated with antigenic variations within *X. albilineans* were detected using three antisera (polyclonal antibodies) method against strains from three different geographical locations [13]. Serovars of 215 strains from 28 locations worldwide are affected by sugarcane leaf scald disease, and the distributed strains are divided into three groups according to serotype: (i) serotype 1 is the largest group, with strains from various geographic locations; (ii) serotype 2 consists of strains from tropical African countries; and (iii) serotype 3 contains strains from Caribbean islands (Fiji and Sri Lanka). This serological characterization of *X. albilineans* strains has been confirmed with monoclonal antibodies of 38 strains from different worldwide locations [14].

#### **2. Pathogenesis**

The pathogen is limited mainly to the leaf and stalk vascular bundles that are often partly or completely blocked with a gum-like substance. The organism may invade the parenchyma cells between the vascular bundles and cause reddened pockets of gum. No symptom of sugarcane plants can therefore constitute inoculum

**121**

(0.5 μg mL<sup>−</sup><sup>1</sup>

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis…*

sources for crop contamination. In addition, various epidemiological factors play a major role in field contamination. Leaf scald is spread by harvesters, hand knives, and infected setts by planting [9]. The pathogen found in the rhizosphere of infected roots has more possible transmission by root contact [15]. Leaf scald disease can also affect many other grasses which are alternate hosts for the disease. High moisture and temperature are the most favorable condition for the disease transmission.

The genome sequence of *X. albilineans* shows that the genes are effectively involved in pathogenesis. These genes include a cluster of genes called regulation of pathogenicity factors (rpf) responsible for the biosynthesis of a small diffusible signaling molecule. Diffusible signaling factor (DSF) encoded by RpfF has more similarities to long-chain fatty acyl CoA ligases [16, 17]. DSF quorum sensing and disruption of gene rpfF

resulted in reduced virulence in different xanthomonads (*Xanthomonas spp.*), such as *X. axonopodis* pv. citri, *X. campestris* pv. campestris, and *X. oryzae* pv. oryzae [17, 18]. The rpf genes only control production of biofilm and other mechanisms involved in surface attachment of *X. albilineans*. A core group of genes, including *rpfF*, *rpfC* and *rpf G*, has played broader roles in gene regulation other than the transduction of DSF signals in *X. axonopodis spp.* [19]. The pathogen of leaf scald disease, *X. albilineans*, lacks type III secretion system (T3SS), which is found in most of pathogenic xanthomonads and acts as pathogenicity effectors in plant cells. *X. albilineans* also lacks all genes involved in the formation of biofilm, an important factor in virulence of plant pathogenic bacteria. TonB-dependent transporters (TBDTs) are the important transporters involved

in nutrient uptake [20] and also involved in iron or vitamin B12 uptake. These transporters are to facilitate the uptake of carbohydrates present in low amounts on the leaf surfaces [21, 22]. Genomes of several species of *Xanthomonas* are known to have high representation of TBDT genes, and it is functionally associated with

During phyllosphere colonization, *Xanthomonas* encounters nutrient-limited environment on the leaf surfaces. Due to these conditions, TBDT has transported sucrose available in the phyllosphere [24]. Similarly, the genome of *X. albilineans* has 35 putative TBDT genes, and it is involved in the transport of plant cell wall

Albicidin is a major phytotoxic compound specifically synthesized by the Gramnegative bacteria *X. albilineans* and plays an important role in pathogenicity [27]. It causes leaf scald in sugarcane [28]. The molecular target of albicidin is DNA gyrase (topoisomerase II) which is essential for DNA replication in bacteria. In planta, albicidin acts as a potent DNA gyrase inhibitor, thus blocking plastid development [29]. Albicidin (**Figure4**) also has potent antibacterial activity which inhibits the growth of several positive and negative bacteria at nanomolar range with low minimal inhibitory

advantage to *X. albilineans* against other bacteria within the xylem vessels of sugarcane. However, the entire 49-kb albicidin biosynthesis gene cluster was cloned and sequenced from *X. albilineans* (Xa23R1) [28]. This cluster is included in a genomic region of XALB1, and it contains three polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes, as well as several putative modifying

*X. albilineans* strain Xa23R1, namely, XALB2 and XALB3, were found to be involved

resistance and regulatory genes. Two additional 3-kb genomic regions of

) [30], *Salmonella enteritidis*

) [31]. Albicidin gives greater

*DOI: http://dx.doi.org/10.5772/intechopen.88333*

**2.1 Genes and diseases**

pathogenicity of the bacterium [23].

derived nutrients like maltose, xylan, pectin, etc. [24–26].

concentration (MIC), e.g., *Escherichia coli* (0.063 μg mL<sup>−</sup><sup>1</sup>

), and *Staphylococcus aureus* (4.0 μg mL<sup>−</sup><sup>1</sup>

**2.2 Albicidin production and pathogenicity**

sources for crop contamination. In addition, various epidemiological factors play a major role in field contamination. Leaf scald is spread by harvesters, hand knives, and infected setts by planting [9]. The pathogen found in the rhizosphere of infected roots has more possible transmission by root contact [15]. Leaf scald disease can also affect many other grasses which are alternate hosts for the disease. High moisture and temperature are the most favorable condition for the disease transmission.

#### **2.1 Genes and diseases**

*Plant Diseases-Current Threats and Management Trends*

CB = number of stalks with leaf chlorosis or bleaching (rating 3), N = number of stalks with leaf necrosis (rating 4), D = number of dead stalks or stalks with side shooting (rating 5), and T = total number of stalks. The rating of 5 was attributed to stalks with

The multilocus sequence analysis (MLSA) of 119 strains of *Xanthomonas* genus is distributed into two uneven groups, with group 2 containing all but five species, namely, *X. albilineans, X. theicola, X. sacchari, X. translucens*, and *X. hyacinthi*, which

Three serovars associated with antigenic variations within *X. albilineans* were detected using three antisera (polyclonal antibodies) method against strains from three different geographical locations [13]. Serovars of 215 strains from 28 locations worldwide are affected by sugarcane leaf scald disease, and the distributed strains are divided into three groups according to serotype: (i) serotype 1 is the largest group, with strains from various geographic locations; (ii) serotype 2 consists of strains from tropical African countries; and (iii) serotype 3 contains strains from Caribbean islands (Fiji and Sri Lanka). This serological characterization of

*X. albilineans* strains has been confirmed with monoclonal antibodies of 38 strains

The pathogen is limited mainly to the leaf and stalk vascular bundles that are often partly or completely blocked with a gum-like substance. The organism may invade the parenchyma cells between the vascular bundles and cause reddened pockets of gum. No symptom of sugarcane plants can therefore constitute inoculum

dead inoculated leaves 1 month after inoculation [11].

**1.4 Phylogenetic**

**Figure 3.**

**2. Pathogenesis**

were clustered into group 1 [12].

*Acute phase of infected plants leads to death.*

from different worldwide locations [14].

**120**

The genome sequence of *X. albilineans* shows that the genes are effectively involved in pathogenesis. These genes include a cluster of genes called regulation of pathogenicity factors (rpf) responsible for the biosynthesis of a small diffusible signaling molecule. Diffusible signaling factor (DSF) encoded by RpfF has more similarities to long-chain fatty acyl CoA ligases [16, 17]. DSF quorum sensing and disruption of gene rpfF resulted in reduced virulence in different xanthomonads (*Xanthomonas spp.*), such as *X. axonopodis* pv. citri, *X. campestris* pv. campestris, and *X. oryzae* pv. oryzae [17, 18]. The rpf genes only control production of biofilm and other mechanisms involved in surface attachment of *X. albilineans*. A core group of genes, including *rpfF*, *rpfC* and *rpf G*, has played broader roles in gene regulation other than the transduction of DSF signals in *X. axonopodis spp.* [19]. The pathogen of leaf scald disease, *X. albilineans*, lacks type III secretion system (T3SS), which is found in most of pathogenic xanthomonads and acts as pathogenicity effectors in plant cells. *X. albilineans* also lacks all genes involved in the formation of biofilm, an important factor in virulence of plant pathogenic bacteria.

TonB-dependent transporters (TBDTs) are the important transporters involved in nutrient uptake [20] and also involved in iron or vitamin B12 uptake. These transporters are to facilitate the uptake of carbohydrates present in low amounts on the leaf surfaces [21, 22]. Genomes of several species of *Xanthomonas* are known to have high representation of TBDT genes, and it is functionally associated with pathogenicity of the bacterium [23].

During phyllosphere colonization, *Xanthomonas* encounters nutrient-limited environment on the leaf surfaces. Due to these conditions, TBDT has transported sucrose available in the phyllosphere [24]. Similarly, the genome of *X. albilineans* has 35 putative TBDT genes, and it is involved in the transport of plant cell wall derived nutrients like maltose, xylan, pectin, etc. [24–26].

#### **2.2 Albicidin production and pathogenicity**

Albicidin is a major phytotoxic compound specifically synthesized by the Gramnegative bacteria *X. albilineans* and plays an important role in pathogenicity [27]. It causes leaf scald in sugarcane [28]. The molecular target of albicidin is DNA gyrase (topoisomerase II) which is essential for DNA replication in bacteria. In planta, albicidin acts as a potent DNA gyrase inhibitor, thus blocking plastid development [29]. Albicidin (**Figure4**) also has potent antibacterial activity which inhibits the growth of several positive and negative bacteria at nanomolar range with low minimal inhibitory concentration (MIC), e.g., *Escherichia coli* (0.063 μg mL<sup>−</sup><sup>1</sup> ) [30], *Salmonella enteritidis* (0.5 μg mL<sup>−</sup><sup>1</sup> ), and *Staphylococcus aureus* (4.0 μg mL<sup>−</sup><sup>1</sup> ) [31]. Albicidin gives greater advantage to *X. albilineans* against other bacteria within the xylem vessels of sugarcane.

However, the entire 49-kb albicidin biosynthesis gene cluster was cloned and sequenced from *X. albilineans* (Xa23R1) [28]. This cluster is included in a genomic region of XALB1, and it contains three polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes, as well as several putative modifying resistance and regulatory genes. Two additional 3-kb genomic regions of *X. albilineans* strain Xa23R1, namely, XALB2 and XALB3, were found to be involved

**Figure 4.** *Structure of albicidin.*

in albicidin production. XALB2 has a single gene coding for a phosphopantetheinyl transferase required for posttranslational activation of PKS or NRPS enzymes [32]. XALB3 has a single gene coding for protein (HtpG) in albicidin production, which has not been elucidated [33].

#### **2.3 Disease cycle**

*Xanthomonas albilineans* can spread from inoculum sources to contaminated field of sugarcane and affect healthy sugarcane under the impact of various climatic conditions (**Figure 5**) [34]. The pathogen then colonizes the surface of the leaf, enters through the stomata, and progresses within the xylem, and symptoms may appear in infected plants. Pathogen can move into the stalk and then infect the stool showing scalding from the leaves [35]. The pathogen can be transmitted mechanically by harvesting equipment (harvesters) and the infected cane setts.

#### **3. Diagnosis**

#### **3.1 Immunoassay**

Various diagnostic methods are employed for detection of *X. albilineans*, including isolation on selective media and biochemical, immunological, and

**123**

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis…*

molecular assays. ELISA was the most sensitive method and resulted in the detection of bacteria at the low titer [36]. Although isolation of *X. albilineans* with selective media is more time-consuming than other methods, it has proven to be very efficient in detecting the pathogen in symptomless and diseased plants [37]. DAC-ELISA and dot blot techniques were standardized to detect the bacterium in

Polymerase chain reaction (PCR) protocols have been developed to detect *X. albilineans* in diseased stalks. The primer sets Ala4/L1 [39] and PGBL1/PGBL2 [40] were designed based on ITS region between the 16S and 23S rRNA genes of *X. albilineans*. PCR-based detection was further improved by *X. albilineans* specific amplification of the region between the 16S rRNA-tRNAaIa -tRNAiIe - 23S rRNA gene by a nested PCR reaction [41]. Loop-mediated isothermal amplification (LAMP) was also employed for the detection of *X. albilineans* [42, 43]. It is an auto-cycling strand displacement DNA synthesis technique that involves the use of large fragment of DNA polymerase and a set of six primers [44], and it enables the synthesis of larger amounts of both DNA and by-products, e.g., hydroxy naphthol blue (HNB) [45, 46]. Quantitative PCR (qPCR) is a highly effective and accurate

Most of sugarcane diseases can be controlled through the use of disease-free seed cane. Hot-water treatments are used to disinfect planting material (seed cane). Before planting, soaking in ambient-temperature running water for 40 h followed by 3–4 h at 50°C is used to manage leaf scald bacteria, and it can provide 95%

This approach is mainly developed to target factors that are responsible for pathogenicity other than toxins. The molecular modification in the host provides them resistance to pathogenicity factors and the factor inactivates by binding of hormones and enzymes. However, foreign genes' inactivation was carried out by gene silencing, nucleases, and coating proteins [48, 49]. The production of host cell surface components interferes with identification of the host and attachment to

CRISPR-Cas systems are involved in phage and plasmid defense, thus limiting HGT. The genome sequence of *X. albilineans* strain (GPE PC73) unveils the presence of two different systems named CRISPR-1 and CRISPR-2 [50]. The CRISPR-2 is associated with six cas genes (cas1, cas3, csy1, csy2, csy3, and csy4) and contains 28-bp repeats. CRISPR-2 is shared by the four strains (GPEPC73 and Xa23R1 from *X. albilineans*, GPE 39 and MUS 060 from *X. pseudalbilineans*) [50, 51]. Although, CRISPR-2 nucleic acid sequences of the repeats are 100% identical among the four

Different resistant mechanisms on albicidin have been reported, including the nucleoside transporter Tsx, for which mutations have been described to import albicidin, or the endopeptidase AlbD from Pantoea dispersa [53–55], which cleaves albicidin

strains, thus confirming the common origin of this locus [52].

*DOI: http://dx.doi.org/10.5772/intechopen.88333*

method for the detection of leaf scald disease [47].

infected canes [38].

**3.2 Molecular method**

**4. Management strategy**

**4.1 Hot water treatment**

control efficacy [10].

**4.2 Molecular approach**

host cells by the pathogen.

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88333*

molecular assays. ELISA was the most sensitive method and resulted in the detection of bacteria at the low titer [36]. Although isolation of *X. albilineans* with selective media is more time-consuming than other methods, it has proven to be very efficient in detecting the pathogen in symptomless and diseased plants [37]. DAC-ELISA and dot blot techniques were standardized to detect the bacterium in infected canes [38].

#### **3.2 Molecular method**

*Plant Diseases-Current Threats and Management Trends*

in albicidin production. XALB2 has a single gene coding for a phosphopantetheinyl transferase required for posttranslational activation of PKS or NRPS enzymes [32]. XALB3 has a single gene coding for protein (HtpG) in albicidin production, which

*Xanthomonas albilineans* can spread from inoculum sources to contaminated field of sugarcane and affect healthy sugarcane under the impact of various climatic conditions (**Figure 5**) [34]. The pathogen then colonizes the surface of the leaf, enters through the stomata, and progresses within the xylem, and symptoms may appear in infected plants. Pathogen can move into the stalk and then infect the stool showing scalding from the leaves [35]. The pathogen can be transmitted mechani-

cally by harvesting equipment (harvesters) and the infected cane setts.

Various diagnostic methods are employed for detection of *X. albilineans*, including isolation on selective media and biochemical, immunological, and

**122**

has not been elucidated [33].

*Infection cycle of leaf scald pathogen.*

**2.3 Disease cycle**

**Figure 5.**

**Figure 4.**

*Structure of albicidin.*

**3. Diagnosis**

**3.1 Immunoassay**

Polymerase chain reaction (PCR) protocols have been developed to detect *X. albilineans* in diseased stalks. The primer sets Ala4/L1 [39] and PGBL1/PGBL2 [40] were designed based on ITS region between the 16S and 23S rRNA genes of *X. albilineans*. PCR-based detection was further improved by *X. albilineans* specific amplification of the region between the 16S rRNA-tRNAaIa -tRNAiIe - 23S rRNA gene by a nested PCR reaction [41]. Loop-mediated isothermal amplification (LAMP) was also employed for the detection of *X. albilineans* [42, 43]. It is an auto-cycling strand displacement DNA synthesis technique that involves the use of large fragment of DNA polymerase and a set of six primers [44], and it enables the synthesis of larger amounts of both DNA and by-products, e.g., hydroxy naphthol blue (HNB) [45, 46]. Quantitative PCR (qPCR) is a highly effective and accurate method for the detection of leaf scald disease [47].

#### **4. Management strategy**

#### **4.1 Hot water treatment**

Most of sugarcane diseases can be controlled through the use of disease-free seed cane. Hot-water treatments are used to disinfect planting material (seed cane). Before planting, soaking in ambient-temperature running water for 40 h followed by 3–4 h at 50°C is used to manage leaf scald bacteria, and it can provide 95% control efficacy [10].

#### **4.2 Molecular approach**

This approach is mainly developed to target factors that are responsible for pathogenicity other than toxins. The molecular modification in the host provides them resistance to pathogenicity factors and the factor inactivates by binding of hormones and enzymes. However, foreign genes' inactivation was carried out by gene silencing, nucleases, and coating proteins [48, 49]. The production of host cell surface components interferes with identification of the host and attachment to host cells by the pathogen.

CRISPR-Cas systems are involved in phage and plasmid defense, thus limiting HGT. The genome sequence of *X. albilineans* strain (GPE PC73) unveils the presence of two different systems named CRISPR-1 and CRISPR-2 [50]. The CRISPR-2 is associated with six cas genes (cas1, cas3, csy1, csy2, csy3, and csy4) and contains 28-bp repeats. CRISPR-2 is shared by the four strains (GPEPC73 and Xa23R1 from *X. albilineans*, GPE 39 and MUS 060 from *X. pseudalbilineans*) [50, 51]. Although, CRISPR-2 nucleic acid sequences of the repeats are 100% identical among the four strains, thus confirming the common origin of this locus [52].

Different resistant mechanisms on albicidin have been reported, including the nucleoside transporter Tsx, for which mutations have been described to import albicidin, or the endopeptidase AlbD from Pantoea dispersa [53–55], which cleaves albicidin into two inactive fragments [56]. Another strategy is drug binding that counteracts the antibacterial effect of albicidin tetracycline-binding protein (TetR family) [57] or thiostrepton-binding protein (MerR family) [58]. The albicidin-binding protein AlbA from *Klebsiella oxytoca* [59] and AlbB from *Alcaligenes denitrificans* [60] provide protective effects for survival of the host strains. However, far-ultraviolet (UV) spectroscopy has indicated a mostly α-helical structure for AlbA [61], and amino acid residue His125 has played a vital role in albicidin binding [61, 62].

#### **4.3 Genetic approach**

The most potent method to prevent or manage leaf scald disease is the development and planting of resistant cultivars [3]. However, accurate determination of the resistant level of genotype against *X. albilineans* is most important in the cultivar selection program for leaf scald by artificial inoculation tests. The erratic symptom expression failed to accurately detect susceptibility, and thus multiple field trials utilizing inoculation are needed. Under this scenario, the marker-assisted selection (MAS) breeding technique, which uses DNA marker(s) linked to useful trait(s), has greater advantage in selecting clones resistant to leaf scald disease [63]. The transgenic sugarcane plants against *Xanthomonas albilineans* were produced by mediating albicidin detoxification (*albD*) gene through microprojectile bombardment [64].

#### **4.4 Alternative control**

#### *4.4.1 Chemotherapy method*

Spray of antibiotics such as streptomycin + tetracycline (60 g/ha/500 l water) at 2-week intervals was found to efficiently manage the pathogen in the field at 2 months after planting [65]. In preliminary stage, spraying of these antibiotics reduces the severity of leaf scald.

#### *4.4.2 Biocontrol method*

*G. diazotrophicus* may play a major role in defense against pathogens of sugarcane. It inhibited in vitro growth of leaf scald pathogen *Xanthomonas albilineans* [66, 67]. In addition, *G. diazotrophicus*-inoculated sugarcane stems were resistant to infection by *X. albilineans* [68]. Lactic acid bacteria (LAB) may be a biological alternative approach for leaf scald disease in sugarcane, and it produces antimicrobial peptides called bacteriocins and other substances, such as hydrogen peroxide, lactic acid, and reuterin, which are effective against several Gram-positive and Gram-negative bacteria. Antimicrobial peptides are alternatives for conventional pesticides and antibiotics, which are also used to treat against *X. albilineans* [69].

#### **4.5 Farmer service program**

Since 2000, healthy seed cane program has been developed by a government agency to protect the sugarcane diseases in China, further following recommendation to control or manage leaf scald disease.


**125**

**Author details**

Muralidharan Govindaraju, Yisha Li and Muqing Zhang\*

\*Address all correspondence to: mqzhang@ufl.edu

provided the original work is properly cited.

Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning, China

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis…*

3.The crop agronomist should employ to cover aspects of growing the crop, such as soils, fertilizer, and herbicides and also to monitor the performance of new

4.The Inspection Department for all growers' fields should ensure that disease levels are acceptable and that remedial action is implemented where required.

5.The efforts should be made to enhance awareness among the sugarcane farmers and strict vigilance by the quality control and regulatory authorities.

The leaf scald disease is becoming one of the serious threats to sugar industries in China. The infected plants should be removed completely to eliminate the seed cane carrying this disease in the field. Healthy seed cane was used from pathogenfree tissue culture plantlets (PTC). Better fertilization and irrigation practices may reduce the occurrence of this disease. A strict phytosanitary measure is needed to manage exchange of materials of sugarcane (seed cane) and propagation of disease.

The monitoring of fields for pest damage is also important.

*DOI: http://dx.doi.org/10.5772/intechopen.88333*

varieties.

**5. Conclusion**

*Emerging Bacterial Disease (Leaf Scald) of Sugarcane in China: Pathogenesis, Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88333*


#### **5. Conclusion**

*Plant Diseases-Current Threats and Management Trends*

**4.3 Genetic approach**

**4.4 Alternative control**

*4.4.2 Biocontrol method*

**4.5 Farmer service program**

tion to control or manage leaf scald disease.

disease-free parent seed material.

care of harvesting, transportation, and processing.

*4.4.1 Chemotherapy method*

reduces the severity of leaf scald.

into two inactive fragments [56]. Another strategy is drug binding that counteracts the antibacterial effect of albicidin tetracycline-binding protein (TetR family) [57] or thiostrepton-binding protein (MerR family) [58]. The albicidin-binding protein AlbA from *Klebsiella oxytoca* [59] and AlbB from *Alcaligenes denitrificans* [60] provide protective effects for survival of the host strains. However, far-ultraviolet (UV) spectroscopy has indicated a mostly α-helical structure for AlbA [61], and amino acid

The most potent method to prevent or manage leaf scald disease is the development and planting of resistant cultivars [3]. However, accurate determination of the resistant level of genotype against *X. albilineans* is most important in the cultivar selection program for leaf scald by artificial inoculation tests. The erratic symptom expression failed to accurately detect susceptibility, and thus multiple field trials utilizing inoculation are needed. Under this scenario, the marker-assisted selection (MAS) breeding technique, which uses DNA marker(s) linked to useful trait(s), has greater advantage in selecting clones resistant to leaf scald disease [63]. The transgenic sugarcane plants against *Xanthomonas albilineans* were produced by mediating albicidin detoxification (*albD*) gene through microprojectile bombardment [64].

Spray of antibiotics such as streptomycin + tetracycline (60 g/ha/500 l water) at 2-week intervals was found to efficiently manage the pathogen in the field at 2 months after planting [65]. In preliminary stage, spraying of these antibiotics

*G. diazotrophicus* may play a major role in defense against pathogens of sugarcane. It inhibited in vitro growth of leaf scald pathogen *Xanthomonas albilineans* [66, 67]. In addition, *G. diazotrophicus*-inoculated sugarcane stems were resistant to infection by *X. albilineans* [68]. Lactic acid bacteria (LAB) may be a biological alternative approach for leaf scald disease in sugarcane, and it produces antimicrobial peptides called bacteriocins and other substances, such as hydrogen peroxide, lactic acid, and reuterin, which are effective against several Gram-positive and Gram-negative bacteria. Antimicrobial peptides are alternatives for conventional pesticides and antibiotics, which are also used to treat against *X. albilineans* [69].

Since 2000, healthy seed cane program has been developed by a government agency to protect the sugarcane diseases in China, further following recommenda-

1.The Pest and Disease Department plays a vital role by ensuring to provide

2.The government agency should arrange training program for farmers and take

residue His125 has played a vital role in albicidin binding [61, 62].

**124**

The leaf scald disease is becoming one of the serious threats to sugar industries in China. The infected plants should be removed completely to eliminate the seed cane carrying this disease in the field. Healthy seed cane was used from pathogenfree tissue culture plantlets (PTC). Better fertilization and irrigation practices may reduce the occurrence of this disease. A strict phytosanitary measure is needed to manage exchange of materials of sugarcane (seed cane) and propagation of disease.

#### **Author details**

Muralidharan Govindaraju, Yisha Li and Muqing Zhang\* Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning, China

\*Address all correspondence to: mqzhang@ufl.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.

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Management of Plant

Disease

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Section 3
