Chapter II: Review of literature-

---------------------- --- --------- ---------------Chapter II: Review of literature- Extensive survey of the literature pertaining to the current r...
Author: Arnold Marsh
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Extensive survey of the literature pertaining to the current research topic has been carried out and review covered the following aspects of the study: chilli anthracnose, C. capsici, host-pathogen interaction and its management using abiotic, biotic inducers and by biological control agents like plant growth promoting rhizobacteria (PGPR). 2.1 Chilli anthracnose The fungus was reported for the first time in India by Sydow on chillies farm Coimbatore of Madras Presidency in 1913. Since then it has been reported and described from several parts of the World (Butler, 1918; Sydow, 1919; Dastur, 1921; Bertus, 1927; Thompson, 1928; Wallace, 1929; Higgins, 1931; Marchionatto, 1935; Hansford, 1938; Ling and Lin, 1944; Ramakrishnan, 1954; Bansal and Grover, 1969; Kenchaiah, 1975; Seaver et al., 1977; Thind and Jhooty, 1985; Mah, 1986; Meon and Nile, 1988; Mridha and Siddique, 1986; Simons, 1991; Sultana et al., 1992; Mannandhar et al., 1995; Roy et al., 1997; Sangchote et al., 1998; Sangchote, 1999; Qing et al., 2002). Later in 1957, Arx coined C. capsici as a synonym of C. dematium. Anthracnose of chilli, caused by C. capsici, is one of the most devastating seed-borne disease that accounts for up to 50% yield loss (Pakdeevaraporn et al., 2005) and during 1980’s the loss was about 84% (Thind and Jhooty, 1985). Choudhury (1957) observed that the disease has recorded 12-30% fruit loss in chilli growing states of India. Bansal and Grover (1969) during their studies on Capsicum varieties reported that the crop loss due to anthracnose disease was the extent of 10-35% of fruits in 1966 and 20-60% of fruits during 1967 in six districts of Punjab and Haryana. Similar field studies were conducted by Patil et al. (1993) in Maharashtra, India, to assess the losses due to dieback and fruit rot on Capsicum varieties, which revealed that cv. CA960 suffered less loss from dieback when compared to cv. Bhiwapur. Mathew et al. (1995) conducted a survey in Vellanikkara, Trichur, Kerala and reported alternaria leaf blight and dieback or fruit rot caused by C. capsici as a serious problem during the rainy season. However, when yield losses were compared, cultivars with large fruit with thick pericarp were affected less and gave a higher yield compared to cultivars with smaller fruits and thin pericarp. Colletotrichum capsici isolated from infected fruits and seeds proved the pathogenicity on chilli plants and fruits (Singh et al., 1977; Khaleeque and Khan, 1991; Manandhar et al., 1995). 15

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2.2 Characterization of anthracnose pathogen Colletotrichum capsici 2.2.1 Morphological variations Pathogenic fungi are known to contain almost invariably sub-specific varieties or strains, which are morphologically undetectable and may have variable infection capacities on host ranges (Western, 1971). Variation in host resistance is mainly due to variability in the pathogen population. Hence, studies on the extent of variation among plant pathogens are necessary for a better understanding of the pathogen as well as the disease they cause. It enables us to evolve better management strategies to control any particular disease. Misra and Dutta (1963) made a comparative study of single spore inoculation of two C. capsici isolates i.e., IB and ID collected from two widely separated localities in Bihar. Morphological differences between these isolates were observed when grown on different solid and liquid media. The mycelial growth of isolate IB on Richards agar was cottony white and fluffy without acervuli formation. In contrast, isolate ID showed entire mycelia growth covered with black stromatic bodies and pinkish masses of acervuli having abundant sporulation. Singh et al. (1977) reported the fungus excelled its vegetative growth on Czapek’s medium. However, the development of reproductive structures was excellent on potato dextrose agar (PDA). They found that Colletotrichum spp. expressed least growth on oat meal agar medium. Kadu (1977) reported that, Coon’s agar being a synthetic medium supported both growth and sporulation to the maximum over and above leaf and fruit decoction followed by potato dextrose agar. Morphological character of C. capsici isolated from brinjal was studied on PDA by Singh et al. (1973). The fungus showed initially white mycelial colonies changing to pinkish and then to brownish color and abundant acervuli within 5-6 days of growth and had numerous dark, septate, straight to bent and pointed setae measuring 105-305 µm, slightly curved conidia pointed at both the ends measuring 25.5-30.5 x 5-6 µm. Earlier work carried out by Solanki et al. (1974) showed that 35 °C as the optimum temperature for conidial germination. At 18 °C, conidia germinated only in hanging drops and did not germinate on 2% agar. Siddique et al. (1975) observed that different species of Colletotrichum showed maximum growth at 25 °C and minimum 16

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at 15 °C. Mazlan and Sariah (1980) observed cultures receiving continuous normal light and normal light alternating with UV showed better growth, whereas those exposed to alternating light and dark treatment produced better sporulation. The conidial germination and appressorial formation in C. capsici and C. gloeosporioides were greater on immature green and ripe fruits of pepper (C. annuum) variety. Conidial germination was significantly higher for both fungi with the increase in the concentration of sucrose and potassium chloride. Appressoria formation for C. capsici was highest when the concentration of sucrose was 10 mM. Appressoria formation was reduced and mycelia were formed for both fungi at higher sucrose concentration but not at 1-100 mM of potassium chloride (Manandhar et al., 1995). Significant variations in colony growth, sporulation rate, length of conidia and also length of setae on oat meal agar (OMA) medium at 25 °C were observed when Mathur et al. (2001) studied morphological variability of 12 isolates of C. graminicola collected from agricultural fields of Maharastra. Based on distinct colony characteristics, growth rate, conidial and appressorial morphology Than et al. (2008a) categorized Colletotrichum into five groups. Group 1 and 2 consisted of C. gloeosporioieds, group 3 and 4 contained C. acutatum and group 5 was C. capsici. The conidia were of 3 types comprising cylindrical, fusiform and falcate. The distinction among the groups in the size of conidia was very little and they were directly correlated with phylogenetic grouping. Oanh et al. (2004) characterized and distinguished 15 isolates of C. capsici and C. gloeosporoides into five groups based on their morphological characters and growth rate. Four groups of C. capsici were similar in conidial shape but differed in growth rate and mycelial color. Based on variations in cultural and morphological traits, Sharma et al. (2005) categorized 37 of isolates C. capsici into five groups. Most of the C. capsici isolates produced cottony, fluffy or suppressed colonies. However, they did not noticed any significant differences in shape and size of conidia among tested isolates. Akhtar and Singh (2007) studied the morphological variability among five monoconidial isolates of C. capsici from Sitargani, Pantnagar, Bilaspur, Kalinagar and Dineshpur of district Uddam singh Nagar, Uttaranchal. Differences in colony characters such as growth, shape, margin, color, texture and zonation were noticed 17

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when grown on 5 different culture media like PDA, RA, CDA, Leonine agar (LA) and Neopeptone agar (NPA). They also observed difference in the germination and appressoria formation among the studied isolates at 2 different temperatures. Growth of Sitargani isolate was slow on PDA compared to other isolates, but on RA medium the growth was intermediate. Only the Pantnagar isolate showed a faster growth. In CDA medium the differences were sharp, the colonies were circular with smooth margins but the pigmentation was different. On NPA medium, the differences were evident; colony color was blackish in Sitaragani isolate whereas it was white in Pantnagar isolate, grayish white in Bilaspur isolate and greenish black in Kalinagar and Dineshpur isolates. Recently, in Thailand ten C. capsici isolates were distinguished into six groups based on morphological variability and cultural characteristics. The isolates of C. capsici showed differences in morphological characteristics (colony color, colony diameter and conidial shape and size). Isolates produced cottony colonies on PDA with grayish-white to dark grey on the ventral surface, colony diameter of different groups was 65-80 mm after 7 days. All grouped isolates from C.C-I to C.C-V produced zigzag cottony colonies, but C.C-VI possessed circular colonies. Average length and width of conidia varied from 23.5 to 35 µm and 2.5 to 3.75 µm, respectively (Sangdee et al., 2011). Based on cultural characteristics on PDA medium Chowdappa et al. (2012) carried out morphological characterization of 25 isolates C. gleosporioides, causal agent of anthracnose in orchids. They reported all the isolates varied in colony color; white to grey, dark orange or pink-grey and reverse side of the colonies varied from white, dark grey, orange or mixture. Mycelium was hyaline, brown or both with floccose, loose or compact growth. Conidia were cylindrical with both apices rounded or with one apex rounded and the other end pointed. Morphological characterization of twelve isolates of C. coccodes, one of the causal agent of pepper anthracnose was carried out on three nutrient media; PDA, sucrose soy protein agar (SSPA) and water agar (WA). In SSPA medium, growth of the fungus was very quick occupying the whole surface of Petri dish in 14 days with abundant microsclerotia followed by PDA and WA (Stojanova et al., 2013). On WA, all colonies were loosely textured, transparent and consisted mainly of microsclerotia dispersed deeply in the media. No conidia were found. 18

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2.2.2 Pathogenicity Pathogenicity is the ability of an organism to cause disease. It is characterized by complex pathogenic properties of microbes develop during their struggle for existence by specific actions. The pathogenicity tests were conducted by Rai and Chohan (1966) on chilli plant by spray inoculating different isolates of C. capsici and examined for percent fruit rot caused by different isolates, number of days taken to produce symptoms, virulence and intensity of spots on the leaves. They found that isolates A, B and O were highly virulent and expressed the symptoms within 15 days. Kenchaiah (1975); Singh et al. (1977) and Khaleeque and Khan (1991) also proved pathogenicity of C. capsici on chilli fruits. The in vitro detached fruit assay using anthracnose susceptible variety CM022 was carried out by Kanchana-udomkan et al. (2004). Among four maturity stages: immature green, mature green, color turning and ripe red, green chilli fruits appear to be more susceptible to anthracnose when analyzed by three inoculation methods: drop, injection and wound/drop. Drop method failed to cause any disease symptoms. They also reported fruit size does not affect the development of the disease. Pathogenicity of fourteen isolates of C. capsici on various Thai chilli varieties (Man Dum, Chee Fah, Mae ping, Kheenhu and Luang) from various places was analyzed by cotyledon inoculation. The results suggested that chilli variety ‘Mae Ping’ was the most susceptible variety among all C. capsici isolates but other four cultivars showed tiny (small) area of necrotic symptom (Oanh et al., 2004). Sharma et al. (2005) pathologically characterized fifteen pathotypes of C. capsici collected from various chilli-growing regions of Himachal Pradesh. Pathotype Cc-I was highly virulent infecting all the cultivars whereas pathotypes Cc-VI, X and XV were least virulent. Colletotrichum spp. isolates from chilli fruit in Thailand showed typical anthracnose symptoms when pathogenicity test was carried out by wound/drop and non-wound/drop inoculation and were identified as C. acutatum, C. capsici and C. gloeosporioides. Interestingly, C. acutatum isolate from strawberry infect and produce anthracnose on PBC-932, a resistant genotype of C. chinense (Than et al., 2008a; Zivkovic et al., 2010).

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Variations in the host reactions of 11 isolates of C. capsici isolated from nine chilli genotypes were investigated by Montri et al. (2009). They reported that, after 9 days of inoculation by microinjection of spores into pericarp of the fruit (detached fruit assay). PC-1 was most virulent infecting genotypes of C. acutatum, C. chinense and C. frutescens; and PC-3 was least virulent on C. annuum and C. frutescens, whereas PC-1, PC-2 and PC-3 showed differential qualitative infection on genotypes PBC932 and CO4714. They concluded that infection occurred in all chilli genotypes except in genotype of C. baccatum, where no infection occurred. Based on the disease scores and time of acervuli development Sangdee et al. (2011) categorized ten isolates of C. capsici into 3 groups. Group CCP-I with two mildly virulent isolates (KR and SK), four isolates (NK, SI, RL and MK) were grouped in CCP-II as moderately virulent and remaining four isolates (UD, NB, KK and KS) were grouped in CCP-III as severely virulent. By employing fruit puncture method and spray inoculation methods Susheela (2012) evaluated four chilli hybrids for resistance to chilli anthracnose. From fruit puncture method under laboratory conditions revealed that, conidial germination and appresorial formation occurs in both green and ripen fruit surfaces, but lesion area was more in green fruits compared to red fruits. The fruits showed tolerant at green stage for anthracnose exhibited tolerant even at ripened red stage. However if fruits were found susceptible at green stage, they found to be susceptible for anthracnose at ripened red stage. In spray inoculation method, significantly higher percentages of anthracnose lesions were observed at red ripe stage over green fruits. Though inoculation was done at green fruit stage too, the expression of the symptom was observed at color turning stage only. Mahmodi et al. (2013) studied the pathogenic variability of Colletotrichum truncatum causal organism of chilli anthracnose by cross inoculation studies. Pathogenicity and cross-inoculation studies with C. truncatum collected from infected pepper crops. Except for the differences in rate of disease incidence and severity, C. truncatum showed they were more pathogenic on the original host (chilli) species from which isolates were obtained compared with other plant species; tomato, eggplant, onion, lettuce and cabbage. The cross-inoculation studies proved potential of C. truncatum isolates for virulence on chilli and other vegetables including tomato, eggplant, onion, lettuce and cabbage. 20

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2.2.3 Biochemical characterization The activities of pathogens against plants are largely chemical in nature i.e., biochemical reactions taking place between enzymes secreted by the pathogen and structural components of plant cells. The main group of enzymes secreted by pathogen seems to be involved either directly or indirectly in the production of disease in plant. 2.2.3.1 Cell wall degrading enzymes (CWDEs) The plant cell wall is the first barrier for the invasion of fungal pathogens. Therefore, during the invasion, the cell wall degrading enzymes (CWDEs) secreted by pathogens is a crucial factor for a successful early infection and establishment of the disease and is known to be responsible for the pathogenesis (Zhao and Zhang, 2002). Pathogenicity of C. capsici was found to depend upon the ability to produce cellulase and pectinase, the two CWDEs (Doyle and Lambert, 2002; Gao, 2003) and which are known to facilitate cell wall penetration and tissue maceration in host plants. The ability of a pathogen to produce cellulolytic and pectinolytic enzymes determines the degree of degradation of cell wall during pathogenesis and these enzymes ultimately influence disease development (Chenglin et al., 1996; Desouky, 2007). The amounts of CWDEs in diseased tissues were higher, and they are extractable from the plants only in the presence of the pathogen (Bateman and Ettern, 1969). Cellulase was found to be the major cell wall degrading enzyme that showed increased percentage of production among the enzymes with progress in the storage period (Padmini Nagaraj, 1987). Production of cellulase enzyme was found to be the major pathogenic factor that influences the degree of pathogenicity of C. capsici in the spoilage of fresh fruits and vegetables (Agostini and Timmer, 1992). Cellulose is a polysaccharide, consists of chains of glucose (1-4) β-d-glucan molecules, occurs in all higher plants as the skeletal substance of cell walls in the form of microfibrils. The spaces between microfibrils and also spaces between cellulose chains within the microfibrils may be filled with pectin and hemicelluloses in plant cell wall. The enzymatic breakdown of cellulose results in the final 21

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production of glucose molecules i.e., cleaving cross-linkages between chains. The glucose is produced by a series of enzymatic reactions carried out by cellulases (Agrios, 2005). Similarly, pectic substances constitute the main components of the middle lamella, i.e., the intercellular cement that holds in place the cells of plant tissues and primary cell wall. Pectic substances are polysaccharides consisting mostly of chains of galacturonan molecules, interspersed with a much smaller number of rhamnose molecules and small side chains of galacturonan. Several enzymes degrade pectic substances and known as pectinases or pectolytic enzymes. Some of them are the pectin methyl esterases, remove small branches off the pectin chains (Agrios, 2005). Many phytopathogenic fungi produce cellulases in culture adaptively that hydrolyze cellulose and its derivatives (Muthulakshmi, 1990). Yakoby et al. (2000) studied the expression of pectate lyase from C. gloeosporioides expressed in C. magna, which promotes pathogenicity. They found more severe maceration ability on avocado pericarp as compared to wild type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, they reported (pel gene) transformed isolates had more severe maceration capacity and damping off ability in watermelon seedling when compared with the two wild-type isolates, which showed no symptoms. Their result clearly suggests that single pel (PL) in C. magna is a pathogenicity factor required for the penetration and colonization of Colletotrichum species. The local modulations of host pH by Colletotrichum species were studied by Prusky et al., 2001. During the growth of Colletotrichum species in acidified medium the ammonia secreted by fungus increases the pH of the medium, which in turn increased the activity of pectate lyase (PL). They finally suggested that secretion of PL is a key virulence factor in disease development. Lakshmesha et al. (2005) studied the change in pectinase and cellulase activity of C. capsici mutants and their effect on anthracnose disease on Capsicum fruit. They exposed the virulent C. capsici isolate to UV radiation at 312 nm wavelength for 15, 30, 45, 60, 75 and 90 min. UV radiation exposed virulent C. capsici showed reduced activity of pectinase and cellulase enzyme resulted in 3-5 days delay in manifestation of anthracnose disease and also reduced the rate of spreading of disease. 22

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The role of CWDEs produced in poplar cultivars infected with Melampsora larici-populina Kleb. was analyzed. The result showed that CWDEs, pectin methyl galacturonase (PMG), pectin methyl-trans-eliminase (PMTE) and β-glucosidase played a role during the infection. Among these, the activities of PMG and PMTE were higher, and the activities of cellulase (CX) and β-glucosidase were relatively lower. They also found that these CWDEs were significantly higher in the susceptible cultivar than in the resistant one (Tian et al., 2009). Babalola (2010) found that pectinolytic and cellulolytic enzymes enhanced Fusarium compactum virulence on tubercles of Egyptian broomrape. These enzymes could facilitate pathogen penetration into plant host and combination of cellulase and pectinase was ascertained by the pathogenicity. 2.2.3.2 Generation of Reactive oxygen species (ROS) and their detoxification during pathogenesis Reactive oxygen species (ROS) have long been known to be a component of the killing response of immune cells to pathogen invasion. ROS includes free radicals such as superoxide anion (O2−), hydroxyl radical (OH), as well as non radical molecules like hydrogen peroxide (H2O2), singlet oxygen (1O2), and so forth (Sharma et al., 2012). The principle pathogen-induced ROS in plants is H2O2 (HammondKosack and Jones, 1996) and has received much attention during the last decade. It is known to participate in many resistance mechanisms, including the reinforcement of plant cell wall, phytoalexin production and enhancement of resistance to various stresses (Dempsey and Klessig, 1995). Accumulation of H2O2 can lead to oxidative stress in plants, trigger cell death. Some virulent pathogens to overcome the toxic effects of ROS in plant tissues produce defensive antioxidant enzymes such as catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11) and superoxide dismutase (SOD) (Chamnogpol et al., 1995; Hammond-Kosack and Jones, 1996; Agrios, 2005; Asada, 1997; Willekens et al., 1997). Catalase plays the role of a specific peroxidase (POX) protecting the cells from the toxic effects of H2O2 (Lebeda et al., 2001). The function of CAT in the cell is to remove the bulk H2O2, whereas APX gets involved mainly in scavenging the H2O2 that is not taken by CAT. Unlike CAT, APX is found throughout the cell (Jespersen et al., 1997) and it has a considerably greater affinity for H2O2 than CAT (Dalton et al., 1987). Studies 23

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on incompatible plant-pathogen interactions suggest that the cytosolic APX isoform is differentially regulated during pathogen infection. APX catalyzes reduction of H2O2 to water with ascorbate as an electron donor. The activity of APX, which is proposed to be predominantly responsible for H2O2 decomposition in plants, is significantly higher in infected leaves. The role of ascorbate in scavenging ROS generated in response to pathogen ingress has only been sporadically examined. Preceding the discovery of the APX catalysed reaction, ascorbate was reported to affect the symptoms of plants invaded by pathogens. In the tobacco, the APX activity increased at the appearance of necrotic lesions, preceded by a minor decrease (Fodor et al., 1997). The induction of APX suggests that it was mobilized as a detoxification mechanism for active oxygen species (AOS) generated during tomato-Botrytis cinerea interaction (Tiedemann, 1997). So far the reports show that, only limited attempts were made in the extraction and characterization of CAT and APX from plant pathogens. El-Zahaby et al. (1995) showed a substantial increase in APX activity, in susceptible barley (Hordeum vulgare) cultivars and less pronounced increase in the resistant cultivar after powdery mildew infection by Blumeria graminis. In contrast, using the same pathosystem, Vanacker et al. (1998) observed that the foliar APX activity decreased in the resistant cultivar whereas it was unchanged in the susceptible during B. graminis infection. Garre et al. (1998) studied the secretion of extracellular CAT from ergot pathogen Clavisceps purpurea in rye (Secale cereale) and its role in pathogenicity and in suppressing host defence. This study reveals C. purpurea secretes catalases activity into the medium and during infection of rye. Further, it was noticed that the CAT activity was located in hyphal walls both axenic culture and infection of rye. Periplasmic spaces and hyphal surfaces showed strong accumulation of CAT activity. The intracellular activity staining in organelles of fungal secretory pathway revealed that CAT secreted by C. purpurea. In addition, the immunogold localization showed that catalases diffuses through the cell wall and accumulate at the cell surface. Migration of fungal CAT into host cell wall takes place exclusively at the host pathogen interface. Zhang et al. (2004) made a similar kind of report, secretion of catalase B (CATB) at germ tube tips of Blumeria graminis causal agent of powdery

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mildew of barley. In this study, increased expression of CATB at times of fungal invasion was correlated with both spatial and temporal clearing of H2O2 indicates the fugal origin of the enzyme. Bussink and Oliver (2001) identified two highly divergent genes cat 1 and cat 2 in fungal tomato pathogen Cladosporium fulvum. These two genes showed differential expression, with the cat 1 mRNA preferentially accumulate in spores and cat 2 mRNA preferentially accumulate in response to external H2O2. This study reports an existence of a complex CAT system in C. fulvum in relation to both structure and regulation of genes. The fungus produces the enzyme CAT and APX, which reacts with and neutralizes the H2O2 that is produced as one of the first defence responses of plants against infecting pathogens. The fungal CAT concentration is greatest at hyphal walls and hyphal surfaces and is secreted by the fungus into the host apoplast at the hostpathogen interface, where the host H2O2 is produced. By inactivating ROS produced by the host through CAT and APX, the fungus suppresses the host defences and finally produces disease (Asada, 1997; Agrios, 2005). The role of CAT enzyme in determining the virulence of Xanthomonas oryzae pv. oryzae (XOO) on susceptible rice cultivar ‘Jaya’ was studied by Choodamani et al. (2009). All the eleven isolates showed variable levels of catalase activity and differential expression of isoforms. Catalase activity was found maximum in isolate XOO2 followed by XOO4 and minimum activity was recorded in isolate XOO3. They suggested that CAT is important for pathogenicity, and it acts as a virulent tool in disease development. Similar study carried out by Chitrashree and Srinivas (2012) suggested the activity of CAT and APX could be used as biochemical markers in determining the virulence. They noticed antioxidant scavenging activity was variable among the 34 isolates studied. Most of the XOO isolates with higher CAT activity exhibited higher APX activity. Also, maximum levels of CAT and APX activities were found in isolate XOO 32, which induced maximum lesion length on cultivar ‘Jaya’ upon clip inoculation in virulence assay.

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Hence, CWDEs (cellulase and pectinase) and H2O2 scavenging enzymes (CAT and APX) are used to characterize pathogens as virulent and hence are the tools for the study of virulence. 2.2.4 Molecular characterization Colletotrichum spp. comprise unique group of microorganisms that express a wide range of variations. These variations are the results of a wide range of adaptability to both natural and man-made ecological situations. Identification of different species is based more on morphological characters, but recently genetic variations by advanced techniques like PCR, random amplified polymorphic DNA (RAPD), Inter simple sequence repeat (ISSR) and Internal Transcribed Spacer region (ITS) sequencing have been done. From the past few decades, researchers used DNA based markers to know to detect and to measure the variability among individuals and such markers have been used successfully in identification of different Colletotrichum spp. infecting different hosts (Casela et al., 1993; Madan et al., 2000). According to Agwanda et al. (1997), molecular methods have been employed successfully to differentiate between populations of Colletotrichum from hosts. Cannon et al. (2000) have stated that data derived from nucleic acid analysis proved the most reliable framework to build a classification of Colletotrichum. The combined application of traditional methods like morphology, biochemistry and pathogenicity with molecular diagnostic tools is an appropriate and reliable approach to know the species complex of Colletotrichum. Several reviews have appeared on the use of RAPD PCR in the study of plant pathogens (Annamalai et al., 1995; Tudzynski and Tudzynski, 1996). Different species of Colletotrichum have been reported to possess a high degree of molecular variability when evaluated by RAPD analysis. Molecular markers such as ISSR have been successfully used as tools in understanding the phylogenetic relationships of fungi (Chadha and Gopalakrishna, 2007; Schwarzenbach et al., 2007). RAPD and ISSR are simple technique and require no sequence information and is carried out using a single primer base on a simple repeat. The results are clearly scorable and reproducible (Nagaoka and Ogihara, 1997). Many workers suggested that RAPD approach is useful for proper identification and categorization of Colletotrichum spp. isolates as it yielded race-specific amplified DNA profiles (Balardin et al., 1997; Mesquita et al., 1998). 26

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Kutcher and Mortensen, (1999) also successfully differentiated the pathogen Colletotrichum gloeosporioides f. sp. malvae infecting velvetleaf (Abutilon theophrasti) from other species of Colletotrichum. Thottappilly et al. (1999) categorized 51 isolates of C. gloeosporioides into four groups following RAPD analysis, which were previously categorized on the basis of morphology and virulence that is pathogenicity screening. Grouping was done by using unweighted pair-group method with arithmetic mean (UPGMA) analysis of with 10-mer random primers in which 95 RAPD bands were obtained. The studies on 5 isolates of C. falcatum with six RAPD primers generated 12 bands per primer. Out of this, only 23% were polymorphic (Madan et al., 2000). Molecular analysis of a subset of 21 isolates from a total of 103 isolates of C. gloeosporoides by RAPD and electrophoretic karyotyping gave comparable results for both markers. Electrophoretic karyotyping detected a higher level of polymorphism. Among the 103 isolates majority were placed into three groups on the basis of RAPD analysis (Chakraborty et al., 1998). The characterization studies done on C. falcatum isolates of sugarcane red rot pathogen using RAPD studies showed that, out of 80 primers, 61 exhibited well-defined polymorphism among the pathotypes. The isolate CO 7717 recorded the highest genetic distance from the other pathotypes with coefficient value of 0.291 and maximum distance from the isolate CO 1148. The grouping of the isolates based on RAPD was similar to the pathogenicity on differential host clones (Mohanraj et al., 2002). Phylogenetic analysis using RAPD markers was carried out by Xiao et al. (2004) to determine the genetic variability among C. gloeosporioides isolates two separate clusters were obtained in relation to isolates obtained from infected strawberry plants and non cultivated hosts. RAPD analysis of Colletotrichum sp. causing chilli anthracnose disease was analyzed by Ratanacherdchai et al. (2007) using 18 isolates including, C. gloeosporioides and C. capsici from three varieties of chilli. PCR amplification of genomic DNA by RAPD-PCR using 90 random 10 mer primers, of which 13 primers produced easily scorable banding patterns. RAPD data showed less variation among C. gloeosporioides isolates than in C. capsici isolates.

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Sharma et al. (2005) have investigated genetic relationship between five previously morphologically grouped C. capsici using RAPD analyses. They found that molecular polymorphism generated by RAPD profile confirmed the variation in virulence of C. capsici and isolates were grouped into five clusters. Classification of C. capsici isolates causing fruit rot of chilli did not correlate with virulence or geographic origin. Shenoy et al. (2007) molecular characterization of C. capsici by PCR based techniques was done using β tubulin primer combination Bt 2A and Bt 2B and ITS4- ITS5 and phylogenetic analysis was conducted in MEGA 4. From this study, a close phylogenetic relationship of epitype of C. capsici strains from Thailand was observed. Phylogenetic analysis of Colletotrichum species; C. capsici, C. gloeosporoides and C. acutatum associated with anthracnose of chilli was done by DNA sequence data of ITS r-DNA and β-tubulin (tub 2) gene regions produced three major clusters representing these three Colletotrichum species. Also, morphological characters like colony growth rate and conidia shape in culture were directly correlated with the phylogenetic grouping. Comparison with the isolates of C. gloeosporioides from mango and C. acutatum from strawberry they found that the host was not important for phylogenetic grouping (Than et al., 2008c). Ratanacherdchai et al. (2010) revealed that C. capsici from three tested hosts expressed highest virulence out of 34 isolates belonging to two different Colletotrichum species (i.e., C. gloeosporioides and C. capsici) causing anthracnose on different host plants (bell pepper, long cayenne pepper and bird’s eye chilli). Six ISSR primers showed multi-band patterns, out of 103 bands from 34 isolates. Dendrogram analysis divided 34 isolates of Colletotrichum spp. into three main groups. Group 1 contained C. gloeosporioides isolates whereas, groups 2 and 3 consisted of C. capsici isolates. There was less similarity between C. gloeosporioides isolates and C. capsici isolates. The characterization of 211 Colletotrichum isolates collected from strawberry plant tissues with/without typical anthracnose symptoms and from symptomless weeds of strawberry fields was carried out by Van Hemelrijck et al. (2010). Classification of isolates was done on the basis of r-DNA-ITS sequencing, which showed that 97% isolates were C. acutatum, 2% C. gloeosporiodes and 1% 28

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C. coccodes. Similarly, Zivkovic et al. (2010) studied and characterized C. gloeosporioides, C. acutatum, C. coccodes and C. dematium which cause tomato anthracnose. PCR analysis was done using species-specific primer CaInt 2 in conjunction with ITS4 primer amplified a 490 bp fragment of C. acutatum, but not the DNA of C. gloeosporioides isolates. These results demonstrated that r-DNA analysis is a reliable method for taxonomic species identification. The genetic characterization of C. capsici was performed using 3 RAPD primers; PELF, URP1F and OPA 03 (Sangdee et al., 2011). Molecular polymorphism generated by RAPD confirmed the variation in different isolates and they were grouped in two clusters. In their study, they did not find any correlation between cluster analysis with cultural morphology and virulence patterns. This work concludes RAPD analysis can be used to classify and study the genetic diversity of C. capsici more rapidly than other methods. Chowdappa et al. (2012) studied molecular characterization of 25 C. gleosporioides isolates collected from diseased leaf samples of orchids in different geographical regions of Sikkim state. PCR amplification of all the isolates with ITS region of r-DNA using ITS1 and ITS4 primers yielded 560 bp product which showed 100 % homology with C. gleosporioides, isolates. Further, PCR assay with species specific primer CgInt together with ITS4 produced 450 bp amplification product. From this study, it was confirmed that this pathogen is responsible for anthracnose of orchids in India. Stojanova et al. (2013) characterized 12 isolates of C. coccodes from fruit and root samples of pepper using gene specific (Cc1F1/Cc2R1) and species specific (Cc1NF1/Cc2NR1) primers. The genus specific primers yielded 450 bp amplicon in all the isolates i.e., C. coccodes, C. acutatum and C. gleosporioides confirms that they belong to the genus Colletotrichum and species specific primer yielded 350 bp size PCR product only in C. coccodes but not in other species. 2.3 Host-pathogen interaction Plants get attacked by a wide variety of phytopathogens. The battle between the plant and an invading pathogen is often depend on the speed. The winner is determined by how quickly the pathogen can proliferate and damage, compared to 29

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how fast the plant can respond with the necessary levels of defence. Common features exist between the signaling processes involved in gene-for-gene mediated resistance during incompatible interactions and the restriction of virulent pathogens during compatible interactions by basal resistance (Feys and Parker, 2000). There are numerous defence mechanisms in plant, many of which are induced by pathogen attack. Plants have a specific resistance (R) gene that interacts with the corresponding avirulence (avr) gene from the pathogen, a rapid defence mechanism known as the hypersensitive response (HR) occurs to prevent infection, i.e., pathogen recognition by plant (Staskawicz et al., 1995; Ellis et al., 2000). Such pathogens that fail to cause disease are termed as avirulent pathogens; the host is called resistant and the interaction is known as incompatible. In the absence of gene-for-gene recognition, due to the absence of the avr gene in the pathogen and/or of the R gene in the host, the pathogen is virulent, the host is susceptible and the interaction is said to be compatible (Glazebrook, 2005). There are two defence responses that are considered hallmarks of R-genemediated resistance. One is a rapid production of reactive oxygen species (ROS) called the oxidative burst. This may have a direct antimicrobial effect as well as serve as a signal for activation of other defence responses through jasmonic acid (JA) and ethylene (ET) pathways (Nimchuk et al., 2003). Another is a form of programmed cell death (PCD) known as HR, which is thought to limit the access of the pathogen. The HR is thought to act against biotrophic pathogens by restricting the pathogen access to water and nutrients. It activates the salicylic acid (SA)-dependent signaling pathway that leads to the expression of certain pathogenesis-related (PR) proteins thought to contribute to resistance (Durrant and Dong, 2004). In addition to localized resistance response, plants have also evolved a mechanism of systemic immunity in which local defences establish a state of heightened resistance throughout the plant against subsequent attack. This phenomenon is known as SAR, which is effective against a broad spectrum of pathogens and requires the phenolic signaling molecule, SA (Dempsey et al., 1999). It has become evident that plants utilize multiple pathways like SA, JA and ET to transfer pathogenic signals to activate HR, SAR and other resistance responses and that SA-mediated SAR is not the only pathway that leads to broad-spectrum disease resistance as early discussed. The production of these signals varies greatly in 30

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quantity, composition and timing, and it results in the activation of differential sets of defence-related genes that eventually determine the nature of the defence response against the attacker to encounter (Rojo et al., 2003). As mentioned in Figure 2.1, the mode of defence signaling in plant is influenced both by the structural type of R protein mediating specific plant-pathogen recognition and also the pathogen’s lifestyles i.e., whether it is biotrophic or necrotrophic (McDowell and Dangl, 2000).

Figure 2.1 Genetically defined relationships between salicylic acid (SA)dependent and ethylene-jasmonic acid (ET-JA)-dependent defence responses in plant system. The SA-dependent response is deployed against a biotrophic pathogen that obtains nutrients from living cells, whereas the ET-JA response is activated by necrotrophic pathogens that kill plant tissue. These pathways appear to be mutually inhibitory (McDowell and Dangl, 2000). The lifestyle of Colletotrichum species is hemibiotrophic (as biotrophic and necrotrophic developmental stages are sequentially established), which combines an initial short biotrophic phase, during which the host cell remains alive followed by highly destructive necrotrophic development characterized by extended areas of killed host tissues (Munch et al., 2008). Most of the Colletotrichum species establish an initial stage of the biotrophic interaction after penetration into the host cell (Bergstrom and Nicholson, 1999). In the biotrophic interaction initial infection structures formed by Colletotrichum species (infection vesicles and primary hyphae), do not kill the host cell but invaginate its plasma membrane (O’Connell et al., 1985). In addition to biotrophic phase, the pathogen tries to avoid defence responses of plant by using such strategies as masking of invading hyphae by converting the hyphal surface-exposed 31

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chitin by deacetylation to avoid degradation of chitin by plant chitinases or transfer of suppressor-active proteins into the plant cell for active suppression of plant defence. The nutrition of Colletotrichum species during the biotrophic phase depends on the living host cell and is coordinated by an efficient transfer system directing nutrients such as hexoses and amino acids from living host cell towards the fungus (Mendgen and Hahn, 2002). Necrotrophic phase that occurs between 48 and 72 h post inoculation depends on environmental conditions. During necrotrophic development, the pathogen may not primarily rely on prevention of defence initiation but may rather actively kill the host tissues. Secondary hyphae representing this parasitic phase will be of smaller diameter, which breach the plasma membrane, kill the host cells and ramify within the tissue. Because of extensive degradation of plant cell walls by a vast array of secreted depolymerases (Herbert et al., 2004) and by secretion of toxins (colletotrichins from pathogen C. nicotina; Thines et al., 2006). However, those toxins have only rarely been reported in Colletotrichum species so far. Alternatively, ROS production has been reported (Govrin and Levine, 2000), but no experimental proof for this strategy to induce death of host cells has yet been reported in case of Colletotrichum species. The observations of Parbery (1996) led to the suggestion that plant defence responses are tailored to the attacking pathogen type. Salicylic acid-dependent defences signal response act against biotrophs; it is easy to imagine the R genemediated resistance and SA signaling could result in resistance. Jasmonic acid and ET-dependent responses act against necrotrophs (McDowell and Dangl, 2000). However, in necrotrophs, programmed cell death of the host would merely make life easier for the pathogen (Thomma et al., 1998). Evidence strongly suggests the importance of JA and ET as alternative signals (to SA signaling) in the induction of resistance against necrotrophic pathogens that are not associated with cell death and are considered to provide alternative defence system. It has been demonstrated earlier that the ET-JA-dependent defence response gets activated by pathogens that kill plant cells to obtain nutrients. In contrast, the SA-dependent response is triggered by a pathogen that gets nutrients from living plant tissue. These studies also suggested that the ET-JA and SA responses are mutually inhibitory (Debener et al., 1991; De Wit, 2002). Jasmonic acid-dependent responses are associated with enhanced expression of several defence genes that encode 32

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antimicrobial proteins such as plant defensin and thionins (Peiterse and Van Loon, 1999). The role of ET signaling lay in the formation of induced structural barriers like cell-wall strengthening and production of antimicrobial secondary metabolites called phytoalexins (Pedras et al., 2000). Phytoalexins derived from the phenylpropanoid pathway can be induced by ET in different plant species. Most extensively documented ET-induced defence-related effector molecules are PR-proteins, majority of which exert direct antimicrobial activity against fungal species (Eyal et al., 1993). The concept of phytoalexins was introduced over 70 years ago (Muller and Borger, 1940). Phytoalexins are low molecular mass secondary metabolites with antimicrobial activity that are induced by stress and are an important part of the plant defence repertoire (Hammerschmidt, 1999; Pedras et al., 2011). Phytoalexins are a heterogeneous group of compounds (Shinbo et al., 2006; Huffaker et al., 2011) that show biological activity towards a variety of pathogens and are considered as molecular markers of disease resistance. Over 350 phytoalexins have been found in over 100 plant species from 30 families of dicotyledons and monocotyledons. Phytoalexins have been isolated from all parts of plants, but different organs may accumulate different phytoalexins. Phytoalexins are thought to be synthesized in cells adjacent to the infection site, in response to a signal produced either by the invading pathogen or infected host cells. They are packaged in lipid vesicles and exported to the infected cell. Consequently, the infected cell becomes a toxic micro-environment or the invading pathogen. Capsidiol is the major phytoalexin produced by inoculation of pepper with pathogenic fungi (Zhang et al., 2005; Literakova et al., 2010). It is a bicyclic sesquiterpene that prevents the germination and growth of several fungal species and has been isolated from many Solanaceae species. Rivera et al. (2002) studied the induction patterns of β-1,3-glucanase in susceptible and resistant melon cultivars in response to infection by cucurbit powdery mildew fungus Sphaerotheca fusca. Both spectrophotometric and enzyme assays indicated a rapid increase in activity of βGlu in the resistant cultivar ‘PMR-6’ than in susceptible cultivar ‘Rochet’. Western blot analysis showed both cultivars were having a 33 kDa βGlu polypeptide. 2-D gel electrophoresis of this polypeptide

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revealed the presence of at least two acidic isoforms of βGlu induced in both cultivars of which one isoform common in both and other was specific for each cultivar. Further, an earlier induction of βGlu transcripts in the resistant cultivar than in susceptible one was observed when Northern blot analysis was carried out with RT-PCR isolation of homologous βGlu cDNA probe from powdery mildew infected leaves. A significant correlation was observed between the degree of host resistance and the enzyme levels when twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease caused by Xanthomonas axonopodis pv. vesicatoria. In highly resistant tomato cultivars total phenols and lignin contents were increased compare to highly susceptible tomato cultivars. In addition, the defence-related enzymes (POX and PPO) were up-regulated in the resistant tomato cultivars upon pathogen inoculation, whereas in the susceptible tomato cultivars, the levels of these enzymes remain unchanged or down-regulated (Kavitha and Umesha, 2008). Wongpia and Lomthaisong (2010) investigated defensive protein response in 2 cultivars of chilli (resistant-Mae Ping 80 and susceptible-Long chilli 455) against wilt pathogen Fusarium oxysporum f. sp. capsici. Protein responses investigated 48 h after infection under in vitro condition analyzed by 2-D electrophoresis showed differential protein patterns in susceptible and resistant cultivars. However the comparison of protein patterns of susceptible and resistant cultivars revealed higher expression of ROS detoxification related proteins in resistant cultivars. The phytopathogen infection leads to changes in secondary metabolism based on the induction of defence programmes as well as changes in primary metabolism, which affect growth and development of the plant. Therefore, pathogen attack causes crop yield losses even in interactions, which may not end up with disease or death of the plant. Although the regulation of defence responses has been intensively studied over the decades, but not much is known about the effects of pathogen infection on primary metabolism (Berger et al., 2007). 2.4 Management In the case of chilli anthracnose seed-borne infection acts as a primary inoculum, but the secondary spread is mainly by air-borne conidia (Kallupurackal and 34

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Ranvindran, 2004). For the control of this pathogen, different management practices have been under practice. Cultivation of resistant varieties, crop rotation and mixed cropping practices are some of the best ways for elimination of this pathogen (Singh and Kaur, 1986). Treating seeds with synthetic chemicals can also reduce the seedborne infection by C. capsici (Ahmed, 1982). The recommendations of integrated management techniques have indicated that no single specific management program could eliminate chilli anthracnose (Bailey, 1987; Agrios, 2005). Effective control of Colletotrichum disease usually involves the use of a combination of cultural, biological and chemical controls along with intrinsic resistance (Wharton and Dieguez-Uribeondo, 2004). 2.4.1 Chemical control measures The use of chemicals is the most common and practical method to control plant diseases. The fruit rot of chilli can be effectively controlled by seed treatments with fungicides like Thiram, Mancozeb, Ziram, Captan and Zithane M-45 (Jharia et al., 1977; Raju and Rao, 1989), and increased yield was also reported (Raju et al., 1982). The fungicide traditionally recommended for the anthracnose management in chilli is Manganese ethylene bisdithiocarbamate (Maneb) (Smith, 2000), though it does not consistently control the severe form of anthracnose on chilli fruit. Spraying of fungicides such as Dithane M-45 and Blitox can effectively control the fruit rot and die-back diseases (Thind and Jhooty, 1990). The strobilurin fungicides such as Azoxystrobin (Quadris), Trifloxystrobin (Flint) and Pyraclostrobin (Cabrio) have recently been labeled for the control of chilli anthracnose. But, only preliminary reports are available on the efficacy of these fungicides against the severe form of the disease (Alexander and Waldenmaier, 2002; Lewis and Miller, 2003). Efficacy of various growth regulators and fungitoxicants against fruit rot of chillies determined by Datar (1996). Chilli fruits dipped in 200 mg/ml indole-3-butyric acid or naphthalic acid for 30 min and 10 min in 100 mg/ml carbendazim solution delayed fruit rot caused by most of the pathogens. In vitro studies were done with five fungicides against C. capsici that had been isolated from diseased chillies in Vellayni and Kerala. Applications of 1% Bordeaux mixture and 0.3% Ziride were equally effective in inhibiting the fungal growth. Agrimycin, Cycloheximide and Zineb were found to be effective in reducing seed35

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borne C. capsici incidence after seed-treatment (Sulochana et al., 1992). The treated seeds showed a higher percentage of germination and lowest percentage of diseased fruits in cv. Rukini following treatment with 300 ppm agrimycin and 100 ppm carbendazim (Azad, 1992). Similar studies were carried out by Biswas (1992) under seed bed conditions. They reported that Bavistin at 0.1% applied once in the nursery bed before transplanting and again one month and two months after transplanting successfully reduced C. capsici incidence on chilli plants. Efficacies of field application of nine commonly used fungicides were tested against fruit rot and dieback in chilli caused by C. capsici in Tamil Nadu, India (Ebenezar and Alice, 1996). Maximum control was achieved with Mancozeb (0.2%) followed by Carbendazim and Copper oxychloride (0.2%). Six antibiotics Cagrimycin, Actidione, Aureofungin, Streptomycin, Grisovin and 4 fungicides (Dithane z-78, Calixin, Bavistin and Vitavax) were evaluated for controlling of C. capsici in chilli. However, Excessive application of chemical fertilizers causes soil sealing, fertility level diminishing and also residual problems. The residue of chemical fertilizers and pesticides create serious damage to the quality of agricultural products, people and animal health, at the same time causing environmental pollution. 2.4.2 Host resistance The use of resistant varieties not only eliminates losses from diseases but also eliminate chemical and mechanical expenses on disease control (Agrios, 2005). Some genetic resources resistant to anthracnose in chilli have been independently reported from different countries and regions (Kim et al., 1986; Kim et al., 1987; Park et al., 1987; Hong and Hwang, 1998; Pae et al., 1998; AVRDC, 2003; Yoon and Park, 2001). The resistance to fruit rot or dieback caused by C. capsici has been reported in certain chilli varieties such as Perennial, Lorai, Bengal green, S20-1, H-4 and H-6 (Singh and Thind, 1980; Kaur and Singh, 1985). The resistance to die-back has also been recorded in BG-1, Shahkote and Laichi-11 (Singh et al., 1990). These varieties serve as donors for incorporating resistance in high-yielding varieties. The inheritance of resistance studied in two resistant varieties viz. Perennial and Lorai was found to be controlled by two recessive complementary genes. A high-yielding genotype developed from a cross between Perennial and Long Red was found to be moderately resistant to fruit rot and die-back (Singh and Kaur, 1986). On the basis of its excellent performance for yield and disease characters, it was released under the name “Punjab 36

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lal” in 1985 for general cultivation in Punjab. The chilli F1 hydrid (CH-1) was recorded as resistant to die-back (Singh et al., 1990) and was released in 1991 for cultivation in the Punjab state. In particular, some lines of C. baccatum showed strong resistance to the pathogen and pathogen inoculation resulted in nil or limited lesions on the chilli fruits (Yoon, 2003). However, even to this day no strong resistance has been found in C. annuum, which is the only major, species grown worldwide (Park, 2007). Mongkolporn et al. (2004) carried out a genetic study of anthracnose resistance to C. capsici, which was expressed in the inter-specific cross of Thai susceptible C. annuum cv. ‘Bangchang’ and anthracnose resistant C. chinense ‘CM 021’. The genetic purity of F1 was proven by using molecular marker analysis. Cultivar Mae Ping showed largest area of necrotic symptom compared with other four chilli varieties; Mun Dam, She Fha, Khee Nhu, and Louang showed tiny areas of necrotic symptom. The variety Mae Ping was highly susceptible when inoculated with C. capsici isolates (Oanh et al., 2004). 2.4.3 Abiotic inducers Induction of plant resistance against pathogen is considered as a potential method to reduce the severity and to control plant diseases. Defence responses occurring in plants exhibiting induced resistance (IR) reflect genetically fixed mechanism typical for plant species. Abiotic inducers are synthetic chemicals, which do not show a direct antibiotic effect by themselves against plant pathogens, but can induce resistance in treated plant system and thereby protect various types of plant species against a wide range of pathogens. Abiotic inducers like acibenzolar-S-methyl (ASM) and β-amino butyric acid (BABA) are known to induce SAR in a range of plant species (Lead beater and Staub, 2007; da Rocha and Hammerschmidt, 2005). Vallad and Goodman (2004) achieved high levels of disease control under controlled environmental conditions but lower levels of disease control are usually observed in the field. Recently, ASM was developed as a potent SAR activator as it has no antimicrobial properties. ASM derivatives have been exhaustively studied for years as resistance inducers and commercial SAR primers (Chinnasri et al., 2006). Induction of resistance by ASM was demonstrated in plant species against a wide spectrum of 37

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fungal, bacterial and viral pathogens by many researchers. Few important reports are presented in Table 2.1. Table 2.1 Abiotic inducers in horticultural crop protection Inducer

Tested plants

Pathogen controlled

Reference

ASM

Banana (Musa spp.)

Mycophaerella fijiensis

Tally et al., 1999

ASM

Chilli (Capsicum annuum)

Colletotrichum spp.

Tally et al., 1999

ASM

Cucumber (Cucumis sativus)

Cladosporium cucumerinum Pythium ultimum

Narusaka et al., 1999, Benhamou and Belanger 1998

ASM

Lettuce (Lactuca sativa)

Bremia lactucae

Tally et al., 1999

ASM

Pea (Pisum sativum)

Mycospherella pinodes,

Dann and Deverall, 2000

Pseudomonas syringae pv. Pisi, Uromyces viciae-fabae ASM

Chilli (Capsicum annuum)

Phytophthora capsici

Matheron and Porchas, 2002

ASM

Rose (Rosa hydrida)

Diplocarpon rosae,

Suo and Leung, 2002

Agarobacterium tumefaciens ASM

Spinach (Spinacia oleracea)

Alburgo occidentalis

Leskovar and Kolenda, 2002

ASM

Tomato (Lycopersicon esculentum)

Cucumber mosaic virus

Anfoka, 2000

ASM

Chilli (Capsicum annuum)

Phytophthora capsici

Baysal et al., 2005

ASM

Cucumber (Cucumis sativus)

Colletotrichum orbiculare

Lin and Ishii, 2009

ASM

Lettuce (Lactuca sativa)

Xanthomonas campestris pv. vitians

Yigit, 2011

ASM

Tomato (Lycopersicon esculentum)

Clavibacter michiganensis

Soylu et al., 2003

BABA

Cucumber (Cucumis sativus)

Sphaerotheca fuliginea

Vogt and Buchenauer, 1997

BABA

Grape (Vitis spp.)

Plasmopora viticola

Cohen et al., 1999

BABA

Lettuce (Lucumis sativum)

Bremia lactucae

Pajot et al., 2001

BABA

Chilli (Capsicum annuum)

Phytophthora capsici

Hwang et al., 1997

BABA

Tomato (Lucumis esculentum)

Meloidogyne javanica

Oka et al., 1999

BABA

Tomato (Lucumis esculentum)

Phytophthora infestans

Cohen et al., 1994

BABA

Potato (Solanum tuberosum)

Phytophthora infestans

Kim and Jeun, 2007

BABA

Pea (Pisum sativum)

Uromyces pisi

Barilli et al., 2010

BABA

Potato (Solanum tuberosum)

Fusarium suphureum

Yin et al., 2010

BABA

Cucumber (Cucumis sativus)

Meloidogyne javanica

Sahebani and Hadavi, 2011 38

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The ASM also can induce multi-component defence response against Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker in tomato. A significant decrease in the disease severity (up to 75%) in correlation with pathogen suppression (up to 68.2%) was observed when tomato seedlings were treated with ASM (Soylu et al., 2003). Acibenzolar-S-methyl treatment exhibited induced resistance to P. capsici in pepper leaves by rapid induction of phenylalanine ammonia lyase (PAL), increase in total phenol and increase in activities of PR proteins, chitinase and β-1,3-glucanase (Baysal et al., 2005). Pathogenicity test showed 45% lower disease incidence after 7 days of challenge inoculation in ASM treated plants compared with water treated control plants. The induction of SAR against bacterial wilt of tomato caused by Ralstonia solanacearum was observed when ASM was applied as foliar spray and soil drench under greenhouse and field conditions. Reduction in disease incidence, increase in plant height, higher fruit production (33%) in moderately resistant plants were observed than untreated plants giving 13% fruit yield (Pradhnang et al., 2005). The ability of ASM to suppress fusarium wilt of Cyclamen (Cyclamen persicum) caused by Fusarium oxysporum f. sp. cyclaminis was investigated by Elmer (2006) in greenhouse conditions. Some of the ASM plants remained asymptomatic for the entire period. Dry mass of plant grown in infested potting mixture was proportional to decrease in ASM. The ASM (50 µg/ml) affected the flower number and quality. It was finally concluded that ASM as one of the useful components of integrated disease management. Efficacy of ASM in controlling bacterial spot of tomato was carried out by spray inoculating the susceptible tomato plants with 0.2 g/l of ASM and after 4 days, challenge inoculated with a virulent strain of X. vesicatoria. The greenhouse data showed 87% of disease protection against X. vesicatoria in ASM treated plants and enhancement of defence related enzymes, PR enzymes like lignin deposition and phenolic compounds in leaves exposed to ASM treatment. Enhancement of POX, PPO and chitinase were recorded from 1 to 72 h after ASM treatment, but an enhancement in PAL activity was observed after 6, 9 and 12 h of treatment (Cavalcanti et al., 2007).

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The activation of resistance in tobacco plants was observed when treated with different concentrations of ASM as 50% active ingredient (a.i.) in wettable powder formulation against tomato spotted wilt virus (TSWV). Reduced systemic infection and increased resistance occurred in 2 days after treatment and high level were reached 5 days onwards, along with the expression of PR gene (PR-3). Different classes of PR protein were detected after 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV (Mandal et al., 2008). Induction of PR-proteins (Friedriech et al., 1996) and accumulation of chitinase and glucanase gene activations in Arabidopsis, tobacco and rose have been recorded after ASM treatment (Suo and Leung, 2002). Acibenzolar-S-methyl as a SAR inducer to C. orbiculare caused powdery mildew disease in young cucumber plants within 3 h of foliar application. H2O2 and LOX assays were carried out after foliar treatment of ASM and challenge inoculation with C. orbiculare and the activity was compared with distilled water control plants. H2O2 accumulation was higher in ASM treated plants after 3 h and LOX activity increased 6 h after ASM treatment compared with control (Lin and Ishii, 2009). Similar finding was confirmed by the studies of Cools and Ishii (2002). Work of Deepak and Ishii (2006) revealed that H2O2 accumulation within xylem might increase the level of POX and NADPH oxidase gene expression after ASM treatment. Efficacy of ASM treatment against bacterial speck disease of lettuce caused by Xanthomonas campestris pv. vitians under greenhouse conditions and inducing defence mechanism using ASM treatment were carried out by Yigit (2011). This study revealed that ASM was effective in reducing the severity of disease and bacterial growth when compared with control and copper hydroxide. Further, 48 h interval before inoculation period was convenient for the control of pathogen by ASM treatment. ASM treatment in inoculated plants has a long-lasting effect in enhancing plant resistance by inducing defence-related enzymes, PR protein (chitinase) and thereby decreasing bacterial growth. β-amino butyric acid is a non-protein amino acid, which occurs rarely in nature. Although BABA is only rarely found naturally in plants, it has proven to be a potent inducer of acquired resistance. The possibility of a direct toxic effect of BABA has been tested repeatedly in vitro, on many plant pathogens by different research 40

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groups and the possibility of any toxic effects have been ruled out (Cohen, 1994; Cohen et al., 1994; Hong et al., 1999; Tosi et al., 1999). β-amino butyric acid is a well known activator in many plants, which induces resistance against a wide range of pathogens in a number of plant species and enhances activation of defence responses on pathogen encounter (Jakab et al., 2001; Cohen, 2002; Van Hulten et al., 2006). Sunwoo et al. (1996) demonstrated the relatively high concentration of BABA to the extent of 1000 µg/ml, which did not show any antifungal activity in vitro but foliar and stem spray treatments induced resistance and controlled the disease completely. The effect of BABA in inhibiting Phytophthora capsici infection on the pepper plant was studied in artificially infested fields. β-amino butyric acid alone treated plants showed more protection against phytophthora blight than its combination with antagonistic bacterial strain Burkholderia cepcia strain N9523 (Lee et al., 1999). Treatment with BABA induced PR-1a, chitinase and glucanase in tobacco, tomato and pepper and other plants. However, induction of PR proteins is not the only mode of action of BABA, which also leads to callose deposition, lignification and hypersensitivity in some plants (Hwang et al., 1997; Cohen et al., 1999; Siegrist et al., 2000). β-amino butyric acid reduced infection of downy mildew fungus Plasmopara viticola in 2 cultivars of grapevine (Vitis vitifera); Chardonnay and Cabernet sauvignon by 57% and 98% respectively (Reuveni et al., 2001). Kamble and Bhargava (2007) studied BABA effect in brassica plants that protected them against necrotrophic pathogen Alternaria brassicae. β-amino butyric acid treated plants achieved higher resistance level than SA and JA pre-treatments. Expression of two marker genes of the SA and JA pathways namely, PR1 and PDF 1.2 was enhanced in response to BABA treatment. BABA induced PAL and chitinase gene expression in grape fruit (Porat et al., 2003) as also phenol and phytoalexins expression in potato, thereby promoting protection against fungal pathogen infection. It has been shown that pre-treatment with BABA decreased disease severity in potato after inoculation with Phytophthora infestans by suppressing the appressorium formation on the infected leaves of potato (Kim and Jeun, 2007) and in cucumber plants after inoculation with C. orbiculare (Jeun et al., 2007). Jeun (2000) and Siegrist et al. (2000) reported increase of SA level in the leaves of BABA treated tomato and tobacco plants. Andreu et al. (2006) also showed similar results with the reduction in foliage infection against P. infestans in moderately susceptible and 41

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moderately resistant potato cultivar after BABA treatment as a result of increase in defence enzyme and phenol content. The mechanism of resistance to Plasmopara halstedii, the downy mildew pathogen of sunflower after treatment with BABA inducer was studied in susceptible, partially resistant and completely resistant sunflower genotypes (Korosi et al., 2011). Their results indicated that BABA improved resistance in all genotypes of sunflower by increasing defence enzyme activities (POX and PPO) as well as accumulation of mRNAs of glutathione S-transferase, defensin and catalase. In addition to this, Marcucci et al. (2010) found increase in the activity of chitinase, β-1,3-glucanase and POX in artichoke (Cynara cardunculus var. scolymus) induced by BABA against white mould caused by Sclerotinia sclerotiorum. BABA treatment induced various defence responses including chitinase, β-1,3-glucanase and POX activities in artichoke. BABA induced the activity of defence enzymes (PAL, PPO and POX) and also increased accumulation of lignin, flavonoids and phenolics. All the defence enzymes were enhanced in susceptible and resistant cultivars. However, resistant cultivar showed stronger resistance than the susceptible cultivar (Yin et al., 2010). Similarly, Sahebani et al. (2011) investigated the effect of BABA on root-knot nematode (Meloidogyne javanica) infecting cucumber by evaluating accumulation of total phenolics, H2O2 and activity of defence enzyme (POX, PPO and CAT) in root infected with nematodes. Seedlings treated with BABA and BABA+nematode significantly increased POX, PPO and CAT activity 1 day after nematode challenge inoculation when compared with nematode control and distilled water control, and it reached the maximum on the 3rd and 4th day respectively after inoculation. Whereas, H2O2 and oxidative stress reached maximum on the 5th day after inoculation in BABA and BABA+nematode and phenol accumulation increased on the 7th day after nematode inoculation. 2.4.4 Biotic elicitor The term biotic elicitor usually refers to macromolecules originating either from the host plant (endogenous elicitors) or from the plant pathogen (exogenous elicitors), which are capable of inducing structural and/or biochemical responses 42

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associated with the expression of plant disease resistance (Dixon et al., 1994). A number of biotic elicitors have been identified including oligosaccharides (Cote and Hahn, 1994), glycoproteins, peptides (De Wit and Spikman, 1982) and phospholipids from fungi (Creamer and Bostock, 1986). Radhajayalakshmi et al. (2009) used the word “hit and stick” rather than “hit and go” mechanism as it could cause prolonged activation of elicitor. One class of signal molecules played a significant role in the signal exchange between plants and pathogen/elicitor (Ebel and Cosio, 1994). Elicitors are capable molecules, which mimic the perception of a pathogen by a plant thereby triggering induction of defence response in plants. To examine induced defence reactions of plants to pathogenic organisms, several workers have successfully used cell suspension cultures instead of whole plants and replaced pathogens by elicitors, the isolated compounds responsible for inducing defence responses in plants (Wojtaszek et al., 1995; Walkes and O’Garro, 1996). One of the earliest events known to occur upon elicitor-receptor recognition is production of reactive oxygen intermediates such as superoxide anion (O-) and H2O2 (Apostol et al., 1989). Hydrogen peroxide (H2O2) has been suggested to be an important regulator of disease resistance mechanisms associated with HR and SAR (Levine et al., 1994). Elicitation of H2O2 production by cell wall extracts and cell death in cultured rice cells and up-regulation of several defence genes have been reported (Ono et al., 2001; Matsumura et al., 2003). Elicitor-receptor interactions are presumed to generate signals that activate nuclear genes involved in plant defence responses leading to the induction of stressrelated enzymes including PAL phenol-oxidizing enzymes including peroxidase (POX) and polyphenol oxidase (PPO) (Pegg, 1985; Ana et al., 2000) and the associated accumulation of high levels of phenolic compounds. Lipoxygenase (LOX) is known to be induced initially during the host-pathogen interaction and later on its products get involved in defence induction and antimicrobial activity (Farmer and Ryan, 1990; Gundlach et al., 1992; Croft et al., 1993; Bhardwaj et al., 2011). Application of elicitors also induces signaling process that begins upstream activation of PR proteins. Expression of elicitor inducible PR proteins has been well correlated with disease resistance (Vidhyasekaran, 1997). Expression of PR-proteins like chitinase and β-1,3-glucanase exhibited antifungal activity against A. solani in 43

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response to the release of HR elicitors from fungal cell wall in tomato (Lawrence et al., 1996; Christopher et al., 2000). Rhizoctonia solani elicitor treated pearl millet leaves produced 45 kDa chitinase (Radhajeyalakshmi et al., 2000; 2004). The crude cell wall preparation from a fungal pathogen, F. moniliforme gave the similar response as that of chitin in rice cell suspension culture and showed that the major elicitor in the preparation was chitin. Additionally, chitin was shown to be much more active than chitosan in rice. Thus, the study suggested that the fungal cell wall component of microbial enzyme activates defence responses indirectly (Ren and West, 1992). Similarly it was reported that cell wall oligogalacturonides increase cytosolic free calcium in carrot protoplasts; Ca2+ cytosolic free calcium helps to stimulate the signal transduction, which activates the induction of various plant cell responses such as activation of host defence mechanism (Messiaen et al., 1993).

Figure 2.2 Binding of fungal elicitor to plant receptors triggers the activation of signal transduction pathway leading to plant defence gene activation (modified from Doke et al., 1996). The elicitor-induced formation of free and cell wall-bound stilbenes in cell suspension cultures of scots pine (Pinus sylvestris) were studied by Lange et al. (1994). Treatment of P. sylvestris cell suspension culture with an elicitor prepared from pine needle pathogen Lophodermium seditiosum resulted in several hundredthousand fold accumulation of stilbenes, pinosylvin and pinosylvin 3-O-methyl ether 44

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in methanolic cell extracts. Simultaneous induction of both the biosynthetic enzymes, PAL and stilbene synthase was noticed. As early discussed in host pathogen interaction (Section 2.6), phytoalexins are thought to be synthesized in cells adjacent to the infection site, in response to a signal produced either by the invading pathogen or pathogen derived compounds or by infected host cells. Capsidiol is the major phytoalexin produced by inoculation of pepper in response to pathogenic fungi or pathogen derivatives or exogenous molecules (Zhang et al., 2005; Literakova et al., 2010). The biosynthesis of sesquiterpenic phytoalexin capsidiol occurs in root cultures of chilli plants. Optimal concentrations of sucrose and cellulose were used as elicitors and induction of capsidiol (an antimicrobial compound) production was established. Maximum amount of elicitor inducible sesquiterpene cyclase was found between 6 and 8 h (Patricia et al., 1996). A glycoprotein of 34 KDa from the mycelium of Phytophthora parasitica var. nicotianae was found that molecule GP 34 had elicitor properties on treatment with roots, where LOX had enhanced the activity, and there was an accumulation of hydroxyproline rich glycoprotein (Sejalon Delmas et al., 1997). Fungal mycelium of barley pathogen Bipolaris sorokiniana derived elicitor induced PAL activity in barley cell suspension culture when it was compared with commercially available yeast glucan, chitosane and distilled water treated control. Increase in PAL enzyme levels was observed 2-8 h after the onset of elicitation along with the different types of elicitors (Peltonen et al., 1997). This finding was supported by Grosskopf et al. (1991) using crude elicitor, which contained a number of different signals or structures recognizable by plant. The isolated and partially purified elicitor from the fungal mycelia walls induces resistance in plants (Vidyasekaran, 1997; Velzhahan et al., 1998). Osman et al. (2001) and Buhot et al. (2001) also reported a proteinaceous elicitor isolated from Phytophthora species, which triggered defence response in tobacco plants. Soluble carbohydrate elicitor extracted from Blumeria graminis f.sp. tritici induced the accumulation of thaumatin-like proteins (TLP) in barley, oat, rye, rice and maize as reported by Schweizer et al. (2000).

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Elicitor from F. oxysporum induced lignin, favored callose deposition, and showed higher phenolics and enzymes involved in cell wall strengthening in banana cultivar, cv. Goldfinger. Three to four fold increase in activity of PAL, POX and PPO and higher accumulation of lignin and callose in root tissue culture after 4 h and 16 h of elicitor treatment was observed. Elicited root tissue also showed a prominent increase in total soluble phenolic acids when compared with control Ana et al. (2000). Umemura et al. (2000) were the first to demonstrate that a cerebroside has elicitor activity when sprayed on the intact rice plants as a purified biotic elicitor. Cerebroside elicitors induced phytoalexins activity when applied to plants by spray treatment and also induced the expression of PR proteins in rice leaves. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus Magnoporthe grisea. The cerebroside elicitors protected rice plants from other diseases, and they were found to occur in a wide range of different phytopathogens indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions. Treatment of suspension-cultured cells of rice (Oryza sativa) with cell wall extract of rice blast fungus (Magnaporthe grisea), elicits a rapid generation of H2O2, alkalinization of culture medium and eventual cell death. The elucidate genes were subjected to SAGE (Serial analysis of gene expression) analysis for the molecular analysis of cell death in suspension culture. Serial analysis of gene expression results further confirmed that the genes for PR proteins like Pbz1 and chitinases were represented among the most strongly induced genes in elicitor treatment when compared to non elicitor treatment (Matsumura et al., 2003). The cerebroside compound as a sphingolipid, which produced in diverse strains of F. oxysporum, and as an agent of wilt disease affected a wide range of plant species. Cerebroside A, B or C was detected in other soil-borne phytopathogens such as Pythium and Botrytis. Treatment with cerebroside B on different plants like tomato, lettuce, melon and potato resulted in resistance to infection by pathogenic strains of F. oxysporum. Cerebroside elicitor does not show any antifungal activity in vitro, but induces PR genes and H2O2 production in elicitor treated plants and also the defence mechanism by indicating resistance against Fusarium disease. Hence, cerebroside elicitor serves as a potential biologically derived control agent and functions as a 46

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non-race specific elicitor in a wide range of phytopathogenic fungus (Umemura et al., 2004). The efficacy of elicitor prepared from baker’s yeast was tested for their ability to induce an oxidative burst in cassava suspension cells. After treating cassava cell suspension with different concentrations of elicitor, enhanced PAL and POX activity and H2O2 accumulation were recorded. POX and PAL showed four-fold increase in activity after elicitation with yeast elicitor at 48 h and 15 h, respectively when compared with control (Vasquez et al., 2004). The induction of resistance in chickpea was investigated by spray inoculating 15 days old seedling with cell wall protein (CWP) of F. oxysporum f. sp. ciceris and Macrophomina phaseolina. Cell wall proteins enhanced synthesis of phenol, PR protein and defence enzymes (PAL, POX) compared with water treated control. The result suggested that CWPs of Fusarium and Macrophomina elicitor effectively induced resistance in chickpea and significantly reduced fusarium wilt and charcoal rot diseases of chickpea (Saikia et al., 2006). Earlier studies of Nicholson and Hammerschmid (1992) also showed that rapid esterification of phenolic compounds into the plant cell wall is a common and early response in the expression of resistance to elicitor. Induction of PR proteins (β-1,3-glucanase and chitinase) after crude glycoprotein elicitor prepared from mycelial mat of A. solani treatment and challenge inoculated with A. solani was observed in both cell suspension and in tomato plant respectively. It was concluded that the induction of PR proteins upon inoculation of glycoprotein elicitor of A. solani is due to the stability in the appearance and the binding sites of elicitor, which may be protected from degradation for several hours (Radhajeyalakhsmi et al., 2009). Effective T. harzianum was used for cell wall glucan elicitor and studied its effect on PR protein (glucanase) and phenol accumulation in red chilli. Elicitor fractions very quickly induced systemic resistance as compared to treatment with fungus and distilled water treated control plants (Sriram et al., 2009). Hanania and Avni (1997) observed that challenging tomato or tobacco varieties with ethylene inducing xylanase (EIX) from T. viride caused rapid induction of plant defence responses leading to PCD. 47

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In vitro studies were carried out to evaluate biochemical changes induced by combined fungal elicitors from T. viride, Penicillium chrysogenum and B. cinerea against pathogens and plant defence in regenerants of Fragaria x Ananassa Duch. Strawberry regenerants responded in gradual forms of stress up to foliar necrosis when treated with elicitors. Mixture of fungal elicitor used in this study acted as a strong external stimulus by expressing the enzymes involved in metabolism of ROS compared to control. In addition increase in the activity of SOD and decrease in CAT and POX activity were recorded. A significant ROS accumulation with complex and opposite effects on plant cells including the activity of hypersensitive cell death were noticed (Cogalniceanu et al., 2010). Elicitor prepared from F. oxysporum f. sp. cubense, which was isolated from infected banana rhizosphere was tested in relation to the accumulation of defencerelated enzymes in leaves of susceptible and resistant varieties of banana (Thakker et al., 2011). After treatment, POX activity increased two fold on the 2nd day and 7th day in susceptible and resistant varieties respectively. PPO activity increased up to three folds in susceptible and two-fold in resistant after 2nd day and 3rd day respectively. Chitinase activity increased up to three folds on the 2nd day in susceptible and 0.75 fold on 5th day in resistant variety when compared with respective control plants treated with distilled water. In susceptible varieties, total phenol content increased by 1.5 fold after elicitor treatment. They concluded that the plants were immunized due to elicitor, and even susceptible variety can also be made to tolerate the pathogen attack. 2.4.5. Biological Control- Plant growth promoting rhizobacteria (PGPR) Biological control involves the use of naturally occurring non-pathogenic microorganisms that are able to reduce the activity of plant pathogens and thereby suppress diseases. Hence, controlling this pathogen using biocontrol agents will help in enhancing the yield of the crop. Among different biological approaches, use of the microbial antagonists like yeasts, fungi, and bacteria is promising and gaining popularity (Droby, 2006; Korsten, 2006). In addition biological control using PGPR represent a potentially attractive, alternative disease management approach, which are known for growth promotion and disease protection against several plant pathogens (Jetiyanon and Kloepper, 2002). 48

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The rhizosphere was described in 1904 by Hiltner as being the volume of soil influenced by the presence of living plant roots, whose extension may vary with soil type, plant species, age and other factors (Foster, 1988). Root exudates rich in amino acids, monosaccharides, organic acids, vitamins, etc. serve as a primary source of nutrients, and support the dynamic growth and activities of various microorganisms within the vicinity of the roots. These root-colonizing microorganisms could be freeliving, parasitic or saprophytic, and their diversity remains dynamic with a frequent shift in community structure and species abundance (Kunc and Macura, 1988). An important group of microbial communities that exert beneficial effects on plant growth upon root colonization was first defined by Kloepper and Schroth (1978) and termed as plant growth promoting rhizobacteria (PGPR). Plant growth promoting rhizobacteria represent a wide variety of soil bacteria, which when grown in association with a host plant, stimulate the growth of their host and protect the plant from various infectious organisms (Vessey, 2003). Bacterial inoculants that help in plant growth are generally of two types, a) symbiotic and b) free-living (Frommel et al., 1991). The PGPR concept has been indicated by the isolation of many bacterial strains that fulfill at least two of the three criteria: aggressive colonization, plant growth stimulation and biocontrol (Lucy et al., 2004; Preston, 2004). However, breakthrough research in the field of PGPR occurred during mid 1970’s. The studies demonstrated the ability of Pseudomonas strains capable of controlling soil-borne pathogens to enhance plant growth and the yield of potato and radish plants (Kloepper and Schroth, 1981; Howie and Echandi, 1983). Mechanism of plant growth promotion and disease protection includes direct and indirect mechanisms. The direct effects are improvement of plant nutrient status (liberation of phosphates and micronutrients from insoluble sources and non-symbiotic nitrogen fixation) (Chabot et al., 1998), iron sequestration by siderophores or depletion of iron from the rhizosphere (Baker et al., 1990), production of bacterial volatiles and phytohormones and lowering of ethylene level in plant. Indirect effects that can be exerted by antibiotic production includes, pyrrolnitrin (Keel et al., 1990), phenazine-1-carboxylic acid (Thomashow and Weller, 1990) and 2, 4-diacetyl phloroglucinol (Keel et al., 1992), cyanide (Ahl et al., 1986) induced systemic resistance (Vessey, 2003), synthesis of antifungal metabolites (Dowling and

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O’Gara, 1994), production of fungal cell wall lysing enzymes, competition for sites and nutrition on the root, stimulation of other beneficial symbioses and degradation of xenobiotics in inhibitor-contaminated soils. Plant growth promoting rhizobacteria stimulate plant growth directly by synthesizing hormones such as indole acetic acid (Loper and Schroth, 1986; Fig. 2.3).

Figure 2.3 Schematic representation of plant growth promoting rhizobacteria (PGPR)–plant molecular signaling and plant-induced effects. T-PGPR; P-pathogen; IAA-indole-3-acetic acid; ACCD-ACC deaminase; ET-ethylene; JA-jasmonic acid; SA-salicylic acid; ISR-induced systemic resistance. (Modified, Hermosa et al., 2012) Induced disease resistance is a phenomenon by which the plant exhibits an increased leve1 of resistance to infection by a pathogen after appropriate stimulation. This resistance response was first characterized by Ross (1961a, 1961b) and expressed systemically throughout the plant and was effective against a broad spectrum of viral, bacterial and funga1 pathogens (Hammerschmidt and Kuc, 1995). A well-characterized system of rhizobacteria-induced resistance between A. thaliana and P. fluorescens strain WCS417 (Van Loon et al., 1998). The fluorescent pseudomonads were isolated from the rhizosphere of rice, tomato and cotton. Among the different isolates tested, Pseudomonas fluorescens Pf1 50

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significantly increased the plant vigor and consistently reduced the Fusarium wilt disease incidence under greenhouse conditions and disease protection offered was comparable to that of fungicide, carbendazim (Ramamoorthy et al., 2002a). Later, Ramamoorthy et al. (2002b) isolated 18 isolates belonging to P. fluorescens and two isolates were identified as P. putida and P. fluorescens. Isolate Pf1 biovar I showed maximum inhibition of mycelial growth of Pythium aphanidermatum and increased plant growth promotion of tomato and hot pepper and also witnessed lowest disease incidence in tomato and pepper. The effect of three plant growth-promoting rhizobacteria (Bacillus pumilus, B. licheniformis and P. fluorescens) was analyzed on the growth of tomato seedlings and pepper in two different sterilized and non-sterilized media. All the three isolates had the capacity to modify the plant growth. They had correlated the better growth in tomato over pepper with root colonization. The growth promoting activity of selected rhizobacteria was not affected by sterilized and non-sterilized peat (Lucas Garcia et al., 2003). The effect of chitin and salicylic acid on biological control activity of Pseudomonas spp. has been investigated against damping off of pepper (Rajkumar et al., 2005). Application of inducers such as salicylic acid enhanced the bacterial colonies and showed an increase in growth of pepper under greenhouse conditions. The maximum disease protection of red pepper was observed in the case of chitin amendment with fluorescent Pseudomonads. The successful control of fusarium wilt of tomato has been carried out by using a different combination of arbuscular mycorrhizal fungus (AMF) Glomus intraradices and four rhizobacteria from tomato and tobacco rhizoplane (Akkopru and Demir, 2005). Rhizobacterial isolates belonging to Bacillus, Pseudomonas, Azotobacter and Rhizobium from the rhizospheric area of chickpea were biochemically characterized and further screened in vitro for their plant growth promoting traits like production of IAA, ammonia, hydrogen cyanide, CAT, siderophore, etc. (Joseph et al., 2007). Intanoo and Chamswarng (2007) reported that two bacterial antagonists, Bacillus amyloliquefaciens strains DGg13 and BB133, controlled chilli anthracnose 51

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caused by C. capsici in Thailand. In India, treatment of chilli seed with P. fluorescens isolated from native soil reduced infection of the seed by C. capsici (Srinivas et al., 2006). In addition, P. fluorescens Pf1 can induce systemic resistance in chilli plants against C. capsici by increasing the activities of specific enzymes related to phenylpropanoid metabolism and PR-protein accumulation (Ramamoorthy and Samiyappan, 2001; Anand et al., 2007). Recently, it has been revealed that certain PGPR having ACC-deaminase enzyme activity that cleaves the plant ethylene precursor ACC into α-ketobutyrate and ammonia, and thereby lowers the level of ethylene in a developing or stressed plant (Glick et al., 1998; Arshad et al., 2007). Plants that are inoculated with rhizobacteria having ACC-deaminase are more resistant to the injurious effects of stress ethylene that is produced as a result of stressed environments such as flooding (Grichko and Glick, 2001), drought (Zahir et al., 2007), heavy metal stress, high salt concentration (Kausar and Shahzad, 2006; Nadeem et al., 2006) and pathogen attack (Glick et al., 2007). Isolation and characterization of phosphate-solubilizing rhizobacteria from tomato rhizosphere, a diverse group of rhizobacteria that belonged to the genus, Bacillus, Psedomonas, Azotobacter, Serratia, Azospirillum, Enterobacter etc. has been studied by Hariprasad and Niranjana (2009). Their analysis revealed that the plant growth promotion exhibited by these isolates was by more than one mechanism that included, phosphate solubilization, IAA production, ACC deaminase, siderophore, chitinase and β-1,3-glucanase production. Bal et al. (2013) evaluated ACC-deaminase producing strains of rhizobacteria from rice rhizospheric soil of coastal rice field of West Bengal for their plant growth promotion activities. In addition, they also estimated ethylene in rice seedlings treated with PGPR having ACC-deaminase activity. This study revealed plant growth parameters in rhizobacteria treated plants were significantly improved compared to non-inoculated seeds. Further, plants inoculated with live ACC-deaminase producing bacteria reduced ethylene production significantly than the negative control and heat killed bacteria treated plants.

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ACC-deaminase could be effective trail in promoting chickpea growth under axenic conditions (Shahzad et al., 2010). They screened rhizospheric soil of chickpea from different districts of Panjab, Pakistan, for effective strains of rhizobacteria for ACC-deaminase production. In their study they noticed that significant improvement in plant growth of ACC-deaminase containing bacteria inoculated chickpea plants over non-inoculated control plants. Various biochemical pathways of plants that are activated by PGPR were reviewed by Van Loon et al. (1998). Plant growth promoting and bioprotecting bacteria trigger ISR fortified plant cell wall strength. They also alter host physiology and metabolic responses leading to enhanced synthesis of plant defence chemicals upon challenge with pathogens and/or abiotic stress factors. The bacterized plant response induced after challenge by a pathogen include the formation of structural barriers such as thickened cell wall papillae due to the deposition of callose (Benhamou et al., 1996). Biochemical or physiological changes taking place in plants include induced accumulation of pathogenesis-related proteins such as PR-1, PR-2, chitinase and some peroxidases. However, some PGPR does not induce PR-proteins but rather increase accumulation of POX, PAL, PPO and phytoalexin (Zdor and Anderson, 1992). Similarly, β-1,3-glucanase, chitinase and TLP were induced to accumulate at higher levels in plants raised from PGPR treated seeds followed by challenge inoculation (Ramamoorthy et al., 2002a). The plant-signaling molecules such as SA, JA and ET play an important role in the signaling network; blocking the response to any of these signals can render plants more susceptible to pathogens (Gaffney et al., 1993; Thomma et al., 1998). In Arabidopsis, SAR and ISR are regulated by distinct signaling pathways. As in many other plant species, pathogen-induced SAR is associated with local and systemic increases in endogenously synthesized SA and a coordinated expression of genes encoding PR proteins (Uknes et al., 1992; Lawton et al., 1995). Salicylic acid is a necessary intermediate in the SAR signal transduction pathway because SA-non accumulating NahG plants, expressing the bacterial SA hydroxylase gene NahG get impaired of SAR (Lawton et al., 1995). During ISR, colonization with rhizobacteria leads to an enhanced expression (priming) of jasmonate-inducible genes (Van Wees et al., 1999). With the help of 53

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various biosynthesis and perception mutants for plant signal molecules, it was possible to demonstrate that ISR requires functional jasmonate and ethylene signaling (Van Loon et al., 1998; Pieterse et al., 2001). In contrast, WCS417r-mediated ISR functions independently of SA and PR gene activation (Van Wees et al., 1997) but requires JA and ethylene signaling. The JA response mutant jar1 and the ethylene response mutant etr1 that express normal level of pathogen-induced SAR (Pieterse et al., 1998) did not express ISR upon treatment with WCS417r indicating that the ISR-signaling pathway requires components of the JA and ethylene response (Pieterse et al., 1998; Knoester et al., 1999). Cross-talk between SA and JA-dependent pathways can result in inhibition of JA-mediated defence responses. Van Wees et al. (2000) investigated possible antagonistic interactions between the SA-dependent SAR pathway, which is induced upon pathogen infection and the JA-dependent ISR pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. They showed that the enhanced level of protection is offered through parallel activation of complementary, NPR1-dependent defence responses both of which are active against P. syringae pv. tomato. Induced systemic resistance was resulting from exposure to volatiles from Bacillus sp. GBO3 was independent of SA, JA and Npr1, but appears to be mediated by ethylene. Induced systemic resistance triggered by volatiles from the bacterial strain IN937 is even independent of ethylene signaling pathways (Pieterse et al., 2002). The ability of chitinolytic bacterial strain BK08 as a biocontrol agent of fusarium wilt of red chilli (C. annuum) was examined by soaking seeds in bacteria for 30 min. Significant (28.57-60.71%) reduction in damping off in bacteria treated seedling was observed. There was an increase in seedling height (7.33-7.87 cm compared with control, which was 6.88 cm) and dry weight (2.7-4.3 mg compared with 2.3 mg in control), but no significant difference in leaf number that indicates chitinolytic bacteria exhibited suppression of fusarium wilt. Further, it was concluded that the chitinolytic isolate BK08 was the potential biological control agent against fusarium wilt in chilli seedlings (Suryanto et al., 2010). Nantawanit et al. (2010) evaluated the ability of the Pichia guilliermondii (yeast) strain R13 in suppressing the growth of C. capsici in chilli. By its multiple modes of action like nutrient competition, tight attachment to the fungus and 54

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hydrolytic enzyme secretion, the pretreatment of chilli with the yeast antagonist significantly reduced disease incidence and lesion diameter caused by C. capsici. The yeast treatment also significantly enhanced the activities of PAL, chitinase and β-1,3-glucanase, and the accumulation of capsidiol phytoalexin in chilli tissue compare to control. Similar kind of evidence was reported by Chanchaichaovivat et al. (2007). They observed yeast antagonist P. guilliermondii strain R13 effectively controlled

C. capsici on chili fruit by competition for nutrients, tight attachment of

the yeast to the fungus and secretion of hydrolytic enzymes including chitinase and β-1,3-glucanase. The activation of defence responses in chilli (C. annuum) after foliar application of biocontrol agent like Burkholderia sp strain TNAU-1 was evaluated by (Madhavan et al., 2011). They found induction of phenolics, POX, PPO, chitinase and TLP in leaves of chilli. Burkholderia sp. strain TNAU-1 treated plant exhibited a major PPO (PPO-1) one day after treatment and subsequently the activity decreased. Western blot analysis of protein extracts from Burkholderia sp. strains TNAU-1 treated chilli leaves revealed that two TLPs with sizes of 23 and 25 KDa and chitinase with molecular weight 28 KDa were induced 3 days after treatment. El-Mabrok et al. (2012) isolated lactic acid bacteria (LAB) from fresh fruit and vegetables and screened for their antifungal activity against C. capsici. Among 324 LAB, 7 isolates showed good inhibitory activity conidia germination of C. capsici in overlay method. Further, fungal growth inhibition and enhancement in seed germination was observed with supernatant of effective strain LAB C5 when evaluated through microtitre plate method and seed treatment respectively. Lamsal et al. (2012) evaluated efficacy of seven rhizobacterial isolates belonging to species of Bacillus and Paenibacillus in suppressing the growth of pepper anthracnose pathogen C. acutatum and its plant growth promotion activities under in vitro and in vivo conditions. AB15 isolate (P. polymyxa) exerted highest mycelial growth inhibition (69%) and the antibiotic of this isolate showed maximum zone of inhibition against pathogen in dual culture assay. Further, greenhouse studies revealed AB15 was identified as effective strain in inhibiting the pathogen growth and isolate AB17 induced the plant growth parameters.

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Suwan et al. (2012) evaluated six effective strains of Streptomyces (biofungicides) against C. capsici. Biofungicides NSP-1, NSP-3, NSP-5 and NSP-6 significantly reduced the disease incidence and increased the plant growth parameters such as plant height, stem fresh/dry weight, root fresh/dry weight, root length and fruit yield at 0.5-2.0 g/l concentration. Seed treatment of chilli with Bacillus species collected from chilli rhizosphere soil was carried out by Ashwini and Srividya (2013) for the control of anthracnose disease caused by C. gleosporioides. Treatment of chilli seeds with Bacillus species culture showed 100% germination as in untreated seeds. The seeds coinoculated with pathogen and Bacillus species culture showed 65% reduction in disease rate as compared to the treated and pathogen alone treated. With this background, interaction of pathogen with the host and its control against anthracnose of chilli were planned and executed with the following objectives; 1. Isolation, identification and molecular characterization of Colletotrichum capsici. 2. Host-pathogen interaction studies. 3. Development of suitable management strategies to control anthracnose disease in chilli.

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