Indian Phytopath. 68 (1) : 8-24 (2015)

AWARDS AND HONOURS

K.C. MEHTA AND MANORANJAN MITRA AWARD - 2013 Chaetomium globosum: A potential biocontrol agent and its mechanism of action

RASHMI AGGARWAL Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India

Introduction as compared to diseases of aerial parts of the plants. It is India with diverse soil and climate comprising several due to the reason that more agro-ecological regions provides ample opportunity to emphasis has been given to control foliar diseases by grow a variety of crops and these crops form a significant fungicides. But fungicides and other toxicants have part of total agricultural production in the country. More negative side effects on ecosystem (Dickson and than 70% of all major crop diseases are caused by fungi. Skidmore, 1976). These pathogens perpetuate through seed, crop residue and soil. Considering the nature of the disease, control Recently, there are reports on the antagonistic measures are directed towards host resistance and behaviour of fungi against foliar pathogens. Butler (1953) fungicidal control. Breeding for disease resistance has observed inhibitory effect of Trichoderma spp. on the also been not successful in many cases due to non- growth of Cochliobolus sativus. The T. viride has also availability of sources of resistance. Chemical control been found to be antagonistic to Bipolaris sorokiniana, measures have shown promise but are not considered which infects wheat (Prasad et al., 1978). Mostafa (1993) economical and eco-friendly. Since their large-scale use succeeded in controlling the seed-borne infections of has been responsible for the undesirable effects such Drechslera teres on coleoptiles by treating barley seed as persistence, bioaccumulation, biomagnification, with inoculums of T. viride. In this context, Trichoderma, toxicity, pathogen resistance, secondary pest outbreak Gliocladium and Chaetomium spp. have been explored and destruction of the non-target organisms, these have for the control of B. sorokiniana (Butler 1953; Prasad et necessitated looking for alternative approaches, such as al., 1978; Mostafa, 1993; Srivastava et al., 2000). In vitro biological control. and in vivo studies have shown that cell free culture filterate of Trichoderma reesei and Chaetomium Biological control of fungal plant pathogens appears globosum inhibited the growth of spot blotch pathogen, as an attractive and realistic approach. A number of B. sorokiniana (Mandal, 1995; Mandal et al., 1999). numerous microorganisms have been identified as biocontrol agents. The aim of biological control is the Foliar spray of purified culture filterate of C. globosum reduction of disease by: (i) reduction of inoculum of the on B. sorokiniana infected wheat leaves revealed pathogen through disease survival between crops, (ii) distortion of conidial wall and non germination of conidia reduction of infection of the host by the pathogen and of the pathogen resulting in the reduced blotching (iii) reduction of severity of attack by the pathogen. (Biswas et al., 2000).

Biological control is a principal eco-friendly cultural The C. elatum strain ChE01, C. lucknowense strain method for controlling the plant diseases, and pests. In CLT01 and Emericella rugulosa strain ER01, which were recent years, biological control strategy has received isolated from soil in Thailand, effectively controlled the tremendous attention in controlling plant diseases due most virulent isolate of Fusarium oxysporum f. sp. to hazardous effects of pesticides and agro-chemicals lycopersici NKSC02, causing wilt of tomato on ecosystem (Lockwood, 1988). As an alternate strategy (Lycopersicon esculentum var Sida (Sibounnavong et al., to agro-chemicals, there are about 35 genera of fungi 2011). Present article reviews in details the biology, and bacteria, which have been used as biocontrol agents bioefficacy and mechanism of action of Chaetomium against various plant pathogens (Cook and Baker, 1983). globosum. Chaetomium spp., Trichoderma spp., Gliocladium spp., Aspergillus spp., Bacillus spp., Pseudomonas spp. and Biology of Chaetomium globosum Actinomycetes represent the major antagonistic microorganisms against several plant pathogens. Most The Chaetomium is one of the extremely familiar genus of the work on biological control is on root-borne diseases of Pyrenomycetes (Ascomycotina) encountered on various agricultural commodities. The genus contains *Corresponding author: [email protected] more than 200 species, all characterised by dark Indian Phytopathology 68 (1) : 8-24 (2015) 9

Later, Tviet and Wood (1955) suggested control of Fusarium blight of oat seedlings by introducing antagonistic species of Chaetomium in soil. Chang and Kommendahl (1968) observations revealed that the competition for nutrients and space led to physical exclusion of F. graminearum by coating maize seeds with C. globosum. Seed infestation with Chaetomium spp., has been successful in controlling seedling blight caused by R. solani (Baker, 1968). Antagonastic effect of C. globosum to rice blast (Pyricularia oryzae) was reported by Soytong and Quimio (1989). Kommendahl and Mew (1975) observed increased field stands of maize hybrids when seeds were coated with C. globosum. Fig. 1. Perithecia and ascospores of Chaetomium globosum Seed treatment with ascospores of C. globosum reduced damping off of sugarbeet caused by seed-borne coloured perithecia with short neck, which are clothed Phoma betae and soil-borne Pythium ultimum or R. solani with irregularly coiled or tightly spiraled hairs or with stiff, (Walther and Gindrat, 1988). When the sugarbeet seeds simple or branched setae. It is common colonizer of soil were planted after treating with C. globosum ascospores, and cellulose containing substrates. the spores germinated rapidly and covered the seed coat with a dense mat of mycelium and suppressed seed rot Perithecia of C. globosum are ostiolate, variable in (Hubbard et al., 1982). Apple scab disease caused by shape, sub globose, somewhat elongated with a bluntly Venturia inaequalis was significantly reduced with foliar pointed base, when young yellow, transluscent, allowing sprays of C. globosum ascospore suspension (Andrews the cellular structure of the wall to be seen. When mature et al., 1983). Boudraeu and Andrews (1989) also opaque, black 200-320 x 200-280µ. Often producing achieved control of apple scab disease with ascospores short, black cirrhi, attached to the substrate with a thick of C. globosum. Vannacci and Harman (1987) reported mass of dark olives to black rhizoids, colour ranging from increased number of healthy radish plants and decreased gray, green, light brown to olive brown (Fig. 1). Terminal pod infection of Alternaria raphani by C. globosum. Vilich hairs numerous, interwoven, forming a bushy or ompact (1988) reported antagonistic effect of C. globosum on head, in age spreading and drooping, the hairs slender, Erysiphe graminis f. sp. hordei causing powdery mildew graceful, undulating, seldom septate, minutely roughened of barley. Treating seeds with C. globosum resulted in an with spines throughout, at base about 3.5µ in diameter increase in fresh weight of root and reductions in disease and dark olive-brown, lighter brown through the greater severity ranging from 16-48% as compared to untreated part of length, tapering and paler to hyaline, at tip, wavy, control. The use of spore suspension for inoculation, led undulate or kinked. Lateral hairs numerous, slender, to better colonization of the tissues. Di Pietro et al. (1992) plainly, or obscurely, and remotely septate, finely reported that C. globosum can produce chetomin, which roughened with spines. Quite dark at base, olive-brown, effectively inhibited Pythium ultimum, which caused about 3.5µ in diameter, light olive, yellow to hyaline at damping-off of sugarbeet. tip, straight or only slightly flexed, or more slender and A significant reduction in pseudothecial initials and undulate or even kinked. Asci irregularly club-shaped, production of asci and ascospores of V. inaequalis was 8-spored, 64 x 13µ pars sporif 37µ. Ascospores filled reported when C. globosum activity was enhanced by with several large, refractive globules, when mature dark, spraying of urea @ 5% at leaf abscission on scab affected rich, olive-brown, varying shaped, with the ends apiculate apple leaves (Thakur and Sharma, 1999). Harrison and or sub-umbonate or nearly rounded, varying in size, 9- Stewart (1988) reported C. globosum as the best 13 x 6-9.5µ, when viewed edgewise frequently antagonistic microorganism against onion white rot, compressed, 7µ broad. caused by Sclerotium cepivorum. Kay and Stewart (1994) also achieved successful disease control of onion white Antagonism of Chaetomium globosum rot and confirmed that use of C. globosum is as effective as T. harzianum and T. viride. Mandal et al. (1999) Chaetomium globosum has been reported effective in reported the inhibitory effect of C. globosum, T. roseum, reducing damage caused by seed rot and damping off, T. recesei, T. hamatum and Talaromyces flavus on of several seed- and soil-borne plant pathogens like mycelial growth of B. sorokiniana causing spot blotch of Pythium ultimum, Alternaria raphani, A. brassicola, wheat. Culture filtrate of these fungi inhibited conidial Fusarium spp. and B. sorokiniana (Harman et al., 1978; germination up to 92%. Soil and foliar application of Vannacci and Harman, 1987; Aggarwal et al., 2004). antagonists reduced the spot blotch lesions and protected Seed coating with selected isolates of C. globosum green leaf area from drying due to infection (Biswas et protected corn, oat and barley from seedling blight al., 2000). caused by Fusarium spp. and Drechslera sorokiniana (Kommedahl and Mew, 1975). Tveit and Moore (1954) The SEM showed lysis of mycelium due to over found that Chaetomium isolated from oat seeds protected growth and penetration through hyphal pegs and tight oat seedlings from infection by seed-borne D. victoriae. coiling produced by antagonists. Selvakumar (2000) 10 Indian Phytopathology 68 (1) : 8-24 (2015) studied the biocontrol potential of C. globosum for the Mechanism of biological control management of spot blotch of wheat. The C. globosum reduce the primary inoculum of Diaporthe phaseolorum Understanding the mechanism (s) of action involved in f. sp. meridionalis in soil surface soybean stubble under biocontrol process is primary importance for establishing field conditions (Dhingra et al., 2003). Aggarwal et al. the effectiveness of biocontrol agents and will provide (2004) tested the role of antibiosis in biological control much into where and when the interaction occurs and of spot blotch (C. sativus) of wheat by C. globosum (Fig. how the pathogens will be affected (Larkin et al., 1996) 2). The C. globosum strain, F0142, which was isolated and also have potential to interfere in the life process of from barnyard grass, showed potent disease control plant pathogens (Cook and Baker, 1983). The efficacy against rice blast (Magnaporthe grisea) and interference may be directly on the pathogen wheat leaf rust (Puccinia recondita). Two antifungal (antagonism) or through the intermediate agency of the substances were purified from broth from this organism host (Baker, 1968). Classically, the antagonism and identified as chaetoviridins A and B. Chaetoviridin A categories are antibiosis, competition and the exploitation exhibited higher antifungal activity than chaetoviridin B (Park, 1960). Many advances have been made in this against plant pathogenic fungi both in vitro and in vivo. subject over the past two decades especially with the advent of molecular biology as a research tool. Many Treatment with chaetoviridin A at 62.5 mg/mL species of Chaetomium with the potential to be biological suppressed the development of rice blast and wheat leaf control agents suppress the growth of bacteria and fungi rust by over 80%. The molecule also exhibited moderate through competition (for substrate and nutrients), control of tomato late blight, resulting in 50% control mycoparasitism, antibiosis, or various combinations of following the application of 125 mg/mL chaetoviridin A these (Marwah et al., 2007; Zhang and Yang, 2007). (Park et al., 2005). Adaptation of C. globosum 3250 kunze ex. Fr to the rhizosphere of spring wheat and its ability to Competition: Competition has been defined as the colonize the root system was studied by Patyka et al. active demand in excess of the immediate supply of the (2007). Biswas et al. (2012) studied the characterization material on the part of two or more organisms (Clark, of antifungal metabolites of C. globosum kunze and their 1965). Competition between microorganisms generally antagonism against fungal plant pathogens. Role of refers to the competitions for nutrients, space for endophyte Chaetomium globosum Lk4 was observed in colonization or infection site on the root or seed surface growth of Capsicum annuum by production of gibberellins (Paulitz, 1990). Chang and Kommendahl (1968) revealed and indole acetic acid by Khan et al. (2012). that the competition for nutrients and space led to physical exclusion of F. graminearum by coating maize seed with C. globosum.

Antibiosis: Antibiosis refer to the inhibition or destruction of pathogens by a metabolic produce of the antagonist such as production of specific toxins, antibiotics or enzymes. Cullen and Andrews (1984) reported evidence of antibiosis by C. globosum against V. inaequalis, which causes apple scab. The positive relation between the production of antifungal metabolite (Chaetomin) by C. globosum isolates in liquid culture or soil and efficacy in suppressing P. ultimum causing damping off has been reported by Di Pietro et al. (1992).

The Chaetomium spp. produce various biological active substances such as chaetomin, chetocin, chaetoglobosins, chaetoglobosin a, chaetochromosins, cochliod, colletodial, mollicellin, oosporein and strerigmatosytin (Powell and Whalley, 1969; Brewer et al., 1970; Udagawa et al., 1979; Sekita et al., 1981). Fig. 2. Antifungal metabolites extracted from different isolates Cullen and Andrews (1984) observed the production of of Chaetomium globosum (Cg1-Cg6) and their bioassay one antibiotic, chaetomin, which was inhibitory to V. against Bipolaris sorokiniana inaequalis. Amemiya et al. (1994) reported an antifungal substance identified as Chaetoglobosin which completely Nematicidal activity of chaetoglobosin A produced inhibited spore germination of Vericillium dahlia, causal by C. globosum NK102 against Meloidogyne incognita agent of tomato wilt. Antagonastic effect of C. globosum had been studied and the results showed that C. to rice blast pathogen was reported by Soytong and globosum NK102 significantly repelled second-stage Quimio (1989). A diffusible antibiotic was isolated from juveniles (J2s) (Hu et al., 2013). Both filtrates and ChA C. globosum (Hubbard et al., 1982). Later, Di. Pietro et demonstrated strong adverse effects on J2 mortality with al. (1992) suggested that antibiotic was responsible for 99.8% at 300 µg ChA/mL (LC(50) = 77.0 µg/mL) at 72 h. inhibition of Pythium spp. The plant disease control ChA and filtrates did not affect egg hatch until 72 h of mechanism may involve in antibiosis, with the exposure. antagonistic fungus releasing antibiotic substances Indian Phytopathology 68 (1) : 8-24 (2015) 11

(Soytong et al., 2001; Kanokmedhakul et al., 2002, 2006; identified four new dimeric spiro-azaphilones; Park et al., 2005). (cochliodones A to D), two new azaphilones; (chaetoviridines E and F), and a new epi-chaetoviridin Ketomium mycofungicide appears to be a promising A. The isolate of the C. elatum strain ChE01 has been preparation maintaining activity over a relatively long reported to produce a chaetoglobosin V, storage period and acts as a new broad spectrum prochaetoglobosin III, chaetoglobosins B to D, F and G, mycofungicide (Soytong et al., 2001). Kanokmedhakal and isochaetoglobosin D, which have been shown to et al. (2002) isolated anti-mycobacterial anthraquinone exhibit cytotoxicity against a human breast cancer cell alkaloid compound and diketoppiperzine alkaloid from line. It should be noted that C. elatum ChE01 can also the fungus C. globosum KMITL-M0802. This strain has produce chaetoglobosin-C as a major compound, been shown to produce chaetoglobosin-C, which inhibits comprising up to 2% of the dried mycelial mat when some pathogens (Kanokmedhakul et al., 2002). grown in liquid culture (Thohinung et al., 2010). The Production of antifungal compounds by C. globosum and antimicrobial activity of chaetoglobosin-C, which is their role in suppression of spot blotch (C. sativus) of produced by C. elatum ChE01, and C. lucknowense wheat caused by this fungus under in vitro and in vivo CLT01, and of tajixanthone, which is produced by E. has been evaluated. Interaction between C. globosum rugulosa ER01, could be involved in the disease control isolates and C. sativus showed mycoparasitism by mechanism of these antagonistic fungi against tomato isolates Cg 1 and Cg 6, whereas isolates Cg 2, Cg 3, Cg wilt fungus F. oxysporum f. sp. lycopersici. 4 and Cg 5 showed antibiosis (Aggarwal et al., 2004). The fungicidal effects of the biological preparation Enzymes: The involvement of enzymes in biocontrol Ketomium® comprising Chaetomium spore suspension blurs the distinction between parasitism and antibiosis, were evaluated for its effect on Siberian isolates of the because the production of cell wall degrading enzymes phytopathogenic fungi, Botrytis cinerea, Didymella by the antagonists may be simultaneously involved in applanata, F. oxysporum and Rhizoctonia solani parasitism and antibiosis or cause only antibiosis (Shternshis et al., 2005). Park et al. (2005) tested the depending upon the pathogen associated. Chester and antifungal activity of chaetoviridins isolated from C. Bull (1963) found that 90% of the 160 fungi produced globosum and concluded that these are effective against cell wall degrading enzymes. A role for the production of rice blast and wheat rusts. cell wall degrading enzymes such as β 1,3- glucanase, Aggarwal et al. (2007) studied the quantitative chitinases, proteases, cellulases, Xylanases, esterases, analysis of secondary metabolites produced by C. alkaline phosphatase and lipase by mycoparasites during globosum. The quantitative estimation of crude extracts interactions with other fungi has frequently been by TLC showed maximum production by isolate Cg3 (47.2 suggested (Elad et al., 1982, 1983; Ridout et al., 1988; mg/mg mycelium) followed by Cg2 (44.2 mg/mg). Isolate Tweddell et al., 1994). Production of cellulases by Cgl produced minimum quantity of metabolites (7.44 mg/ different isolates of C. globosum Krunze ex. Fr. has been mg). Further, characterization of antifungal metabolites studied by Ahammed et al. (2008a) and concluded that of C. globosum was studied by Biswas et al. (2012) and maximum activity of cellulases was obtained in Cg2 they reported that five metabolites like chaetoglobosin, isolate after nine days of incubation. chaetomin, BHT, Mollicelin G and cochliodinol of C. Extracellular cellulase production was studied by globosum of which two metabolites, viz. chaetoglobosin estimating filter paper activity (FPase), carboxy methyl and chaetomin proved effective in suppressing the growth cellulase (CMC-ase) and cellobiase activity in the aliquots of B. sorokiniana, F. graminearum, P. ultimum, of the medium by the method of Mandels et al. (1976). Macrophomina phaseolina and R. solani under in vitro Filter paper activity (FPase) of C. globosum isolates conditions. The antagonism test demonstrated the revealed that maximum activity was observed after 9 days antagonistic activity of C. elatum ChE01, C. lucknowense of incubation. The isolate Cg2 had the maximum activity CLT01 and E. rugulosa ER01 to inhibit the conidial (0.79 IU/ml) while Cg5 had least activity (0.48 IU/ml). production of F. oxysporum f. sp. lycopersici NKSC02 Similarly, the carboxy methyl cellulase (CMC-ase) activity between 63 to 77% (Sibounnavong et al., 2011). was maximum in case of isolate Cg2 and productivity of Soytong (1992) and Soytong et al. (2001) showed cellobiase was also maximum in case of isolate Cg2 and that a specific isolate of C. cupreum produced secondary least in Cg1 on ninth day of incubation. Production, partial metabolites that significantly suppressed tomato wilt purification and characterization of extracelluar xylanase caused by F. oxysporum f. sp. lycopersici in tomato fields from C. globosum was studied by Ahammed et al. in Thailand, and later found that this isolate of C. cupreum (2008b). Study revealed that both the purified xylanase produced rotiorinols A to C and rotiorin, which also and culture filtrate have shown the antifungal activity exhibited antifungal activity against Candida albicans against Bipolaris sorokiniana, a causal organism of spot (Kanokmedhakul et al., 2006). The C. cochlioides-strains, blotch of wheat. Purified xylanase at 100 µg ml”1 VTh01 and CTh05, have been shown to exhibit concentration caused 100 per cent inhibition of conidia antimicrobial activity against a Phytophothora spp. that germination of B. sorokiniana, whereas the culture filtrate causes root rot, the anthracnose fungus Colletotrichum was able to inhibit germination up to 67.5%. Recently, gloeosporioides, and F. oxysporum f. sp. lycopersici. an extracellular β-1, 3-glucanase produced by C. Among the compounds isolated from C. Cochlioides globosum (Cg 2) was purified and characterized by strains, VTh01 and CTh05, Phonkerd et al. (2008) Ahammed et al. (2012). 12 Indian Phytopathology 68 (1) : 8-24 (2015)

Characterisation of Chaetomium globosum exoxylanases, b D xylosidase. Endoxylanes of different types have been described based upon the cleavage Cultural and morphological characterization: points of xylan (Reilly, 1981). Xylanases have been Chaertomium can produce an Acremonium like state isolated, characterized and crystallized from various (imperfect stage) on fungal media and is characterized mesophillic fungi (Fournier et al., 1985; Wong et al., by superficial flask shaped perithecia and they are 1986). Ganju et al. (1989) isolated two xylanases (I and clothed with dark, stiff hairs. The perithecial hairs can II) out of several extracellular xylanases produced by the take a variety of forms depending upon the species, thermophillic fungus, Chaetomium thermophile var. which in turn enclose lemon shaped ascospores. C. coprophile. globosum is one of the commonest species growing saprophytically in rhizosphere and phyllosphere. In this The production of xylanase by different isolates of species the ascospores mature and are released inside C. globosum under different periods of incubation the perithecium and are then squeezed out through an indicated that xylanase activity was maximum in isolate opening at the top (ostiole). C. globosum colonies are Cg2 after 9 days of incubation at pH 5.5 and temperature rapidly growing, cottony and white in colour initially. 30°C when the medium was amended with xylan @ 1.5% Mature colonies become grey to olive in colour. C. as carbon source and ammonium dihydrogen phosphate globosum is reported to produce mycotoxins. Setika et @ 0.6% as nitrogen source. Further purified xylanase al. (1981) screened Chaetomium spp. fraction on SDS PAGE showed a unique band of MW 32KDa. The specific activity of the enzyme was 18.36IU/ For mycotoxin production on rice culture by a mg protein and optimim pH and temperature were combination of cytotoxicity tests using HeLa cells and reported to be 5.5 and 35C respectively (Ahammed et thin layer chromatography and repoted the production al., 2008b) (Fig. 3). of sterigmatocystin, O-methyl cochliodinols and Mollecilin G. Ahammed et al. (2008a) studied on nine isolates of M1 2 C. globosum and found that isolate Cg1, Cg6, Cg7, Cg8 97.4 and Cg9 were fast growing but produced less number of perithecia, while isolates Cg2, Cg3, Cg4 and Cg5 were 66 slow growing showing profuse perithecia formation. Morphology of perithecia as observed under light 43 microscope indicated differences among different isolates. Isolate Cg1 showed egg-shaped perithecia while 29 KDa Cg7 produced flask shaped perithecia. 29 Nutritional requirements: Nutritional requirement of biocontrol agent revealed that nitrogen source was essential for the spore germination and carbon sources 20.1 for both sporulation, growth and biomass production (Monga, 2001). Monga (2001) studied the effect of carbon 14.3 and nitrogen sources on spore germination, biomass production and antifungal mertabolites by the species Fig. 3. Purified β 1, 3 glucanase from C. globosum through SDS- of Trichoderma and Glocladium. Barnett and Ayers (1981) PAGE. Lane M: Molecular weight marker; Lane 1 and 2; studied nutritional and environmental factors affecting purified enzyme growth and sporulation of Sporidesmium sclerotivorum, a mycoparasite of sclerotia that grew well on an agar medium containing mineral salts, glutamine or casamino β 1,3 glucanases: Chitin and β 1,3 glucans are the main acid as nitrogen source, glucose as carbon source, structural components of fungal cell walls (except those thiamine as growth factor within the range of pH 5.0-5.5 from members of class oomycetes, which contain and optimum temperature was 22.5-25.0°C. cellulose). These enzymes have been shown to be Standardization of nutritional requirements for axenic produced by several fungi and bacteria and may be an culture of C. globosum revealed that sucrose as C source important factor in biological control (Artigues and Davet, @ 40g/l, casamino acid as N-source @ 10g/l and vitamin, 1984; Elad et al., 1982). Fungal cells walls contain thiamine @ 100µg/l supported maximum growth of the different kinds of β glucans as structural components. fungus, when amended in basal medium having pH 5.0 These b glucans are generally composed of a major and incubated at 28°C. component of predominantly β 1,3 linked glucans with branches of β 1,6 glycosidic linkages. Some types of b Biochemical characterization glucan degrading enzymes classified according to the type of β glucosidasidic linkages that they cleave and Xylanase: Xylanase play an important role in degradation the mechanism of substrate attacked. of xylan, which are the only hemicelluloses in grasses (monocots) and the major hemicelluloses in hard woods They can hydrolyse the substrate by two possible (dicots). Xylans possess a backbone composed of b (1- mechanisms, identified as the products of hydrolysis (i) 4) linked xylosyl residues. Xylanases are composed of exo β glucanases hydrolyse sequentially cleaving three groups of enzymes: endo 1,4 b D xylanases, glucose residue from non reducing end or (ii) endo β 1,3 Indian Phytopathology 68 (1) : 8-24 (2015) 13 glucanases cleave b linkages at random sites along the main clusters with only 65.3% similarity between them. polysaccharide chain, releasing smaller The dendrogram showed high genetic similarity among oligosaccharides. Degradation of β glucans by fungi is different isolates of C. globosum obtained from different often accomplished by the synergistic action of both endo sources. Cluster I consisted of isolates Cg1, Cg9, Cg4, and exo glucanases. Recently, an extracellular β-1, 3- Cg5, Cg6, Cg7 and C. perlucidum and C. reflexum. The glucanase produced by C. globosum (Cg2) was purified other C. globosum isolates were grouped into cluster II, and characterized by Ahammed et al. (2012). Purified β in which Cg2 and Cg3 showed a high bootstrap value 1,3 glucanase fractionon SDS PAGE showed a band of (98.2%), forming a separate subgroup within this cluster. MW 29 Kda indicating homogeneity. The enzyme showed This group showed >63% genetic similarity among the specific activity of 19.14 IU/mg of protein and its pH and isolates. temperature optima were reported to be 5 and 40°C respectively. A PCR-based marker has been developed for detection of C. globosum, which will help to detect the fungus at place of its application. The specific marker Molecular characterization of Chaetomium species also can detect the presence of C. globosum in soil, root Since the discovery of DNA fingerprinting in 1985, there and leaves. The detection limit of marker in conventional has been tremendous progress made in methodology PCR assay is 75pg. The sensitivity and utility of SCAR comparing DNA samples. The invention of DNA marker is further enhanced by developing q-PCR using databases has brought about some major improvements primer set SCCgQF/SCCgQR designed from to DNA fingerprinting technology in general, and helped SCCgRA1900 (Aggarwal et al., 2014). in authentic identification and taxonomic positioning of Ribosomal DNA (rDNA) in eukaryotes typically new fungi where there are limitations based on present in several hundred tandemly repeated multiples morphological character of identification. The DNA copies. They are one of the most extensively molecular fingerprinting techniques broadly classified into two markers. Syed et al. (2009) took the phylogenetic analysis categories: In type I DNA fingerprinting, a known gene to discuss the genetic relationship of plant endophytic or known DNA sequence are visualized by hybridization and free-living Chaetomium species distributed in South of the restricted DNA fragments with known labeled East Asia, Europe and Australia. They pointed out that probes e.g., RFLP (Zhang et al., 2005; Klemsdal and the Chaetomium species isolated from various substrates Elen, 2006; Lockhart et al., 2006). In type II DNA in South East Asia and Europe shared similar identity fingerprinting, several anonymous genomic sequences with isolates from leaf, seeds and soil collected from are amplified in the genome, using different primers e.g. central and south eastern NSW (Australia). There was MLSTs, RAPDs, URPs AFLPs, repetitive PCR, ITSs and close genetic similarity among species of Chaetomium SNPs etc. which potentially inhabit many environments. Recently, the use of molecular markers has given a Recently, Aggarwal et al. (2013) observed that the boost to the analysis of accurate variation among various Indian Chaetomium isolates are genetically more similar isolates of the bioagents. By using the RAPD (Random to American, than to Australian, European and Oceanian amplified polymorphic DNA) it is possible to distinguish (New Zealand) isolates. Most of the Chaetomium isolates among strains of bioagents. C. globosum isolates have in India showed a close relationship with the Chaetomium been characterized by the PCR-RAPD technique isolates in America. On the other hand, very few isolates (Ahammed et al., 2005). The AFLP analysis is based on from India shared similarity with isolates from Australia. selective amplification of DNA restriction fragments (Vos Nearly 39% of the Indian isolates could form a separate et al., 1995). Aggarwal et al. (2010a) studied the genetic group though they clustered with two European and an diversity of C. globosum, a potential biocontrol agent by Australian isolates. Genetic similarity among isolates amplified fragment length polymorphism (AFLP). The within the groups suggests that some isolates of dendrogram obtained made a clear distinction among Chaetomium potentially inhabit many environments, from the 5 isolates of C. globosum into 2 separate groups, the tropics to temperate regions. However, the separation one having isolates Cg1 and Cg5 and other having of a European isolate MU-2009 and an Australian isolate isolates Cg6, Cg7 and Cg8 in which Cg6 was further NA26 from the rest of the isolates suggests that the separated from two very closely related isolates Cg7 and middle type of evolution, developing from a higher grade Cg8. These distinctions were further confirmed through to a lower grade, possibly have occurred. principal component analysis of the AFLP DNA fragment data. Proteomics studies Repeat sequences from Korean weedy rice, originally referred to as universal rice primer (URP), have Fungi can be distinguished by their protein composition. been used for the fingerprinting of diverse genomes of The whole cell protein is extracted from mycelial tissue, plants, animals and microbes (Kang et al., 2002). denatured and negatively charged with mercaptoethanol and sodium-dodecylsulphate (SDS). The proteins are Aggarwal et al. (2008) analysed C. globosum and separated according to size by polyacrylamide gel other Chaetomium isolates based on URP markers. They, electrophoresis (SDS-PAGE). Proteomics studies of observed that phylogenetic analysis of a combined data biological control agents especially of filamentous fungi set obtained from nine URP’s showed formation of two is of importance for the reason that they secrete a large 14 Indian Phytopathology 68 (1) : 8-24 (2015) number of antimicrobial proteins (Peberdy, 1994). refers to the complete set of proteins that are specified Understanding the mechanisms of action involved in by the genome, and proteomics describes the study and biocontrol process is of primary importance for characterization of the complete set of proteins present establishing the effectiveness of biocontrol agent and in a cell, organ or organism at a given time (Wilkins et will provide much insight into where and when the al., 1995). Genome level studies (genomics) reveal what interaction occurs and how the pathogens will be affected could theoretically happen, whereas the proteome level (Larkin et al., 1996). For this protein content of the fungi, investigations provides insights into the key components including the cell wall, is required to identify determinants involved in mediating specific cellular processes. Effective playing a role in biological control. protein separation and identification can provide targets for antifungal activity for biological and therapeutic uses. Production of extracellular proteins by the biocontrol Recent improvements made to two dimensional gel fungus Gliocladium virens was studied by Tilburg and electrophoresis (2DE), including narrow range IPGs, Thomas (1993) and the study revealed that several of sample pre fractionation (Righetti et al., 2000; Pedersen the polypeptides present in the culture fluid during the et al., 2003), improved protein extraction techniques first 24 h disappeared completely by 48 h. Consequently, (Luche et al., 2003) and highly sensitive staining it appears that extracellular proteins in cultures of G. techniques (Yan et al., 2000) make 2 DE highly virens are regulated by a combination of gene regulation reproducible and sensitive. There are evidences to show and protein degradation. Relationship between the that Trichoderma spp. secrete a range of cell wall thermotolerance and the contents of hydrophobin-like degrading enzymes that break down the cell wall of or formic-acid-extractable (FAE) proteins in aerial conidia phytopathogenic fungi leading to their death. Besides of Beauveria bassiana and Paecilomyces fumosoroseus these, antibiotics are also secreted by fungal biocontrol as fungal biocontrol agents was studied by Ying and Feng agents. Therefore, an understanding the protein content (2004) and they concluded that approximately 80% of of fungal biocontrol agents, including the cell wall is variability in conidial thermotolerance was attributed to required to identify determinants playing a role in either 15.0- or 17.5-kDa FAE protein or both. Earlier biological control. Earlier, some initiatives have been extracellular protein based variability was also done in taken to identify proteins through proteomics approaches nine isolates of C. globosum and result revealed that all in Trichoderma harzianum (Grinyer et al., 2004a, b), and the isolates were found to be different with respect to T. atroviride (Grinyer et al., 2005; Marra et al., 2006). the profile of extracellular proteins. The dendrogram obtained after complete linkage cluster analysis of Recently, Sharma et al. (2014) studied the protein Jaccard’s similarity showed that isolates Cg2 and Cg3 content of a mycoparasitic strain of C. globosum under resembled each other, while Cg5 and Cg6 and Cg7, Cg8 normal and heat shock conditions in order to identify and Cg9 were similar and showed high degree of differentially expressed proteins through 2D gel similarity forming a separate group. Isolates Cg1 and electrophoresis (Fig. 4). Forty-eight proteins were Cg4 did not resemble any other isolate (Ahammed et identified by a combination of matrix-assisted laser al., 2008a). desorption/ ionization time-of-flight mass spectrometry (MALDI-ToF) and liquid chromatography mass Proteomics has emerged as an indispensible tool spectrometry (LCMS/MS). Out of total proteins identified, for understanding the cellular mechanisms of fungal 79% were hypothetical proteins and 21% proteins were pathogenicity, potential impact in plant pathology and the functionally characterized. Out of total 79% hypothetical study of plant fungal interaction. The term proteome proteins 24% proteins matched with C. globosum while

7 4 4 7

97 Kd 97 Kd 66 Kd 66 Kd

41 Kd 41 Kd

31 Kd 31 Kd

21 Kd 21 Kd

14 Kd 14 Kd

Fig. 4. Two dimensional protein reference map of whole cell protein extract of Chaetomium globosum in (a) normal condition (b) temperature shocked condition. This gel was run on 11 cm, 4-7 IPG strips in the first dimension and 12% SDS-PAGE in second dimension. (Spots no. 1-68 identified in normal conditions; spots no. 69-95 identified in induced conditions and spots A-W differentially expressed in both the conditions) Indian Phytopathology 68 (1) : 8-24 (2015) 15

18% proteins matched with Aspergillus spp., 13% with 93.5% conidial germination of B. sorokiniana, wheat spot Coprinopsis cinerea, 10% with Giberrella zaea, 8% with blotch pathogen whereas, the culture filtrate could inhibit Magnaporthe grisea and 5% with Neurospora crassa and conidial germination only up to 59.9%. Lodderomyces elonisporus. Some of the functionally characterized proteins included MAP kinase, maltose Chitinases from Trichoderma spp. have been permease, GTP binding protein, dyenin heavy chain, characterized (Harman et al., 1993) with molecular HET-C2, vacuolar Dig A protein, polyketide synthase, weight within the array of 33Kda (Haran et al., 1996; Lima peptide prolyl cis trans isomerase and translation et al., 1998; Jebakumar et al., 2000). Even the paternal elongation factor. This study has generated a protein role of chitinase gene successfully expressed in reference map for C. globosum, and being the first report transgenic plants and explored in different biocontrol on proteomics studies would greatly help to unravel systems (Shapira et al., 1989; Broglie et al., 1991). biocontrol mechanism and its survival under heat stress Recently, cloning and characterization of a novel conditions. chitinase gene (chi46) from C. globosum has been done and its role in biological activity has been analysed (Liu et al., 2008). The studies on cellulytic enzymes are not Biocontrol related genes extensive like chitinases. But they have an important role The involvement of enzymes in biocontrol blurs the particularly in plant pathogenic oomycetes, which contain distinction between parasitism and antibiosis, because cellulose as the main cell wall component. An early work the production of cell wall degrading enzymes by the on biocontrol of Pythium debaryanum mycelium (Mitchell antagonists may be simultaneously involved in parasitism and Hurwitz, 1965). The seed colonization and protection and antibiosis or causes only antibiosis depending upon of bean seedlings against Pythium splendens by T. the pathogen associated (Boosalis, 1956; Davis et al., koningii was related to high level of carboxy methyl 1986). cellulose, i.e β1,4 glucanase activity of biocontrol agent (Cotes et al., 1996). The possible involvement was A role for the production of cell wall degrading reported by Ridout et al. (1986). Recently production of enzymes such as β 1, 3 glucanases, chitinases, cellulases by different isolates of Chaetomium globosum proteases, cellulases, xylanases, esterases, alkaline Krunze ex. Fr. has been studied by Ahammed et al. phosphatase and lipase by mycoparasites during (2008a). interactions with other fungi has frequently been suggested (Elad et al., 1982, 1983; Ridout et al., 1988; Cellulase and xylanase are the major type of Tweddell et al., 1994). β-glucan degrading enzymes are enzymes that involve in the cell wall degradation of the produced by a wide variety of organisms, although the pathogens. Xylanase producing Trichoderma strain SY fungi are the most predominant producers. Different types was isolated from the soil. The gene coding for xylanase, of β -glucan degrading enzymes are classified according Xyl was cloned by RT-PCR. Xyl was highly expressed to the type of β -glucosidic linkages that cleave and when it was grown in cellulose as an only source of mechanisms of substrate attack. carbon (Min et al., 2002). Earlier, xylanase has been partially purified and characterized in C. globosum They can hydrolyse the substrate by two possible isolates by Ahammed et al. (2008b). Xylanase activity mechanisms, identified by the product of hydrolysis: was maximum after 9 days of incubation when amended (i) exo-β glucanases hydrolyse the substrate by in medium with 1.5% xylan as carbon source and 0.6% sequentially cleaving glucose residues from the reducing NH4H2PO4 as nitrogen source. Both the purified xylanase end, or (ii) endo-β-glucanases cleave β-linkages at and culture filtrate have shown the antifungal activity random sites along the polysaccharide chain, releasing against B. sorokiniana, a causal organism of spot blotch smaller oligosaccharides. Cell walls of phytopathogenic of wheat. Purified xylanase at 100 µg ml”1 concentration fungi are composed of either chitin or cellulose along caused 100 per cent inhibition of conidia germination of with high amount of β-glucans (Bartnicki-Garcia, 1973). B. sorokiniana, whereas the culture filtrate was able to Six endoglucanases, 3 exoglucanases and β-glucosidase inhibit germination up to 67.5%. Li et al. (2007) purified were purified from T. viride and characterized by Beldman and characterize two thermostable proteases from the et al. (1988). The chitinases and β 1,3 glucanses from T. thermophillic fungus C. thermophillum. harzianum were found to be responsible for cell wall degradation of R. solani (Ridout et al., 1986) and their Liu et al. (2007) isolated and characterized a heat a presence in germling extract of the antagonist caused shock protein hsp 22.4 from C. globosum and confers leakage of soluble protein, carbohydrate, aminoacids and heat and Na2Co3 tolerance to yeast. Mitogen activated salts from hyphae of these pathogens (Lewis and protein kinase, mpkC plays an essential role in Papavizas, 1987). osmoreulatory pathways in yeast and many eukaryotes (Jin et al., 2005). MAP Kinases also have significant role An extracellular β-1, 3-glucanase produced by C. in heat and oxidative stress and play a major role in globosum (Cg 2) was purified and characterized by maintaining fungal cell wall integrity (Miyazaki et al., Ahammed et al. (2012). β-1, 3-glucanase activity was 2010). maximum 9 days after incubation at pH 5.5 and 30 C in a medium amended with laminarin @1mg ml-1 as carbon- The ESTs of biocontrol related genes of Trichoderma source and NH4NO3 @ 0.25% as nitrogen source. and Chaetomium spp. were studied by construction of Partially purified glucanase fraction at 100 µg/ ml inhibited cDNA library (Yang et al., 2007a). This EST studies on 16 Indian Phytopathology 68 (1) : 8-24 (2015) these plant disease bio-control fungi supplied huge Dienelactone hydrolase gene is involved in amount of gene resources for promoting the bio-control seconadary metabolite biosynthesis, transport and mechanisms study and new bio-fungicides development. degradation of chlorocatechol compounds. This enzyme Cloning and expression analysis of Functional genes in catalyze the hydrolysis of dienelactone to maleylacetate. Chaetomium globosum was studied by Liu (2006). The The gene was amplified in all the isolates of C. globosum, gene, ThPG1 which encodes for endopolygalacturonase C. reflexum except Cg8, Cg10, Cg11, Cg13 and Cg14 was isolated from T. harzianum and characterized. This and C. perlucidum. Very few studies has been done in enzyme is involved in the cell wall degradation of the the fungi however earlier dienelactone hydrolase gene pathogens like R. solani and P. ultimum and helps in the was purified, characterized and expressed in E.coli from plant beneficial interactions (Moran-Diez et al., 2009). thermoacidophillic archean Sulfolobus salfataricus P1 Xylanase, chitinase, β-1,3 glucanase, MAPK, isolate and concluded that his gene is having glyceraldehydes 3 phosphate dehydrogenase, β tubulin carboxylesterase activity (Park et al., 2010). were present uniformly in all the 15 isolates of C. globosum, C. perlucidum and C. reflexum taken for the With regards to mapkinase gene characterisation study. Sequence based phylogenetic tree of xylanase in Chaetomium isolates, analysis showed 100% identity gene showed that two major clusters were formed with with hypothetical protein of C. globosum 20% similarity between them. Xylanases are the major (XM001223027.1), 88% with hypothetical protein of type of enzymes that involve in the cell wall degradation Myceliophthora thermophila (XM003665750.1), 85% with of the pathogens. A role for the production of cell wall hypothetical protein Thielavia terrestris degrading enzymes such as β 1,3-glucanase, chitinases, (XM003657195.1) and 85% with mitogen activated proteases, cellulases, xylanases, esterases, alkaline protein kinase of Neurospora crassa (XM9570702). In phosphatase and lipase by mycoparasites during many fungi, signaling pathways, including mitogen- interactions with other fungi has frequently been activated protein kinase (MAPK) cascades, have been suggested by many workers (Elad et al., 1982, 1983; implicated in parasitism of host plants as well as in the Ridout et al., 1988; Tweddell et al., 1994). production of asexual spores. MAPK loss-of-function mutants had reduced antagonistic properties in The BLASTP search of endochitinase gene confrontation assays and failed to parasitize the sclerotia. indicated that the product from Cg2 isolate shared the TmkA-dependent and independent pathways are thus highest similarity 84% with a known chitinases involved in antagonism of biocontrol fungi against (XP_955945) from Neurospora crassa, this was in different pathogens (Prasun et al., 2003). accordance with the study Yang et al. (2008) in a different isolate of C. globosum. Family 18 chitinases from Mitogen activated protein kinase, mpkC, plays an biocontrol fungi enable the mycoparasite to penetrate essential role in osmoreulatory pathways in yeast and other fungi and degrade the host cell wall (Klemsdal et many eukaryotes (Jin et al., 2005). MAP Kinases also al., 2006). Earlier studies revealed that the chitinases have significant role in heat and oxidative stress and play and β 1,3 glucanses from T. harzianum were found to be a major role in maintaining fungal cell wall integrity responsible for cell wall degradation of R. solani (Ridout (Miyazaki et al., 2010). β-tubulin gene and et al., 1986) and their presence in germling extract of glyceraldehydes 3 phosphate dehydrogenase genes the antagonist caused leakage of soluble protein, were amplified in all the isolates taken for the study. carbohydrate, amino acids and salts from hyphae of these Tubulins are structural proteins made of microtubules and pathogens (Lewis and Papavizas, 1987). they help in study the cell wall composition of the pathogens and interact with benzimidazole fungicides Endopolygalactouronase gene is another important (Li et al., 2010). This β-tubulin gene was isolated and gene having role in cell wall degradation of pathogens, characterized from T. harzianum (Li and Yang, 2007), thus helps in biocontrol mechanism. The blast result while glyceraldehydes 3 phosphate dehydrogenase gene showed that endopolygalactouronase gene of C. is involved conidiation and mycoparasitism (Puyesky et globosum showed matching with different fungi, viz. al., 1997). 100% with endopolygalactouronase gene of Rhizoctonia solani AG-1 IA strain A1 (HQ197932.1), 99% with Recently, cloning and expression analysis of small Thanatephorus cucumis PG1 mRNA (FJ544455.1) and heat and salt tolerant protein (HSp 22) from Chaetomium 99% with Rhizoctonia solani AG-1IA strain E67 globosum had been done by Aggarwal et al. (2012). This endopolygalactouronase mRNA (HQ197933.1). By gene was further transformed into pET28a (+) and exploring the NCBI database we came to conclusion that transformed E.coli BL21 cells were induced by IPTG, this gene is not characterized in C. globosum, while and the expressed protein of 30Kda was analysed by sequence blast result revealed that this gene showed SDS PAGE. The IPTG induced transformants displayed homology with hypothetical protein of C. globosum. So, significantly greater resistance to NaCl and Na2CO3 this is the first report for the identification of this gene in stresses (Aggarwal et al., 2012). C. globosum. The gene, ThPG1 which encodes for endopolygalacturonase was isolated from T. harzianum Molecular diagnostics and characterized by Moran-Diez et al. (2009). This enzyme involves in the cell wall degradation of the In recent years, several PCR-based molecular pathogens like R. solani and P. ultimum and helps in the techniques have been used to detect and discriminate plant beneficial interactions. among microorganisms. Although conventional PCR has Indian Phytopathology 68 (1) : 8-24 (2015) 17 become an attractive tool for the detection of specific pathogens present in soil. This method can be used for microorganisms in microbial systems, this technique diagnosis of biocontrol agent in small diagnostics does not allow accurate quantification of DNA because laboratories via sampling of plant leaf and soil from field. of the variability in the efficiency of amplification between Thus, farmers can monitor population of biocontrol agent PCR reactions (Raeymaekers 1998). This limiting factor and can apply appropriate doses of it, and contribute to has been overcome by the emergence of Real-time PCR. good decision-making for disease management. This method is highly sensitive, with reliable detection of even 1 pg or less fungal DNA in treated soil or plant Competetive saprophytic ability tissues. Real time PCR is based on the use of TaqMan probes or SYBR Green I dyes. Both systems measure According to Garrett (1970), “the share of a substrate the intensity of a fluorescent signal proportional to the obtained by any particular fungal speies will be quantity of DNA generated during the PCR amplification. determined partly by its intrinsic competitive saprophytic Quantitative real-time PCR is widely used in medicine ability and partly by the balance between its inoculums for the diagnosis of clinical pathogens (Kimura et al., potential and that of competing species”. In our study 1999; Ke et al., 2000). In plant sciences, it is used for out of the three antagonists and one pathogen (B. detection of fungal pathogens (Atallah et al., 2007; sorokiniana) tested, hybrid of C.globosum (CgH6) have Gayoso et al., 2007). However, there are few references higher competitive saprophytic ability (Sharma, 2013). regarding its application to quantify fungi from soil (Filion Antagonists are also helping in plant growth promotion et al., 2003). parameters like root length, shoot length and per cent germination. All the three parameters increased in all The identification of particular strains of fungi on the the three antagonist, but there was highest increase in basis of their DNA requires the characterization of shoot and root length in CgH , followed by M and Cg2 discriminating DNA targets. Molecular technologies are 6 1 one of the most important tools for identification and respectively either present alone or in combination with detection of fungal isolates. Accurate identification and pathogen. Same results were also reported by Singh et monitoring of biocontrol agent is important for the al. (2008), that after colonization BN1 isolate showed biological control of soil-borne plant-pathogens. A distinct stimulatory effect on all vegetative parameters. The most marker had been developed for detection of C. globosum consistent effect of B. subtilis was observed as increased which can differentiate this fungus from isolates of root branching and root dry weight. Earlier 35% increase different species of Chaetomium and other soil fungi in pine root dry weight after inoculation by B. polymyxa (Aggarwal et al., 2014). This marker was also able to L6-16 strain has also been reported (Holl et al., 1988; detect C. globosum in infected wheat tissues and infested Chanway et al., 1991). soil samples. A SCAR marker from an anonymous unique Real time PCR will provide a valuable tool in region of genomic DNA of Chaetomium spp. identified monitoring fungal spatio -temporal population changes after wide screening of different species of Chaetomium in response to exogenous inputs, including change in and other pathogens using 12, URP primers. Primers crops, farm practices, chemical inputs and the presence URP2R, URP6R and URP7R produced monomorphic of pathogen in soil and environment. This will provide a bands of different sizes in all C. globosum isolates tested. more accurate assessment of the influence of the Out of these selected primers, URP2R produced a environment on the activity of this biological control agent. unique 1900 band only in C. globosum, which was Conclusively, CgH6 exhibited good plant growth converted to SCAR marker (Fig. 5). Primer pair promotion activities and strongly inhibited the plant SCARCGF1/ SCARCGR2 amplified a distinct band of pathogens B. sorokiniana, R. solani, T. indica under in 1900 bp with genomic DNA of only C. globosum and vitro conditions. In vivo studies showed enhanced was absent in all other pathogens as well as in genomic vegetative parameters and suppression of the growth of DNA of healthy wheat plant. It detected C. globosum from the pathogen. It showed excellent root colonization ability. wheat leaves and soil samples at the time of its This attributes of CgH6 an improved strain of C. globosum application and also after 15 days. It also detected C. qualifies it as a potent biocontrol agent against various globosum in presence of B. sorokiniana and other plant pathogens.

Fig. 5. Agarose gel showing specificity of primer set SCCgRAF4/ SCCgRAR4 to C. globosum. M-1Kb Molecular marker, Fermentas; (1) C. globosum; (2) C.perlucidum; (3) C.reflexum; (4) C.cupreum; (5) C.cochlioides; (6) Bipolaris sorokiniana; (7) Fusarium moniliforme; (8) Alternaria triticina; (9) Tilletia indica; (10) Epiccocum purpurecens; (11) Puccinia striiformis; (12) Trichoderma virens; wheat leaf (13) DNA; (14) Distilled water 18 Indian Phytopathology 68 (1) : 8-24 (2015)

Strain improvement Earlier studies revealed that among the different mutants ethidum bromide treated A. niger EB-3 (treated The science and technology of manipulating and for 90 min) was the best mutant for enhanced production improving microbial strains, in order to enhance their of citric acid (Javed et al., 2010). Papavizas and Lewis metabolic capacities for biotechnological applications, (1983) selected UV-induced mutants from T. viride and are referred to as strain improvement. Improved some of the mutants proved to be more efficient than bioefficacy of potential strain can be regarded as the original strain. Rhizosphere competence of benomyl- the heart of biocontrol industry, so improvement of the tolerant mutants of Trichoderma spp. was studied by bioefficacy offers the greatest oppourtinities for Ahmad and Baker (1988). Protoplast fusion was also cost reduction and ecofriendly management of the attempted to produce superior biocontrol strains (Harman diseases. and Hayes, 1993). Efforts were made to get the hybrid cultures of two parental strains Cg1 and Cg2. In all eight An improved strain should have certain properties hybrid cultures were established (CgH1-CgH8). These like rapid growth, genetic stability, non-toxicity to humans hybrids were stable for morphological characters by and ability to use cheaper substrates. In addition to repeated subculturing. Total 10 pinhead colonies were antifungal activity, a potential biocontrol agent should obtained using UV and ethyl methane sulphonate, of possess rhizosphere competence and should be capable which three colonies were stable and named as M1, M2 of inducing growth responses by either controlling the and M3. minor pathogens or producing growth stimulating factors. To meet these criteria superior strains must be developed. Out of two improved strains CgH6 and M1 raised The strategies for strain improvement can be through hybridization and mutagenesis respectively, CgH showed better efficacy against various plant hybridization, transformation, protoplast fusion or raising 6 new mutants using mutagenesis. pathogens under in vitro conditions and against B. sorokiniana under in vivo. Real time PCR data revealed

Today, strain improvement can be performed by two that population of improved strain (CgH6) increased four alternative strategies: 1) classical genetic methods times in 21 days and also it showed better competitive (including genetic recombination); and 2) molecular saprophytic ability in comparison to wheat pathogen, genetics methods. Each has distinct advantages, and in Bipolaris sorokiniana than M1 followed by wild type parent some cases all these approaches can be used in concert Cg2. to increase the bioefficacy potential. Fungi produce a diversity of metabolites, many of Classical genetic methods: Strain development by this which appear unnecessary for the primary function of strategy has typically relied on mutation, followed by the host. However, many metabolites appear indirectly random screening. Mutation can be carried out with important, influencing fungal growth and survival. The physical mutagens like UV light or chemical mutagens structure of secondary metabolites varies enormously. like ethyl methane sulphonate (Baltz, 1999). This The function of secondary metabolites is as varied as empirical approach has a long history of success; best their structure. Some appear to directly or indirectly exemplified by the improvement of penicillin production, benefit the fungus, while the function of many others in which modern reported titles are 50 g/I, an remains obscure. Secondary metabolites play an improvement of at least 4,000 fold over the original parent extremely important role in the biological control of (Peberdy, 1985). Other examples include fungal or pathogens. actinomycetal cultures capable of producing metabolites An improved strain of biocontrol fungus, should be in quantities as high as 80 g/L (Rowlands, 1984; Vinci highly stable and must contain high antagonistic and Byng, 1999). properties, have better rhizosphere competence and help in plant growth promotion. The efficacy of secondary Molecular genetics methods: To carry out these metabolite of antagonist against all the three test strategies, some biochemical and molecular genetics pathogens, T. indica, F. graminearum and Bipolaris tools, including identification of the biosynthetic pathway, sorokiniana, at 1000ppm concentration showed that adequate vectors and effective transformation protocols maximum zone of inhibition against all the three test for the particular species have to be developed or made pathogen was formed by CgH6 and minimum zone of available. After this, the biosynthetic gene or genes have inhibition was formed by Cg1. to be cloned and analyzed. Molecular biology of actinomycetes and fungi has been successfully Bioformulations and their efficacy developed to a degree that its application to industrial strain improvement is not a reality. Haq et al. (2001) Chaetomium globosum has also been reported to be a reported that chemical mutagenesis gave better results potential antagonist of Bipolaris sorokiniana (Mandal, when compared to UV irradiation. Studies revealed that 1999; Aggarwal et al., 2004). Recent findings on the chemically treated GCM-7 as the best mutant which biological control of spot blotch of wheat have revealed produced 86.1 1.5 mg/mL citric acid after 168 h of LSF that the antagonist C. globosum, besides reducing the fermentation of potassium ferricyanide + H2SO4 lesion formation on leaf, also improves the growth of the pretreated black strap molasses (containing 150g sugars/ host plant. Increase in green leaf area was noticed when L) in Vogel’s medium. the seedlings were given pre-inoculation treatment at the Indian Phytopathology 68 (1) : 8-24 (2015) 19

rate of 0.2%, and also an increase in growth of shoot was observed in T3 w.r.t to control, which was 13.5% and root of wheat seedlings was observed. The beneficial gain in yield over control in treatment 1: soil amended effect of foliar spray of antifungal compound in reducing with biopellets (Anonymous, 2013). the spot blotch lesions and protection of green leaf area suggests that application of biocontrol agents may be The efficacy of C. globosum as a biocontrol agent against the late blight pathogen Phytophthora infestans useful in IPM system because of its yield enhancing has been reported in potato plants. Among eight property. Mandal et al. (1999) tested the antagonistic Chaetomium isolates evaluated C. globosum isolate Cg- behaviour of 16 different microorganisms and found that 6 showed greater inhibition to mycelial growth of P. Trichoderma reesei and C. globosum significantly infestans in vitro. C. globosum Cg-6 was formulated as a reduced the radial growth of B. sorokiniana. Culture filtrate liquid and applied as a tuber, soil and foliar treatment of C. globosum effectively suppressed spot blotch either individually or in combination against Phytophthora disease in green house when sprayed at 1:1 dilution infection in potato plants. Among different treatments, (Aggarwal et al., 2004). combined application of C. globosum as a tuber treatment A potential strain Cg-2 of this biocontrol agent has @ 1 ml/kg of tubers, as a soil application @ 1 ml/kg of been identified, which showed inhibitory effect on Farm Yard Manure (FYM) and foliar spray @ 0.7% germination of conidia and mycelial growth. Foliar spray resulted in significantly less late blight infection (72%) with this biocontrol agent @ 106cfu/ml is effective in compared to untreated control (100%) under field controlling the disease (Aggarwal et al., 2004). This conditions. The application of C. globosum resulted in isolate produced 13 secondary metabolites when greater tuber yield by reducing late blight infection in two extracted by solvent extraction method (Aggarwal et al., field trials when compared to untreated controls. The 2007), out of which five characterized through NMR and study clearly demonstrated the potential use of C. GC-MS had antifungal activity. It was clearly observed globosum as a biocontrol agent in the management of that the potential antagonistic strain Cg2 of C. globosum late blight disease in potato plants (Shanthiyaa et al., inactivated BS- toxin produced by the pathogen, B. 2013). A formulated product from C. globosum potential sorokiniana and the inactivated toxin caused significantly strain (Cg2WP), which was found effective against late less leakage of electrolytes and less necrosis of wheat blight of potato during 2009-10 was tested again during leaf tissues (Aggarwal et al., 2011). 2010-11 at CPRI, regional station, Modipuram. Three sprays of the bioformulation proved effective in controlling Two bioformulations (Cg2WP (0.2%) for spray and the disease upto 30% and increased the yield up to 10%. Cg2BP (@5g/m2) for soil application) proved effective (Anonymous, 2011). against spot blotch of wheat caused by B. sorokiniana under field conditions. Multilocation trials under All India Induction of defense responses in plants Coordinated wheat programme conducted at Faizabad, Coochbihar and Karnal showed significant reduction in Interactions between plants and pathogens can lead disease and increased yield. Maximum disease severity either to a successful infection (compatible response) or (78.2%) was observed at Coochbihar in untreated resistance (incompatible response). In incompatible control, which reduced to 67.7% in soil amended and interactions, infection by viruses, bacteria or fungi will spray treatment, showing 13.4% reduction in disease elicit a set of localized responses in and around the severity. At Faizabad and Karnal, 35% and 8.5% infected host cells. These responses include an oxidative reduction in disease severity was observed. A significant burst (Lamb and Dixon, 1997), which can lead to cell increase in shoot length and root length was also death (Kombrink and Schmelzer, 2001). Thus, the observed at Faizabad and Coochbihar, when both soil pathogen may be ‘trapped’ in dead cells and appears to treatment and sprays were applied. Significant increases be prevented from spreading from the site of initial in 1000 grain wt. was observed in soil amended and spray infection. Further local responses in the surrounding cells treatment (T3) over control (T5) at all three centers, include changes in cell wall composition that can inhibit however increase in yield (g/plot) was Non significant at penetration by the pathogen, and de novo synthesis of Coohbihar in same treatments (Anonymous, 2012). antimicrobial compounds such as phytoalexins (Hammerschmidt, 1999b) and pathogenesis related (PR) Recently, in 2013-14, these formulations were tested proteins caused by or at least regularly following these as soil amendment and spray against spot blotch of wheat local responses, a signal spreads through the plant and at IARI, Regional station, Pusa, Samastipur (Bihar) under induces subtle changes in gene expression in yet natural disease conditions. Wheat genotype HUW 243, uninfected plant parts. The systemic response involves which is moderately susceptible to spot blotch disease the de novo production, in some cases, of phytoalexins was selected for the study. Maximum disease severity and of PR proteins (Van Loon, 1997; Neuhaus, 1999; (61.75) was observed in untreated control, which reduced Van Loon and Van Strien, 1999). While phytoalexins are to 12.5 in soil amended and spray treatment, showing mainly characteristic of the local response, PR proteins 20.24% reduction in disease severity. A significant occur both locally and systemically. increase in shoot length and root length was also observed when both soil treatment and sprays were Induced systemic resistance (ISR) of plants against applied. A significant increase in 1000-grain weight was pathogens is a widespread phenomenon that has been observed in soil amendment and spray treatment (T3) intensively investigated with respect to the underlying over control (T5). However, 30.3% gain in yield (g/plot) signalling pathways as well as to its potential use in plant 20 Indian Phytopathology 68 (1) : 8-24 (2015)

protection. Elicited by a local infection, plants respond fragment length polymorphism. Indian Phytopath. 63(1): with a salicylic dependent signalling cascade that leads 2-5. to the systemic expression of a broad spectrum and long Aggarwal, Rashmi, Tiwari, A.K., Dureja, P. and Srivastava, lasting disease resistance that is efficient against fungi, K.D. (2007). Quantitative analysis of secondary metabolites produced by Chaetomium globosum Krunze ex Fr. J. Biol bacteria and viruses. Changes in cell wall composition, Cont. 21(1): 163-168. de novo production of pathogenesis related proteins such Ahammed, S.K., Aggarwal, R. and Renu (2005). Use of PCR as chitinases and glucanases, and synthesis of based RAPD technique for characterization of phytoalexins are associated with resistance, although Chaetomium globosum isolates. Acta Phytopathologica et further defensive compounds are likely to exist but remain Entomologica Hungarica 40: 303-314. to be identified. Recently we investigated on important Ahammed, S.K., Aggarwal, R. and Srivastava, K.D. (2008a). parameters of induced resistance in wheat (Triticum Production of extracellular proteins and cellulases by aestivum) against B. sorokiniana and Puccinia triticina different isolates of Chaetomium globosum. Indian using biocontrol agent C. globusum. Enhanced activities Phytopath. 61(4): 437-440. of defense related enzymes polyphenol oxidase, Ahammed, S.K., Aggarwal, R., Sneh and Kapoor, H.C. peroxidase, phenyl alanine lyase and catalase revealed (2008b). Production, partial purification and the role in Induction of systemic resistance. The results characterization of extracellular xylanase from Chaetomium globosum. J. Plant Biochem. Biotechnol. 17: indicate that the biocontrol agent induced effective 95-98. defense responses in wheat plants against B. sorokiniana Ahammed, S.K., Aggarwal, Rashmi, Sharma, Sapna, Gupta, and P. triticina. The reduced disease incidence in wheat Sangeeta and Bashyal, B.M. (2012). Production, partial by C. globusum may be a result of cell wall strengthening purification and characterization of extra-cellular B-1,3- through deposition of lignin and induction of defense glucanase from Chaetomium globosum and its antifungal enzymes. It is concluded that C.globusum may be used activity against Bipolaris sorokiniana causing spot blotch as potential resistance inducer against the fungal of wheat. J. Mycol. Plant Pathol. 42: 146-152. pathogens, B. sorokiniana and P. triticina. The science Ahmad, J.S. and Baker, R. (1988). Rhizosphere competence accumulated on the chemical and physiological functions of benomyl-tolerant mutants of Trichoderma spp. Canadian J. Microbiol. 34: 694-696. of C. globusum associated with plant defense suggests it has a bright future in many aspects. In conclusion, prior Amemiya, Y., Kondo, A., Hirano, K., Hirukawa, T. and Kato, T. (1994). Antifungal substances produced by Chaetomium treatment of wheat plants with biocontrol agents triggered globosum. Tech. Bull. Faculty of Hort. Chiba Univ. 48: 13- the plant defense mechanism in response to infection 18. by B. sorokiniana and P. triticina. Andrews, J.H., Barber, F.M. and Nordheim, E.V. (1983). Microbial antagonism to the imperfect stage of the apple REFERENCES scab pathogen Venturia inaequalis. Phytopathology 73: 228-234. Aggarwal, R., Gupta, S., Sharma, S. and Renu (2014). Anonymous (2011). National Fellow Project, IARI, pp. 169. Development of conventional and real time PCR assay for rapid detection and quantification of a biocontrol agent, Anonymous (2012). Annual report (2012-13), IARI, pp. 169. Chaetomium globosum. J. Pl. Pathol. 96(3): 477-485. Anonymous (2013). National Fellow Project Report (2013-14) Aggarwal, R., Gupta, S., Sharma, S., Banerjee, S. and Singh, pp. 18. P. (2012). Cloning and expression of a small heat and salt Artigues, M. and Davet, P. (1984). β -1, 3-glucanase and tolerant protein (Hsp22) from Chaetomium globosum. Ind. chitinase activities of some fungi in Relation to their J. Expt. Biol. 50: 826-832. capacity to destroy sclerotia of Corticium rolfsii in strerile Aggarwal, R., Lalit L. Kharbikar, Sharma, Sapna, Gupta, soil. Soil Biol. Biochem. 16: 527-528. Sangeeta and Yadav, Anita (2013). Phylogenetic Attallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I. and relationships of Chaetomium isolates based on the internal Stevenson, W.R. (2007). Multiplex real-time quantitative transcribed spacer region of the rRNA gene cluster. African PCR to detect and quantify Verticillium dahliae colonization J. Biotechn. 12: 914-920. in potato lines that differ in response to Verticillium wilt. Aggarwal, R., Sharma V., Lalit L., Kharbikan and Renu (2008). Phytopathology 97: 865-872. Molecular characterization of Chaetiminum spp. using Baker, R. (1968). Mechanism of biological control of soil borne URP-PCR. Gen. Mol. Bio. 31(4): 943-946. pathogens. Annu. Rev. Phytopath. 6: 263-294. Aggarwal, R., Sharma, S., Gupta, S. and Shukla, R. (2014). Baltz, R.H. (1999). Mutagenesis. In Encyclopedia of bioprocess Development of conventional and real time PCR assay for technology: Fermentation, biocatalyst and separation, the rapid detection and quantification of a biocontrol agent, edited by MC Flickinger and S W Drew. Wiley, New York. Chaetomium globosum. J. Plant Pathol. 96(3): 477-485. pp. 307-311. Aggarwal, R., Tiwari, A.K., Srivastava, K.D. and Singh, D.V. Barnett, E.A. and Ayers, W.A. (1981). Nutritional and (2004). Role of antibiosis in the biological control of spot environmental factors affecting growth and sporulation of blotch (Cochliobolus sativus) of wheat by Chaetomium Sporidesmiumsclerotivorum. Canadian J. Microbiol. 24: globosum. Mycopathol. 157(4): 369-377. 685-691. Aggarwal, Rashmi, Gupta, S., Banerjee, S. and Singh, V.B. Bartnicki-Garcia, S. (1973). Fungal cell wall composition In: (2011). Development of a SCAR marker for detection of Handbook of Microbiology, Chemical Rubber Co., Bipolaris sorokiniana causing spot blotch of wheat. Cleveland, OH 2: 201-214. Canadian J. Microbiol. 57: 934-942. Beldman, G., Voragen, A.G.J., Rombouts, F.M., Searle-van- Aggarwal, Rashmi, Renu, Srinivas, P. and Malathi, V.G. Leeuwen, M.F. and Pilnik, W. (1988). Specific and non (2010a). Assessment of genetic diversity in Chaetomium specific glucanases from Trichoderma viride. Biotech. globosum, a potential biocontrol agent by amplified Bioeng. 31: 160-167. Indian Phytopathology 68 (1) : 8-24 (2015) 21

Biswas, S.K., Aggarwal, R., Srivastava, K.D., Gupta, S. and Dickson, C.H. and Skidmore, A.M. (1976). Interactions Dureja, P. (2012). Characterization of antifungal between germinationg spores of Septoria nodorum and metabolites of Chaetomium globosum Kunze and their phylloplane fungi. Trans. Brit. Mycol. Soc. 66: 45-56. antagonism against fungal plant pathogens. J. Biol. Cont. Elad, Y., Barak, R. and Chet, I. (1983). The possible role of 26(1): 70-74. lectins in mycoparasitism. J. Bacteriol. 154: 1431-1435. Biswas, S.K., Srivastava, K.D., Aggarwal, R., Dureja, P. and Elad, Y., Chet, I. and Henis, Y. (1982). Degradation of plant Singh, D.V. (2000). Antagonism of Chaetomium globosum pathogenic fungi Trichoderma harzianum. Canadian J. to Drechslera sorokininana, the spot blotch pathogen of Micribiol. 28: 719-725. wheat. Indian Phtytopath. 53: 436-440. Filion, M., St-Arnaud, M. and Jabaji-Hare, S.H. (2003). Direct Boosalis, M.G. (1956). Effect of soil temperature and green quantiûcation of fungal DNA from soil substrate using real- manure amendment of unsterilised soil on parasitism of Rhizoctonia solani by Penicillium vermiculatum and time PCR. J. Microbiol. Methods 53: 67-76. Trichoderma sp. Phytopathology 46: 473-478. Fournier, A.R., Frederick, M.M., Frederick, J.R. and Reilly, P. Boudreau M.A. and Andrews, J.H. (1989). Factors influencing (1985). Purification and characterization of endo-xylanases antagonism of Chaetomium globosum to Venturia from Aspergillusniger. III. An enzyme of pl 3.65. Biotechnol. inaequlais: a case study in field biocontrol. Phytopathology Bioeng. 27: 539-546. 77: 1470-1475. Ganju, R.K., Vithayathil, P.J. and Murthy, S.K. (1989). Brewer, D., Jerram, W.A., Meier, D. and Taylor, A. (1970). The Purification and characterization of two xylanases from toxicity of cochliodinol, a metabolite of Chaetomium spp. Chaetomium thermophile var. coprophile. Canadian J. Canadian J. Microbiol. 16: 433-440. Microbiol. 35: 836-842. Broglie, K., Chet, I. Holiday, M., Cressman, R., Biddle, P., Garrett, S.D. (1970). Pathogenic Root-Infecting Fungi. Knowlton, S., Mauvais, C.J. and Broglies, R. (1991). Cambridge mutants not only produced greater amounts Transgenic plants with enhanced resistance to the fungi of 8-1,4-glucanases. University Press, London, 294 pp. pathogen Rhizoctonia solani. Science 254: 1194-1197. Gayoso, C., Martínez De, I.O., Pomar, F. and Merino De, C.F. Butler, F.C. (1953). Saprophytic behavior of some cereal root- (2007). Assessment of real time PCR as a method for rot fungi. II. factors influencing saprophytic coloniaztion of determining the presence of Verticillium dahliae in different wheat straw. Ann. Appl. Biol. 40: 298-304. Solanaceae cultivars. European J. Plant Pathol. 118: 199- 209. Chang, I. and Kommedahl, T. (1968). Biological control of seedling blight of corn by coating kernels with antagonistic Grinyer, J., Hunt, S., McKay, M., Herbert, B.R. and Nevalainen, microorganisms. Phytopathology 58: 1395-1401. H. (2005). Proteomic response of the biological control Chanway, C.P., Radley, R. and Holl, F.B. (1991). Inoculation fungus Trichoderma atroviride to growh on the cell walls of conifer seed with plant growth promoting Bacillus strains of Rhizoctonia solani. Curr. Genet. 47: 381-388. causes increased seedling emergency and biomass. Soil Grinyer, J., McKay, M., Herbert, B. and Nevalainen, H. (2004a). Biol. Biochem. 23: 575-580. Fungal proteomics: mapping the mitochondrial proteins of Chesters, C.G.C. and Bull, A.T. (1963). The Enzymic a Trichoderma harzianum strain applied for biological Degradation of Laminarin. Biochem. J. 86: 31-38 control. Curr. Genet. 45: 170-175. Clark, F.E. (1965). The concept of competition in microbial Grinyer, J., McKay, M., Nevalainen, H. and Herbert, B.R. ecology. In: Ecology of Soil Borne Plant Pathaogens. Baker, (2004b). Fungal proteomics: initial mapping of biological K.F. and Synder, W.C. (Eds). University of California Press, control strain Trichoderma harzianum. Curr. Genet. 45: 163- Berkeley, California, pp. 339-345. 169. Cook, R.J. and Baker, K.F. (1983). The Nature and Practice of Hammerschmidt, R. (1999). PHYTOALEXINS: What Have We Biological Control of Plant Pathogen. American Learned After 60 Years? Ann. Rev. Phytopathol. 37: 285- Phytopathological Society, St. Paul, Minn., 539 pp. 306. Cotes, A.B., Lepoivre, P. and Semal, J. (1996). Correlation Haq, I., Khurshid, S., Ali, S., Ashraf, M., Qadeer, M.A. and between enzyme activities measure in bean seedling after Rajoka, M.I. (2001). Mutation of Aspergillus niger for Trichoderma koningii treatment combine with hyperproduction of citric acid from black strap molasses. pregermination and the protective effect against pythium J. Microbiol. Biotechnol. 17: 35-37. splendens. European J. Pl. Pathol. 102: 497-506. Haran, S., Schickler, H., Oppenheim, A. and Chet, I. (1996). Cullen, D. and Andrews, J.H. (1984). Evidences for the role of Differential expression of Trichoderma harzianum chitinase antibiosis in the antagonism of Chaetomium globosum to during mycoparasitism. Phytopathology 86: 980-985. the apple scab pathogen Venturia inaequalis. Canadian J. Harman, G.E. and Hayes, C.K. (1993). The genetic nature and Bot. 62: 1819-1823. biocontrol ability of progeny from protoplast fusion in Davis, J.R., Fravel, R., Marois, J.J. and Sorensen, L.H. (1986). Trichoderma. In: Biotechnology in Plant Protection. (Chet, Effect of soil fumigation and seed piece treatment with I. Ed). J. Wiley-Liss, pp. 237-255. Talaromyces flavus on wilt incidence and yield. In: Biological and Cultural Tests for Control of Plant Diseases, Harman, G.E., Eckenrode, C.J. and Webb, D.R. (1978). Vol. I. Harman, J.R. (Ed), St. Paul Minn American Alteration of spermosphere ecosystems affecting Phytopathological Society, pp. 84. oviposition by the bean seed fly and attack by soil borne fungi on germinating seeds. Ann. Appl. Biol. 90: 1-6. Dhingra, O.D., Mizubuti, E.S.G and Santana, F.M. (2003). Chaetomium globosum for reducing primary inoculum of Harrison, Y.A. and Steward, A. (1988). Selection of fungal Diaporthe phaseolorum f. sp. Meridionalis in soil surface antaonists for biological control of onion white rot in New soybean stubble in field conditions. Biol. Control 26: 302- Zealand. New Zealand. J. Exp. Agric. 16: 249-256. 310. Holl, F.B., Chanway, C.P., Turkington, R. and Radley, R. (1988). Di Pietro, A., Gut-Rella, M., Pachlatko, J.P. and Schwin, F.J. Growth response of crested wheatgrass (Agropyron (1992). Role of antibiotics produced by Chaetomium cristatum L.) white clover (Trifolium repens L.) and globosum in biocontrol of Pythium ultimum, a causal agent perennial ryegrass (Lolium perenne L.) to inoculation with of damping off. Phytopathology 82: 131-135. Bacillus polymyxa. Soil Biol. Biochem. 20: 19-24. 22 Indian Phytopathology 68 (1) : 8-24 (2015)

Hu, Y., Zhang, W., Zhang, P., Ruan, W. and Zhu, X. (2013). Larkin, R.P., Hopkins, D.L., Martin, F.N. (1996). Suppression Nematicidal activity of chaetoglobosin a poduced by of Fusarium wilt of watermelon by nonpathogenic Fusarium Chaetomium globosum NK102 against Meloidogyne oxysporum and other microorganisms recovered from a incognita. J. Agric. Food Chem. 61: 41-46. disease suppressive soil. Phytopathology 86: 812-819. Hubbard, J.P., Harman, G.E. and Eckenrode, C.J. (1982). Lewis, J.A. and Papavizas, G.C. (1987). Permeability changes Interaction of a biological control agent, Chaetomium in hyphae of Rhizoctonia solani induced by germling globosum, with seed coat microflora. Canadian J. Microbiol. preparation of Trichoderma and Gliocladium. 28: 431-437. Phytopathology 77: 699-703 Javed, S., Asgher, M., Sheikh, M. A. and Nawaj, H. (2010). Li, A.N., Ding, A.Y., Chen, J., Liu, S.A., Zhang, M. and Li, D.C. Strain improvement through UV and chemical mutagenesis (2007). Purification and characterization of two for enhanced Citric acid production in Molasses-based thermostable proteases from the thermophilic fungus solid state fermentation. Food Biotech. 24: 165-179. Chaetomium thermophilum. J. Microbiol. Biotechnol. 17(4): 624-31. Jebakumar, R.S., Georage, G.L., Babu, S., Raghuchander, T., Gopalaswamy, G., Vidhyasekaran, P., Samiyappan, Li, M. and Yang, Q. (2007). Isolation and characterization of a R., Raja, J.A.J. and Balasubramanian, P. (2000). β-tubulin gene from Trichoderma harzianum. Biochem. Purification and N-terminal sequencing of a 42 kDa Genet. 45: 529-534. Trichoderma viride chitinase isoform effective against Li, M., Yang, Q. and Song, J. (2010). Three tubulin genes of Rhizoctonia solani. Indian Phytopath. 53: 157-161. Trichoderma harzianum: Alpha, Beta, and Gamma. Braz. Jin, Y., Weining, S. and Nevo, E. (2005). A MAPK gene from Arch. Biol. Technol. 53(4): 811-816. Dead Sea fungus confers stress tolerance to lithium salt Lima, L.H.C., De Marco, J.L., Ulhoa, C.J. and Felix, C.R. and freezing-thawing: Prospects for saline agriculture. Proc. (1998). Synthesis of a Trichoderma chitinase which affects Natl. Acad. Sci. USA 102(52): 18992-18997. the Sclerotium rolfsii and Rhizoctonia solani cell wall. BGF Bulletin 62: 45-49. Kang, H.W., Park, D.S., Park, Y.J., You, C.H., Lee, B.M., Eun, M.Y. and Go, S.J. (2002). Fingerprinting of diverse Liu, Z.H., Yang, Q. and Ma, J. (2007). A heat shock protein genomes using PCR with universal rice primers generated gene (hsp22.4) from Chaetomium globosum confers heat from repetitive sequence of Korean weedy rice. Mol. Cells and Na2CO3 tolerance to yeast. Appl. Microbiol. Biotechnol. 13: 281-287. 77(4): 901-908. Kanokmedhakul, S., Kanokmedhakul, K., Nasomjai, P., Liu, Z.H., Yang, Q., Hu, S., Zhang, J.D. and Ma, J. (2008). Loungsysouphanh, S., Soytong, K., Sobe, M., Cloning and characterization of a novel chitinase gene Kongsaeree, K., Prabpai, S. and Suksamran, A. (2006). (chi46) from Chaetomium globosum and identification of Antifungal Azaphilones from the fungus, Chaetomium its biological activity. Appl. Microbiol. Biotechnol. 80(2): 241- cupreum CC3003. J. Natural Products 69: 891-895. 252. Kanokmedhakul, S., Kanokmedhakul, K., Phonkerd, N., Lockhart, R.J., Van Dyke, M.I., Beadle, I.R., Humphreys, P. Soytong, K., Kongsaree, P. and Suksamrarn, A. (2002). and Mc-Carthy, A.J. (2006). Molecular biological detection Antimycobacterial anthraquinone-chromanone compound of anaerobic gut fungi. Appl. Environ. Microbiol. 72: 5659- and diketopiperazine alkaloid from the fungus Chaetomium 5661. globosum KMITL-N0802. Planta. Medica. 68: 834-836. Lockwood, J.L. (1988). Evalution of concepts associated with soil borne plant pathogens. Annu. Rev. Phytopath. 26: 93- Kay, S.J. and Stewart, A. (1994). Evaluation of fungal 121. antagonists of control of onion white rot in soil box trials. Plant Pathol. 43: 371-377. Luche, S., Santoni, V. and Rabilloud, T. (2003). Evaluation of nonionic and zwitterionic detergents as membrane protein Ke, D., Menard, C., Picard, F.J., Boissinot, M., Ouellete, M., solubilizers in two-dimensional electrophoresis. Proteomics Roy, P.H. and Bergeron, M.G. (2000). Development of 3: 249-253. conventional and real-time PCR assays for the rapid detection of group Bacillus Streptococci. Clin. Chem. 46: Mandal, S. (1995). ‘Effect of some antagonist on Drechslera 324-331. sorokiniana, causal agent of spot blotch of wheat’. M.Sc. Thesis, IARI, New Delhi. Khan, A.L., Shinwar, Z.K., Kim, Y., Waqas, M., Hamayun, M., Kamran, M. and Lee, I. (2012). Role of endophyte Mandal, S., Srivastava, K.D., Aggarwal, R. and Singh, D.V. Chaetomium globosum lk4 in growth of Capsicum annuum (1999). Mycoparacitic action of some fungi on spot blotch by producion of gibberellins and indole acetic acid. Pakistan pathogen (Drechslera sorokiniana) of wheat. Indian J. Bot. 44(5): 1601-1607. Phytopath. 52: 39-43. Kimura, H., Morita, M., Yabuta, Y., Kuzushima, K., Kato, K., Mandels, M., Andreotti, R. and Roche, C. (1976). Kojima, S., Matsuyama, T. and Morishima, T. (1999). Measurement of saccarifying cellulose. Biotechnol. Bioeng. Quantitative analysis of Epstein-Barr virus load by using a Symp. 13: 299-314. real-time PCR assay. J. Clin. Microbiol. 37: 132-136. Marra, R., Patrizia, A., Virginia, C., Francesco, V., Sheridan, Klemsdal, S.S. and Elen, O. (2006). “Development of a highly L., Woo, M., Ruocco, R., Ciliento, S., Lanzuise, S., sensitive nested-PCR method a single closed tube for Ferraioli, I., Soriente, S., Gigante, David, Turrà, detection of Fusarium culmorum in cereal samples. Lett. Vincenzo, F., Felice, S. and Matteo, L. (2006). Study of Appl. Microbiol. 42: 544-548. the three-way interaction between Trichoderma atroviride, plant and fungal pathogens by using a proteomic approach. Kombrink, E. and Schmelzer, E. (2001). The hypersensitive Curr. Genet. 50: 307-321. response and its role in local and systemic disease Marwah, R.G., Fatope, M.O., Deadman, M.L., Al-Maqbali, Y.M. resistance. European J. Plant Pathol. 107: 69-78. and Husband, J. (2007). Musanahol: a new aureonitol- Kommedahl, T. and Mew, I.C. (1975). Biocontrol of corn root related metabolite from a Chaetomium sp. Tetrahedron 63: infection in the field by seed treatment with antagonists. 8174-8180. Phytopathology 65: 296-300. Min, Y.S., Kim, B.G., Lee, C., Ho, Gil, H. and Joong Hoon, Lamb, C. and Dixon, R.A. (1997). The oxidative burst in plant A.H.N. (2002). Purification, Characterization, and cDNA disease resistance. Ann. Rev. Plant Physiol. Plant Mol. Biol. cloning of Xylanase from fungus Trichoderma strain SY. J. 48: 251-275. Microbiol. Biotechnol. 12: 1-5. Indian Phytopathology 68 (1) : 8-24 (2015) 23

Mitchell, R. and Hurwitz, E. (1965). Suppression of Pythium Powell, J.W. and Whalley, W.B. (1969). The chemistry of fungi. debaryanum by lytic rhizosphere bacteria. Phytopathology Part LVIII. Structure of colletodial, metabolite of 55:156-158. Chaetomium funicola. J. Chem. Society(C) 1969: 911-912. Miyazaki, T., Inamine, T., Yamauchi, S., Nagayoshi, Y. and Prasad, K.S.K., Kulkarni, S. and Siddarmaiah, A.L. (1978). Saijo, T. (2010). Role of the Slt2 mitogen-activated protein Antagonistic of Trichoderma sp. and Streptomyces spp. on kinase pathway in cell wall integrity and virulence in Drechslera sativum (Helminthosporium sativum Pam. King Candida glabrata. FEMS Yeast Res. 10(3): 343-52. and Bakke). Subram and Jain. Curr. Res. 12: 1202-213. Monga, D. (2001). Effect of carbon and nitrogen sources on Prasun, K. Mukherjee, Jagannathan, Latha, Ruthi, Hadar and spore germination, biomass production and antifungal Benjamin, A., Horwitz (2003). TmkA, a Mitogen-Activated metabolites by species of Trichoderma and Gliocladium. Protein Kinase of Trichoderma virens, Is Involved in Indian Phytopath. 54: 435-437. Biocontrol Properties and Repression of Conidiation in the MoranDiez, E., Hermosa, R., Ambrosino, P., Cadoza, R.E. Dark. Eukaryot Cell. 2(3): 446-455. and Gutierrez, S. (2009). The ThPG1 Puyesky, M., Noyola, P.P., horwitz, B.A., Estrella, A.H. (1997). endopolygalacturonase is required for the Trichoderma Glyceraldehyde -3-phosphate dehydrogenase expression harzianum-Plant beneficial interaction. Am. Phytopathol. in Trichoderma harzianum is repressed during conidiation Soc. 22(8): 1021-1031. and mycoparasitism. Microbiol. 143: 3157-3164. Mostafa, M.M. (1993). Biocontrol of Drechslera teres: abiity of Raeymaekers, L. (1998). Quantitative PCR. In: Methods in antagonists to reduce conidia formation, coleoptile infection Molecular Medicine: Clinical application of PCR. Lo, Y.M.D. and leaf infection in barley (Hordeum vulgare). (Ed). Humana Press, Totowa, NJ, USA 16: 27-38. Cryptogamie Mycologie 14: 287-295. Reilly, P.J. (1981). Xylanases: structure and function. Basic Life Neuhaus, J.M. (1999). Plant chitinases (PR-3, PR-4, PR-8, PR- Sci. 18: 111-129. 11). In: Pathogenesis-related Proteins in Plants, Datta, S.K. and Muthukrishnan, S. (Eds). CRC, Boca Raton, pp. 77- Ridout, C.J., Coley, J.R. and Lynch, J.M. (1988). Fractionation 105. of extracellular enzymes from a mycoparasitic strain of Trichoderma harzianum. Enzymol. Technol. 10: 180-187. Papavizas, G.C. and Lewis, J.A. (1983). Physiological and biocontrol characteristics of stable mutants of Trichoderma Ridout, C.J., Coley-Smith, J.R. and Lynch, J.M. (1986). viride resistant to MBC fungicides. Phytopathology 73: 407- Enzyme activity and electrophoretic profiles of extracellular 411. protein induced in Trichoderma spp. By cell walls of Park, D. (1960). Antagonism – the background of soil fungi. In: Rhizoctonia solani. J. Gen. Microbiol. 132: 2345-2352. The Ecology of Soil Fungi. Parkinson, D. and Waid, J.S., Righetti, P.G., Castagna, A. and Herbert, B. (2000). Eds), Liverpool University Press, Liverpool, pp. 148-159. Prefractionation technique in proteome analysis. Anal. Park, D.S., Kang, H.W., Lee, M.H., Park, Y.J., Lee, B.M., Hahn, Chem. 73: 320A-326A. J.H. and Go, S.J. (2003). DNA Fingerprinting Analysis of Rowlands, R.T. (1984). Industrial strain improvement: the Genus Phytophthora in Korea. Mycobiology 31: 235- mutagenesis and random screening procedures. Enzymes 247. Microbiol. Technol. 6: 3-10. Park, Joong-Hyeop, Gyung, J., Choi, Kyoung, Soo, Jang, Sekita, S., Yoshihira, K., Natori, S., Udagawa, S., Muroi, T., He, Kyoung Lim, Heungm, Tae Kim, Kwang, Yun Cho Sirgiyama, Y., Kurata, H. and Umeda, M. (1981). and Jin-Cheol Kim (2005). Antifungal activity against plant Mycotoxin production by chaetomium spp. And related pathogenic fungi of chaetoviridins isolated from fungi. Canadian J. Microbiol. 27: 766-772. Chaetomium globosum. FEMS Microbiol. Lett. 252(2): 309- SelvaKumar, R., srivastava, K.D., Aggarwal, Rashmi, Singh, 313. D.V. and Dureja, Prem (2000). Studies on development Park, Y.J., Yoon, S.J. and Lee, H.B. (2010). A novel dienelactone of Trichoderma viride mutants and their effect of Ustilago hydrolase from the thermoacidophilic archaeon Sulfolobus segetum tritici. Indian Phytopath. 53(2): 185-189. solfataricus P1: purification, characterization, and expression. Biochim. Biophys. Acta 1800: 1164-1172. Shanthiyaa, V., Saravanakumar, D., Rajendran, L., Karthikeyan, G., Prabakar, K. and Raguchander, T. Patyka, V.P., Kopylov, le P. and Nadkernychnyi, S.P. (2007). (2013). Use of Chaetomium globosum for biocontrol of Adaptation of Chaetomium globosum 3250 Kunze ex Fr. potato late blight disease. Crop Prot. 52: 33-38. to the rhizosphere of spring wheat and its ability to colonize the root system. Mikrobiol. Z. 69(4): 54-62. Shapira, R., Orden tilich, A., Chet, I. and Oppenheim, A.B. (1989). Control of plant diseases by chitinase expressed Paulitz, T.C. (1990). Fungal cell walls: a review, In: Biochemistry from cloned DNA in Escherichia coli. Phytopathology 79: of Cell Walls and Membranes in Fungi. Kuhn, P.J., Trinci, 1246-1249. A.P.J., Jung, M.J., Goosey, M.W. and Copping, L.G. (Eds). Springer Verlag, Heidelberg, Germany, p. 5-24. Sharma, S. (2013). Molecular and biochemical analysis of antagonistic potential of Chaetomium globosum. Ph.D Peberdy, J.F. (1985). Biology of Penicillins. In: Biology of Industrial Microorganism, Demain A and Solomon N. thesis (Kurukshetra University, Haryana). Benjamin Cummings, Menlo Park, pp. 407-431. Sharma, S., Aggarwal, R., Yadav, A. and Gupta, S. (2014). Peberdy, J.F. (1994). Protein secretion in filamentous fungi- Protein mapping of Chaetomiumglobosum, a potential trying tounderstand a highly productive black box. Trends biological control agent through proteomics approach. J. Biotechnol. 12: 50-57. Plant Biochem. Biotechnol. 23(3): 284-292. Pedersen, S.K., Harry, J.L. and Sebastian, L. (2003). Unseen Shternshis, M., Tomilova, O., Shpatova, T. and Soytong, K. proteome: mining below the tip of the iceberg to find low (2005). Evaluation of Ketomium myco fungicide on Siberian abundance and membrane proteins. J. Protein Res. 2: 303- isolates of phytopathogenic fungi. J. Agric. Technol. 1(2): 311. 247-253. Phonkerd, N., Kanokmedhakul, S., Kanokmedhakul, K., Sibounnavong, P. Charoenporn, C., Kanokmedhakul, S. and Soytong, S., Prabpai, S. and Kongsearee, P. (2008). Bis- Soytong, K. (2011). Antifungal metabolites from spiro-azaphilones and azaphilones from the fungi antagonistic fungi used to control tomato wilt fungus Chaetomium cochliodes VTh01 and C. cochliodes CTh05. Fusarium oxysporum f. sp. Lycopersici. African J. Biotech. Tetrahedron 64: 9636-9645. 10(85): 19714-19722. 24 Indian Phytopathology 68 (1) : 8-24 (2015)

Singh, N., Pandey, P., Dubey, R.C. and Maheshwari, D.K. van, Loon L.C. and van, Strien E.A. (1999), The families of (2008). Biological control of root rot fungus Macrophomina pathogenesisrelated proteins, their activities, and phaseolina and growth enhancement of Pinus roxburghii comparative analysis of PR-1 type proteins. Physiol. Mol. (Sarg.) by rhizosphere competent Bacillus subtilis BN1. Plant Path. 55: 85-97. World J. Microbiol. Biotechnol. 24: 1669-1679. Vannacci, G. and Harman, G.F. (1987). Biocontrol of seed Soytong, K. (1992). Biological control of tomato wilt caused by borne Alternaria raphani and A. brassicicola. Can. J. Fusarium oxysporum f. sp. Lycopersici using Chaetomium Microbiol. 33: 850-856. cupreum. Kasetsart J. (Nat. Sci.) 26: 310-313. Vilich, V. (1988). Effect of benzoxazolinone allelochemicals from Soytong, K. and Quimino, T.H. (1989). Antagonism of wheat on selected soil borne pathogens and antagonists. Chaetomium globosum to the rice blast pathogen Proceedings of 50th International Symposium on Crop Pyricularia oryzae. Kasetsart J. (Nat. Sci.) 23: 198-203. Protection Gent. 63: 971-976. Soytong, K., Kanokmedhakul, S., Kukongviriyapa, V. and Vinci, V. and Byng G. (1999). Strain improvement by non Isobe, M. (2001). Application of Chaetomium species recombinant methods. In: Manuals of Industrial (Ketomium®) as a new broad spectrum biological fungicide Microbiology and Biotechnology. Demain, A.L. and Davies, for plant disease control: A review article. Fungal Divers. J.E. (Eds). American Society for Microbiology, Washington 7: 1-15. DC, pp. 103-113. Srivastava, K.D., Biswas, Samir, Mandal, S. and Aggarwal, Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de, Lee T., R. (2000). Biological control of spot blotch (Drechslera Hornes, M., Frijters. A. Pot, J., Peleman, J., Kuiper. M. sorokiniana) of wheat. Proceedings of Conference on et al. (1995). AFLP: a new technique for DNA fingerprinting. Integrated Plant Disease Management for Sustainable Nucleic Acids Res. 23: 4407-4414. Agriculture, Indian Phytopathological Society, New Delhi, Walther, D. and Gindrat, D. (1988). Biological control of pp. 1052-1053. damping off of sugar beet and cotton with Chaetomium Syed, N.A., David, J.M., Pearl, K.C. Ly, Jennifer, A.S. and globosum or a fluorescent Pseudomonas sp. Canadian J. Peter, A. McGee (2009). Do plant endophytic and free- Microbiol. 34: 631-637. living Chaetomium species differ? Australas. Mycol. 28: Wilkins, M.R., Sanchez, J.C., Gooley, R.D., Appel, I., 51-55. Humphery-Smith, Hochstrasser, D.F. and Williams, K.L. Thakur, V.S. and Sharma, R.D. (1999). Effect of urea on (1995). Progress with proteome projects. Biotechnol. microbial degradation of apple leaf litter and its relationship Genet. Eng. Rev. 13: 19-50. to the inhibition of pseudothecial development of Venturia Wong, K.K.Y., Tan, L.U.L., Saddler, J.N. and Yaguchi M. (1986). inaequalis. Indian J. Agric. Sci. 69: 147- 151. Purification of a third distinct xylanase from the xylanolytic Thohinung, S., Kanokmedhakul, S., Kanokmedhakul, K., system of Trichodermaharzianum. Canadian J. Microbiol. Kukongviriyapan, V., Tusskorn, O. and Soytong, K. 32: 570-576. (2010). Cytotoxic 10-(indol-3-yl)-[13] cytochalasans from Yan, J.X., Harry, R.A., Spibey, C. and Dunn, M.J. (2000). Post the fungus Chaetomium elatum ChE01. Arch. Pharm. Res. electrophoretic staining of proteins separated by two- 33: 1135-1141. dimensional gel electrophoresis using SYPRO dyes. Tilburg Van, A.U.B. and Thomas, M.D. (1993). Production of Electrophoresis 21: 3657-3665. Extracellular Proteins by the Biocontrol Fungus Yang, Qian, Cong, Hua, Zhang, Haiyan, Yao, Lin, Liu, Pigang Gliocladium virens. Appl. Environ. Microbiol. 59(1): 236- and Jin, Hongxing (2007). Study on Bio-Control Related 242. Genes of Trichoderma sp. and Chaetomium spp. Kmitl. Sci. Tveit, M. and Moore, M.B. (1954). Isolates of Chaetomium that Tech. J. 7(1): 8-15. protects oats from Helminthosporium victoirae. Yang, S.Y., Qiaojuan, Z., Jiang, G.F. and Wang, L. (2008). Phytopathology 44: 686-689. Biochemical characterization of a novel thermostable β-1, 3-1, 4-glucanase (lichenase) from Paecilomyces Tveit, M. and Wood, R.K.S. (1955). The control of Fusarium thermophila. J. Agric. Food Chem. 56: 5345-5351. blight in oat seedling with antagonists species of Chaetomium. Ann. Appl. Biol. 43: 538-552. Ying, S.H. and Feng, M.G. (2004). Relationship between thermo tolerance and hydrophobin-like proteins in aerial conidia Tweddell, J.R., Jabaji-Hare, S.H. and Charest, P.M. (1994). of Beauveria bassiana and Paecilomyces fumosoroseus Production of chitinase and β 1,3- glucanases by as fungal biocontrol agents. J. Appl. Microbiol. 97: 323- Stachybotrys elegans, a Mycoparasite of Rhizoctonia 331. solani. Appl. Envir. Microbiol. 60: 489-495. Zhang, H. and Yang, Q. (2007). Expressed sequence tags Udagawa, S. Muroi, T., Kurata, H., Sekita, S., Yoshihira, H., based identification of genes in the biocontrol agent Natori, S. and Umeda, M. (1979). The production of Chaetomium cupreum. Appl. Microbiol. Biotechnol. 74(3): chaetoglobosins, sterigmatocystins, O-methyl 650-658. sterigmatocystin and chaetocin by Chaetomium spp. and Zhang, Y., But, P.P., Wang, Z. and Shaw, P. (2005). Current related fungi. Can. J. Microbiol. 25: 170-177. approaches for the technique for detecting genetic variation van, Loon L.C. (1997). Induced resistance in plants and the of Xanthomonas axonopodis pv. Manihotis”.using a single role of pathogenesis-related proteins. European J. Plant closed tube for detection of Fusarium culmorum in cereal Pathol. 103: 753-765. samples”. Letters in Plant Genetic Res. 3: 144-148.