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Turkish Journal of Biology Turk J Biol(2013) 37: 323-328© TÜBİTAKdoi:10.3906/biy-1209-20

Enhancement of mycolytic activity of an antagonistic Bacillus subtilisthrough ethyl methane sulfonate (EMS) mutagenesis

Ashwini NARASIMHAN, Samantha SURESH, Deepak BIST, Srividya SHIVAKUMAR*Department of Microbiology, Center for PG Studies, Jain University, 18/3,9th Main, Jayanagar 3rd block, Bangalore, India

* Correspondence: sk.srividya@jainuniversity.ac.in

1. IntroductionMycolytic enzyme-based formulations consisting of chitinases, proteases, and glucanases have been used to control fungal phytopathogens (1–4). The genus Colletotrichum and its teleomorph Glomerella contain an extremely diverse number of fungi, including both plant pathogens and saprophytes. Plant pathogenic species are important worldwide, causing pre- and postharvest losses of crops. These fungi cause diseases commonly known as anthracnose of grasses, legumes, vegetables, fruits, and ornamentals. The disease can occur on leaves, stems, and fruit of host plants (5). Disease outbreaks can occur rapidly and losses can be severe, especially under prolonged warm and wet weather conditions. Effective control of anthracnose disease involves the use of one of, or a combination of, the following: resistant cultivars, cultural control, and chemical control. The intensive use of fungicides has resulted in the accumulation of toxic compounds potentially hazardous to humans and the environment and the build-up of resistance by the pathogens. In view of this, investigation and application

of biological control agents (BCAs) seems to be one of the promising approaches (6).

Strain improvement is a lengthy and laborious job, where we have to screen the better isolates among a mutagen-treated population. Classical mutagenesis with physical and/or chemical agents followed by titer test of a large number of isolates has been used successfully to improve the productivity of several fungal metabolites and enzymes (7–9). Chemical mutagens may induce mutations within a sequence, originating mutagen-specific patterns of mutations. Still, mutagenesis and selection are cost effective procedures for reliable short term strain improvement (10,11). The greatest advantage of screening methods is simplicity, as these methods do not require any profound understanding of the molecular biology and physiology of the microorganisms being manipulated (12). B. subtilis JN032305, isolated from the chili rhizosphere, produced appreciable levels of 3 mycolytic enzymes (chitinase, glucanase, and cellulase), and showed broad spectrum antagonism against potent bacterial and fungal phytopathogens (13). The production

Abstract: Bacillus subtilis, isolated from the chili rhizosphere, capable of producing 3 mycolytic enzymes (chitinase, β-1,3-glucanase, and cellulase), was mutated by the chemical mutagen ethyl methane sulfonate (EMS). Mutants were screened based on their antifungal ability on dual plate assay against Colletotrichum gloeosporioides OGC1, the causal agent of anthracnose disease in fruit crops. EMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4, and M24) showed loss of antagonism against C. gloeosporioides OGC1 and 6 (M8, M21, M22, M57, M58, and M59) exhibited increased antagonism. The remaining mutants did not show any difference in their antagonistic property. In liquid culture, the mutants exhibited varied levels of the 3 enzymes. These mutants were studied for their mycolytic enzyme activities under shake flask conditions. Of all these mutants, M57 showed a 36-fold and 4.71-fold increase in β-1,3-glucanase and cellulase activities, respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59, with a 5.68-fold and 1.57-fold increase in β-1,3-glucanase and cellulase activities, respectively, and a 2.23-fold increase in hydrolytic activity as compared to the wild type B. subtilis strain. M24 exhibited a complete loss of β-1,3-glucanase and a decrease in chitinase, with a concomitant decrease in levels of hydrolytic activity with C. gloeosporioides mycelia as compared to the wild type strain. The study clearly indicated the mycolytic enzyme mediated antagonism of this strain and yielded 2 hyperproducing mutants for β-1,3-glucanase and cellulase, 2 important enzymes for various agricultural and industrial uses.

Key words: Bacillus subtilis, mycolytic enzymes, ethyl methane sulfonate (EMS) mutagenesis, Colletotrichum gloeosporioides

Received: 10.09.2012 Accepted: 04.11.2012 Published Online: 16.05.2013 Printed: 17.06.2013

Research Article

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of all 3 enzymes of this strain has been optimized using the Plackett–Burman approach (14,15). The objective of the present study was to ascertain the concerted role of these 3 mycolytic enzymes (chitinase, β-1,3-glucanase, and cellulase) in mediating antagonism of B. subtilis against C. gloeosporioides and strain improvement for the hyperproduction of these mycolytic enzymes for agricultural or industrial purposes.

2. Materials and methods2.1. Organism and culture conditionsThe mycolytic enzymes producing bacteria was isolated from the chili rhizosphere. It was identified as Bacillus subtilis by morphological, biochemical, and 16S rDNA sequence analysis. The GenBank accession no. for the nucleotide sequence is JN032305.2.2. Preparation of colloidal chitinTen grams of crab shell chitin was slowly added into 100 mL of cold concentrated hydrochloric acid (HCl), with vigorous stirring, and then kept overnight at 4 °C. The mixture was precipitated with 1000 mL of ice cold ethanol at 4 °C with rapid stirring. The chitin suspension was then centrifuged at 10,000 × g for 20 min and the resultant chitin pellet was washed repeatedly with a 0.1 M phosphate buffer (pH 7) until the pH became neutral (16). 2.3. Enzyme productionThe culture was grown at 30 °C with 120 rpm for 24 h. Then 200 µL of the culture inoculum was transferred to a 250-mL Erlenmeyer flask containing 100 mL of broth medium with (g/L): CMC, 10; peptone, 5; Beef extract, 3; and NaCl, 5. The pH of the medium was adjusted to 7.0 using 1 N NaOH before autoclaving. All the experiments were performed in duplicate. The culture broth, after 72 h of incubation, was centrifuged at 10,000 × g for 10 min at 4 °C to separate the cells. The cell-free supernatant was analyzed for enzyme activity (17).2.4. Chitinase assayChitinase activity was assayed following the release of N-acetylglucosamine according to the method described by Monreal and Reese (18). A reaction mixture (3 mL), containing 1 mL of 1% colloidal chitin in 0.1 M phosphate buffer (pH 6.5), was incubated with 1.0 mL of culture filtrate at 37 °C for 45 min. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 µg of glucose per min. 2.5. Glucanase (β-1,3 and β-1,4) assay The specific activity of β-1,3 and β-1,4 glucanase was determined by measuring the amount of reducing sugars liberated using dinitrosalicylic acid solution (DNS) (19). The culture broth was centrifuged and 1 mL of enzyme solution was added to 1.0 mL of substrate solution, which

contained 1 mL of yeast cell wall extract (YCW, 1 %, v/v) or carboxymethyl cellulose solution (CMC, 1 %, v/v), respectively. The mixture was incubated in a water bath at 50 °C for 30 min, after which the reaction was terminated by adding 1 mL of DNS solution and incubating the mixture in a boiling water bath for 10–15 min until the development of the color of the end product. Reducing sugar concentration was determined by optical density at 540 nm (17). 2.6. EMS mutagenesisChemical mutagenesis of B. subtilis was carried out with ethyl methane sulfonate by the method of Khattab and Bazaraa (20). The B. subtilis cells (108 cfu/mL) were washed once with 0.1 M tris-HCl buffer (pH 7.5) and resuspended in 1 mL of the same buffer. To this 100 μL of EMS was added and the mixture incubated in a shaker water bath for 45 min. The cells were washed 3 times with 0.1 M tris-HCl buffer (pH 7.5) and serial dilutions were plated on nutrient agar plates. The mutants were tested for their antagonistic activity using the dual plate assay. The mutants with loss or increase in this property were tested for their ability to produce the mycolytic enzymes under shake flask conditions. These mutants were also checked for their hydrolytic activity using C. gloeosporioides mycelia as substrate (21).2.7. PhytopathogensThe following 6 phytopathogens were obtained as a kind gift from the Indian Institute of Horticultural Research (IIHR), Hessarghatta, Bangalore: Alternaria brassisicola (OCA1), Alternaria brassicae (OCA3), Alternaria alternata (OTA36), Fusarium solani, Colletotrichum gloeosporioides (OGC1), and Phytophthora capsici (98-01). Verticillium theobromae, Fusarium oxysporum, and Rhizoctonia solani (MTCC 4633) were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh. These 9 pathogens were used in the study as they were potent pathogens of chili plants, causing leaf spot, wilt, anthracnose, and root rot, thereby severely constraining the yield of chili plants.2.8. Dual plate assayThe wild and mutant strains of B. subtilis were evaluated for their antagonistic potential against Colletotrichum gloeosporioides OGC1 by the dual culture technique of Huang and Hoes (22). One 5-mm disk of a pure culture of the pathogen was placed at the centre of a petri dish containing PDA. The B. subtilis (wild type) and EMS mutants were inoculated at 4 opposing corners. Plates were incubated for 72 h, at 28 °C, and the growth diameter of the pathogen was observed and compared to control growth, where the bacterial suspension was replaced by sterile distilled water. Each experiment using a single pathogen isolate was run in triplicate.

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2.9. Preparation of hyphal wallThe pathogenic fungal culture (C. gloeosporioides) was inoculated into 50 mL of PDB broth and incubated at 30 °C for 5 days under shaking conditions. After incubation, the mycelia were collected by filtration. The mycelia were thoroughly washed with autoclaved distilled water and homogenized on ice with a homogenizer for 5 min. The mycelia suspension was centrifuged at 10,000 × g for 20 min at 4 °C (Remi-C24, Remi Laboratory Instruments, India). The pellet was resuspended in sodium phosphate buffer (0.1 M, pH 7.0). This preparation was used as substrate for the hydrolytic assay (17). 2.10. Hydrolytic activity of B. subtilis (wild type and EMS mutants) culture filtrateFor assessing the hydrolytic activity, the reaction mixture (1 mL) containing 1 mg/mL of fungal mycelia with 1.0 mL of crude enzyme (from B. subtilis grown on NB+CMC) was incubated at 30 °C for 24 h. The released total reducing sugars (19) in the control and treated fungal cell wall was estimated using the DNS method. The hydrolytic activity was performed using the culture filtrate of both the WT B. subtilis strain and the EMS mutants of B. subtilis.2.11. Statistical analysisAll the experiments were done 3 times in triplicate and the values represented statistically are in analysis of variance (ANOVA) form.

3. Results and discussionThe present organism under study, B. subtilis (JN032305), exhibited a broad spectrum inhibition of potent chili pathogens by the production of mycolytic enzymes. As reported earlier, on dual plate assay, the wild type strain of B. subtilis JN032305 showed broad spectrum antagonism against Alternaria (3) spp. (55%), Colletotrichum gloeosporioides (57%), Phytophthora capsici (55%), Rhizoctonia solani (42%), Fusarium solani (42%), Fusarium oxysporum (40%), and Verticillium theobromae (36%) (13,14).3.1. EMS mutagenesisEMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4, and M24) mutants showed loss of antagonism against C. gloeosporioides and 6 mutants (M8, M21, M22, M57, M58, and M59) exhibited increased antagonism against C. gloeosporioides. The remaining mutants did not show any difference in their antagonistic property (Figure 1). These mutants were studied for their mycolytic enzyme activities under shake flask conditions. Mutants M4 and M8 showed a complete loss of chitinase and β-1,3-glucanase activity; mutants M21 and M22 showed a complete loss of chitinase activity; mutants M3, M24, M58, and M59 showed decrease in chitinase activity; mutant M24 showed a complete loss of β-1,3-glucanase activity. All the mutants except M21

Figure 1. Dual plate assay showing different antagonistic levels of EMS mutants against C. gloeosporoides OGC1 on PDA plates. Plates A & C showing mutants 21, 22, 23, and 24, and mutants 1, 2, 3, and 4, respectively, with varied levels of inhibition; Plate B showing mutants 57, 58, and 59 with increased inhibition; Plate D showing C. gloeosporioides OGC1 control.

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showed varied levels of increase in cellulase levels. These mutants were further checked for their antifungal activity using C. gloeosporioides mycelia and it was found that M8, M22, M57, M58, and M59 showed increased hydrolytic activity with concomitant increases in one or more mycolytic enzyme levels as compared to wild type (Figure 2). Of all these mutants, M57 showed 36-fold and 4.71-fold increases in β-1,3-glucanase and cellulase activities, respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59 with 5.68-fold and 1.57-fold increases in β-1,3-glucanase and cellulase activities, respectively, with a 2.23-fold increase in hydrolytic activity as compared to the wild type B. subtilis strain (Figure 3). The mutant M24 exhibited a complete loss of β-1,3-glucanase and decrease in chitinase with concomitant decreases in levels of hydrolytic activity with C. gloeosporioides mycelia as compared to the wild type strain (Figure 2). All these observations clearly indicated the mycolytic enzyme mediated antagonism of this strain. In a similar study, Kandasamy and Saleem (23) showed the role of the Bacillus strain BC121 in suppressing the fungal growth in vitro when studied in comparison with a mutant of that strain that lacks both antagonistic activity and chitinolytic activity. Lorito et al. (24) reported chitinolytic enzymes contributing to the ability of Trichoderma sp. to act as biocontrol agents.

ANOVA has been performed for hydrolytic enzyme production by the wild type and mutants of B. subtilis. P-value was found to be very low at both P = 0.05 and P = 0.01, which indicated that there are significant differences in mycolytic enzyme production between the strains (Table).

Strain improvement can generally be described as the use of any scientific techniques that allow the isolation of cultures exhibiting a desired phenotype. The technology has been utilized for more than 50 years in conjugation with modern submerged culture fermentations (25). EMS is a well-known mutagenic agent, whose mode of action is attributed to alkylation at nitrogen position 7 of guanosine of the DNA molecule, leading to transversion or transition type of mutations (26).

Biocontrol efficacy of the mutants and wild strain were tested against phytopathogens such as Fusarium oxsporum, Bipolaris oryzae, Rhizoctonia solani, and Alternaria sp. by dual culture assay on PDA medium (27). The UV H11 mutant and adapted mutant showed increased biocontrol activity when compared to the wild strain. The overall mycelia growth inhibition of F. oxysporum, B. oryzae, R. solani, and Alternaria sp. were 69.9%, 75.5%, and 82.7% by wild, adapted, and UV mutant, respectively. Graeme-Cook et al. (28) reported that high antibiotic production by two

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Figure 2. Mycolytic activity of Bacillus subtilis wild type and EMS mutants. Values are mean ± SE of 3 replicates.

Figure 3. Comparison of mycolytic enzyme profile of Bacillus subtilis wild type and hyperproducing EMS mutants M57 and M59. Values are mean ± SE of 3 replicates.

Table. ANOVA for hydrolytic enzymes production by wild type and mutant strains of B. subtilis.

Source of variation Degree of freedom Sample square Mean square F-statistics P-value

Between samples 9 291.92 32.43 853.42 2.45 (S*)

Within samples 20 0.76 0.038

(S*)- Significant

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T. harzianum mutant strains, BC10 and BC63, increased inhibition of hyphal growth of R. solani and P. ultimum, while Papavizas et al. (29) have shown UV-induced benomyl resistant mutants suppress the saprophytic activity of R. solani more effectively than the wild strain. Bapiraju et al. (30) reported mutation induced enhanced lipase production from Rhizopus sp. using UV radiation and NTG. Kadam et al. (31) successfully employed UV mutagenesis to improve a strain of Lactobacillus delbruekii for lactic acid production. Successful use of EMS in induced mutations has been reported for many bacterial strains (31–33). Increase in chitinolytic enzyme production was reported in Pseudomonas stutzeri YPL M26 after UV and NTG mutagenesis (34).

Mutagenesis increased enzyme production successfully in Trichoderma (35) and some other fungi in an industrial process (36). G. virens mutants have shown differences in their ability to produce the antibiotic gliovirin (37). Mutagenesis alters the production of antibiotics and mycolytic enzymes in biocontrol agents in T. viride (38).

In summary, these results suggest that B. subtilis M59 showed a high level of mycolytic enzyme production (chitinase, β-1,3-glucanase, and cellulase) and enzyme mediated inhibition of the growth of plant fungal pathogens as compared to the wild type strain and other mutants of B. subtilis. Although naturally occurring organisms provide a major source of mycolytic enzymes, genetic improvement plays an important role in their biotechnological applications. Of the different methods available for strain improvement to increase enzyme production, random mutagenesis through physical (UV, gamma etc.) and chemical (EMS) agents have been employed to obtain improved biological strains, including Pantoea dispersa (22) and Alcaligenes xylosoxydans (39). Thus, B. subtilis mutants obtained in this study by EMS mutagenesis have good potential for use as an antifungal biocontrol agent upon appropriate formulation and can be exploited for other industrial uses based on the chitinase and cellulases produced by this strain.

References

1. Vyas P, Deshpande MV. Chitinase production by Myrothecium verrucaria and its significance for fungal mycelia degradation. J Gen Appl Microbiol 35: 343–50, 1989.

2. Singh G, Sharma JR, Hoondal GS. Chitinase production by Serratia marcescens GG5. Turk J Biol 32: 231–236, 2008.

3. Okay S, Özcengiz G. Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni. Turk J Biol 35: 1–7, 2011.

4. Çömlekçioğlu U, Özköse E, Akyol İ, Yazdıç FC, Ekinci MS. Expression of β-(1,3-1,4)-glucanase gene of Orpinomyces sp. GMLF18 in Escherichia coli EC1000 and Lactococcus lactis subsp. cremoris MG1363. Turk J Biol 35: 405–414, 2011.

5. Sutton BC. The genus Glomerella and its anamorph Colletotrichum. In: Colletotrichum: Biology, Pathology and Control (eds. Bailey JA, Jeger MJ). CAB Int. Wallingford; 1992: pp. 1–26.

6. Cook RJ. Biological control of plant pathogens: theory to application. Phytopathology 75: 25–29, 1985.

7. Bai Y, Feng X, Van der Hulst R et al. A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12. Mol Breeding 13: 281–287, 2004.

8. Pandey A, Nigam P, Soccol VT et al. Advances in microbial amylases. Biotechnol Appl Biochem 31: 135–152, 2000.

9. Rubinder K, Chadha BS, Singh S et al. Amylase hyper producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion. Can J Microbiol 46: 669–673, 2000.

10. Rowland JH, Glidewell OJ, Sibley RE et al. Effects of different forms of central nervous system prophylaxis on neuropsychologic function in childhood leukemia. J Clin Oncol 2: 1327–1335, 1984.

11. Iftikhar T, Niaz M, Anjum ZM et al. Mutation induced enhanced biosynthesis of lipases by Rhizopus oligosporus var Microsporus. Pak J Bot. 42: 1235–1249, 2010.

12. Gromada A, Fiedurek J. Selective isolation of Aspergillus niger mutants with enhanced glucose oxidase production. J Appl Microbiol 82: 648–652, 1997.

13. Srividya S, Sasirekha B, Ashwini N. Multifarious antagonistic potentials of rhizosphere associated bacterial isolates against soil borne diseases of Tomato. Asian J Plant Sci Res 2: 180–186, 2012.

14. Ashwini N, Srividya S. Optimization of chitinase produced by a biocontrol strain of Bacillus subtilis using Plackett-Burman design. European J Experimental Biology 2: 861–865, 2012.

15. Ashwini N, Srividya S. Optimization of fungal cellwall degrading glucanases produced by a biocontrol strain of Bacillus subtilis using Plackett-Burman design. Proc Intl Conf Biol Active Mol, (Excel India Publisher, New Delhi) 2012. 430–433.

16. Roberts WK, Selitrennikoff CP. Plant and  bacterial chitinases differ in antifungal activity. J Gen Microbiol 134: 169–176, 1988.

17. Moataza MS. Destruction of Rhizoctonia solani and Phytophthora capsici causing tomato root-rot by Pseudomonas fluorescences lytic enzymes. Res J Agri Biol Sci 2: 274–281, 2006.

18. Monreal J, Reese ET. The chitinase of Serratia marcescens. Can J Microbiol 15: 689–96, 1969.

19. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426–428, 1959.

20. Khattab AA, Bazaraa WA. Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production. J Ind Microbiol Biotechnol 32: 289–294, 2005.

NARASIMHAN et al. / Turk J Biol

328

21. Gohel V, Chaudhary T, Vyas P et al. Isolation and identification of chitinolytic bacteria and their potential in antifungal biocontrol. Indian J Exp Biol 42: 715–720, 2004.

22. Huang HC, Hoes JA. Penetration and infection of. Sclerotinia sclerotiorum by Coniothyrium minitans. Can J Bot 54: 406–410, 1976.

23. Basha S, Ulaganathan K. Antagonism of Bacillus species (strain BC121) towards Curvularia lunata. Curr Sci India 82: 1457–1463, 2002.

24. Lorito M, Harman GE, Hayes CK et al. Chitinolytic enzymes produced by Trichoderma harzianum: Antifungal activity of purified endochinitinase and chitobiosidase. Phytopathology 83: 302–307, 1993.

25. Victor AV, Graham B. Strain Improvement by Nonrecombinant Methods. In Demain and Devis (eds) Manual of Industrial Microbiology and Biotechnology. ASM Press, Washington, D.C.; 1999: pp.103–113.

26. Freese EB. Transitions and transversions induced by depurinating agents. Proc Natl Acad Sci USA 47: 540–545, 1961.

27. Balasubramanian N, Thamil Priya V, Gomathinayagam S et al. Effect of chitin adapted and ultraviolet induced mutant of Trichoderma harzianum enhancing biocontrol and chitinase Activity. Aust J Basic Appl Sci 4: 4701–4709, 2010.

28. Graeme-Cook KA, Faull JL. Effect of ultraviolet induced mutants of Tricoderma harzianum with altered antibiotic production of selected pathogens in vitro. Can J Microbiol 37: 659–664, 1991.

29. Papavizas GC, Lumsden RD. Improved medium for isolation of Trichoderma spp. from soil. Plant Dis 66: 1019–1020, 1982.

30. Bapiraju KV, Sujatha P, Ellaiah P et al. Mutation induced enhanced biosynthesis of lipase. Afr J Biotechnol 3: 618–621, 2004.

31. Kadam S, Patil SS, Bastawde KB et al. Strain improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid production. Process Biochem 41: 120–126, 2006.

32. Shanthamma MS, Joseph R, Rao TNR. Quantitative assessment of the mutagenic effect of ethyl methane sulfonate in Micrococcus glutamicus. Folia Microbiol 17: 81–87, 1972.

33. Haq IU, Ali S, Saleem A et al. Mutagenesis of Bacillus licheniformis through ethyl methane sulphonate for alpha amylase production. Pak J Bot 41: 1489–1498, 2009.

34. Lim HS, Kim SD, Kin SD. Pseudomonas stutzeri YPL-1 genetic transformation and antifungal mechanism against Fusarium solani, an agent of plant root rot. Appl Environ Microbiol 57: 510–516, 1991.

35. Mandels M, Weber J, Parizek R. Enhanced cellulase production by a mutant of Trichoderma viride. Appl Microbiol 21: 152–154, 1971.

36. Mäntylä A, Paloheimo M, Suominen P. Industrial mutants and recombinant strains of Trichoderma reesei. In Trichoderma and Gliocladium, Harman GE, Kubiceck CP eds. Taylor & Francis; 1998: pp. 289–310.

37. Howell CR, Stripanovic RD. Gliovirin an antibiotic from Gliocladium virens and its role in the biocontrol of Pythium ultimum. Can J Microbiol 29: 321–324, 1983.

38. Pandey A, Ashakumary L, Selvakumar P. Coconut oil cake—a potential raw material for the production of α-amylase. Bioresource Technol 93: 169–174, 2004.

39. Vaidya RJ, Macmil SLA, Vyas PR et al. The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans. Lett Appl Microbiol 36: 129–134, 2003.