Chitinolytic efficacy and secretion of cell wall degrading enzymes from Trichoderma spp. in response to phyto-pathological fungi

Dinesh K Khatri; Durgesh Nandini Tiwari; Himanshu S Bariya

doi:10.7324/JABB.2017.50601

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Abstract

Chitinolytic activity and major antifungal enzyme secretion form Trichoderma spp. was studied. Soil samples were collected from different environmental niche of North Gujarat Region, India and 12 different species of Trichoderma were obtained and identified. Among 12 isolates, 4 isolates were identified as T. harzianum, 5 isolates were identified as T. viride and remaining 3 isolates were as of T. hamantum. These isolates were identified by using species specific primers amplification by PCR. All identified isolates were screened for chitinase activity using colloidal chitin derived from commercial chitin on the media supplemented with bromocresol purple. According to results of chitinase activity screening assay, T. viride was found to be more potential isolate for chitinase production. From biocontrol assay by using duel culture method, T. viride was found to be more potent antagonist against fungal plant pathogens like A. niger, F. oxysporum and S. rolfsii. T. viride was selected for further study of biocontrol potential and production of cell wall degrading enzymes. T. viride was inoculated in media containing basal media and mycelia of fungal pathogens for cell wall degrading enzyme production. It was found that T. viride secrete three major cell wall degrading enzymes i.e. chitinase, protease and β-Glucanase. Optimum production of all three enzymes was found at 96 hr incubation. Details of antifungal protein secretion are mentioned in this paper.

Keyword: Trichoderma Chitinolytic Chitinase Protease Glucanase Biocontrol.

Citation:

Khatri DK, Tiwari DN, Bariya HS. Chitinolytic efficacy and secretion of cell wall-degrading enzymes from Trichoderma spp. in response to phytopathological fungi. J App Biol Biotech. 2017;5(6):1-8. DOI: 10.7324/JABB.2017.50601.

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Reference

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8 Gajera H, Domadia R, Patel S, Kapopara M, Gloakiya B. Molecular mechanism of Trichoderma as bio-control agents against phytopathogen system - a review. Current research in Microbiology and Biotechnology. 2013; 1(4):133-142.
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1 Andre S, Monika S. Biology and biotechnology of Trichoderma. Applied Microbiology and Biotechnology. 2010; 87:787-799. https://doi.org/10.1007/s00253-010-2632-1
2 Agrawal T, Kotasthane AS. Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India, Spinger Plus. 2012; 1:73-84. https://doi.org/10.1186/2193-1801-1-73
3 Beak JM, Howell CR, Kenerely CM. The role of extracellular chitinase from Trichoderma viriens Gv 29-8 in the biocontrol of Rhizoctina solani. Current Genetic. 1999; 35:41-50. https://doi.org/10.1007/s002940050431
4 Ahmed AS, Ezziyyani M, Sanchez CP, Candela ME. Effect of chitin on biological control activity of Bacillus Spp and Trichoderma harzianum against root rot disease in peeper (Capsicum annuum) plants. European Jounral of Plant Pathology. 2003; 109:633-637. https://doi.org/10.1023/A:1024734216814
5 Benitez T, Rincon AM, Limon MC, Codon AC. Biocontrol mechanisms of Trichoderma strains. International Journal of Microbiology. 2004; 7:249-260.

6 Navazio L, Baldan B, Moscatiello R, Zuppini A, Woo SL, Mariani P, Lorito M. Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biology. 2007; 7:41-46. https://doi.org/10.1186/1471-2229-7-41

7 Sharma P, Kumar VP, Ramesh R, Saravanan K, Deep S, Sharma M, Mahesh S, Dinesh S. Biocontrol genes from Trichoderma species- A review. African Journal of Biotechnology. 2011; 10(86):19898-19907.

8 Gajera H, Domadia R, Patel S, Kapopara M, Gloakiya B. Molecular mechanism of Trichoderma as bio-control agents against phytopathogen system - a review. Current research in Microbiology and Biotechnology. 2013; 1(4):133-142.
9 Beier S, Bertilsson S. Bacterial chitin degradation- mechanisms and ecophysiological strategies. Frontier in Microbiology. 2013; 4:149-160. https://doi.org/10.3389/fmicb.2013.00149
10 Ruiz-Herrera J. Fungal cell wall: structure, synthesis, and assembly. CRC Press: Boca Raton; 1992.
11 Schirmbock M, Lorito M, Wang YL, Hayes CK, Arisan-atac I et al. Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Applied Environmental Microbiology. 1994; 60: 4364-4370.

12 Kubicek CP, Mach RL, Peterbauer CK, Lorito M. Trichoderma: from genes to biocontrol. Journal of Plant Pathology. 2001; 83:11-23.

13 Viterbo A, Ramot O, Chernin L Chet I, Signiï¬cance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal plant pathogens, Antonie van Leeuwenhoek, 81 (2002) 549-556. https://doi.org/10.1023/A:1020553421740

14 Gajera HP, Bambharolia RP, Patel SV, Khatrani TJ, Goalkiya BA. Antagonism of Trichoderma spp. against Macrophomin aphaseolina: Evaluation of coiling and cell wall degrading enzymatic activities. Journal of Plant Pathology and Microbiology. 2012; 3(7):149-156.
15 Elad Y, Chet I. Improved selective media for isolation of Trichoderma spp. or Fusarium spp. Phytoparasitica. 1983; 11:55-58. https://doi.org/10.1007/BF02980712

16 Meena SN, Development of species SCAR markers for the identification of Trichoderma species. 2009. Ph.D. thesis. University of Agricultiural Science, Dharwad.

17 Roberts WK, Selitrennifoff CP. Plant and bacterial chitinase differ in antifungal activity. Journal of General Microbiology. 1998; 134:169-176.

18 Tseng SC, Liu SY, Yang HH, Lo CT, Peng KC. Proteomic study of biocontrol mechanism of Trichoderma harzianum ETS 323 in response to Rhizoctina solani. Journal of Agricultural and Food Chemistry. 2008; 56:6914-6922. https://doi.org/10.1021/jf703626j
19 Lowry OH, Rosebrogh NJ, Farr AL, Randall RJ. Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry. 1951; 193:265-276.
20 Almeida FB, Cerqueira FM, Silva RN, Ulhoa CJ, Lima AL. Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production. Biotechnology Letter. 2007; 29:1189-1193. https://doi.org/10.1007/s10529-007-9372-z
21 Charney MS, Tomarelli RM. A colorimetric method for the determination of the proteolytic activity of duodenal juice. Journal of Biological Chemistry. 1947; 171:501-505.
22 Lee YP, Takahashi T. An improved colorimetric determination of amino acids with the use of ninhydrin. Anals of Biochemistry. 1966; 14:71-77. https://doi.org/10.1016/0003-2697(66)90057-1

23 Gomez RM, Rojas AL, Rojas AN, Cruz CR. Colloidal chitin stained with Remazol Brilliant Blue R®, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. Journal of Microbiological Methods. 2004; 56:213-219. https://doi.org/10.1016/j.mimet.2003.10.011

24 Papavizas GC. Trichoderma and Gliocladium: Biology, Ecology and potential for biocontrol. Annuual Review of Phytopathology. 1985; 23:23-54. https://doi.org/10.1146/annurev.py.23.090185.000323
25 Sivan A, Chet I. Biological control of Fusarium spp in cotton, wheat and muskmelon by Trichoderma harzianum. Journal of Phytopathology. 1986; 116:39-47. https://doi.org/10.1111/j.1439-0434.1986.tb00892.x
26 Calvet C, Pera J, Bera JM. Interaction of Trichoderma spp with Glomus mossaeae and two wilt pathogenic fungi. Agricultural Ecosystem and Environment. 1990; 9:59-65. https://doi.org/10.1016/0167-8809(90)90255-C

27 Elad Y, Zimand G, Zaqs Y, Zuriel S, Chet I. Use of Trichoderma harzianum in combination or alternation with fungicides to control Cucumber grey mold (Botrytis cinerea) under commercial greenhouse condition. Plant Pathology. 1993; 42:324-332. https://doi.org/10.1111/j.1365-3059.1993.tb01508.x

28 Spiegel Y, Chet I. Evaluation of Trichoderma spp as biocontrol agent against soil borne fungi and plant parasitic nematodes In Israel. Integrated Pest Management Review. 1998; 3:169-175. https://doi.org/10.1023/A:1009625831128

29 Elad Y. Biological control of foliar pathogens by means of Trichoderma harzianum and potential modes of action. Crop Protection. 2000; 19:709-714. https://doi.org/10.1016/S0261-2194(00)00094-6
30 Ashrafizadeh A, Etebarian HR, Zamanizadeh HR. Evaluation of Trichoderma isolates for biocontrol of Fusarium wilt of melon. Iranian Journal of Phytopathology. 2005; 41:39-57.

31 Dave N, Prajapati K, Patel A, Patel Z, Nandini D, Bariya H. Trichoderma harzianum elicits defense response in Brassica juncea plantlets. International Research Journal of Biological Science. 2013; 2(11):1-10.

32 Bhale UN, Wagh PM, Rajkonda JN. Antagonist confrontation of Trichoderma spp. against fruit rot pathogens on sapodilla (Manilkarazapota L.). Journal of Yeast and Fungal Research. 2013; 4(1):5-11.

33 Amin F, Razdan VK, Mohiddin FA, Bhat KA, Banday S. Potential of Trichoderma species as biocontrol agents of soil borne fungal propagules. Journal of Phytology. 2010; 2(10):38-41.

34 Mishra BK, Mishra RK, Mishra RC, Tiwari AK, Yadav RS, Dikshit A. Biocontrol efficacy of Trichoderma viride isoaltes against fungal plant pathogens causing disease in Vigna radiate L. Archives of Applied Science Research. 2011; 3(2):361-369.
35 Alwathnani HA, Perveen K, Tahmaz R, Alhaqbani S. Evaluation of biological control potential of locally isolated antagonist fungi against Fusarium oxysporum under in vitro and pot condition. African Journal of Microbiology Research. 2012; 6(2):312-319. https://doi.org/10.5897/AJMR11.1367

36 Shalini S, Kotasthane AS. Parasitism of Rhizoctonia Solani by strains of Trichoderma spp. Electronic journal of environmental, agricultural and food chemistry. 2007; 6:2272-2281.
37 McIntyre M, Nielsen J, Arnau J, Brink VH, Hansen K et al. Proceedings of the 7th European Conference on Fungal Genetics. 2004. Copenhagen. Denmark.
38 El-Katany MH, Gudelj M, Robra KH, Enaghy MA, Gubitz GM. Characterization of a chitinase and an endo-1,3-glucanase from Trichoderma harzianum Rifae T24 involved in control of the phytopathogen Sclerotium rolfsii. Applied Microbiology and Biotechnology. 2001; 56:137-143. https://doi.org/10.1007/s002530100646
39 Monteiro VN, Silva RN, Steindorff AS, Costa FT, Noronha EF et al. New insights in Trichoderma harzianum antagonism of fungal plant pathogens by secreted protein analysis. Current Microbiology. 2010; 61:298-305. https://doi.org/10.1007/s00284-010-9611-8

40 Wirth SJ, Wolf GA. Dye-labeled substrates for the assay and detection of chitinase and lysozyme activity. Journal of Microbiological Methods. 1990; 12:197-205. https://doi.org/10.1016/0167-7012(90)90031-Z

41 Cody RM. Distribution of chitinase and chitobiase in Bacillus. Current Microbiology. 1989; 19:201-205. https://doi.org/10.1007/BF01570162

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