Korean Journal of Soil Science and Fertilizer (한국토양비료학회지)

Korean Society of Soil Science and Fertilizer (한국토양비료학회)
권호 표지

Biocontrol of Damping-Off(Rhizoctonia solani) in Cucumber by Trichoderma asperellum T-5

Trichoderma asperellum T-5를 이용한 오이 모잘록병(Rhizoctonia solani)의 생물학적 제어

  • Ryu, Ji-Yeon (Division of Applied BioScience and Biotechnology, and Environmental-Friendly Agriculture Research Center, Chonnam National University) ;
  • Jin, Rong-De (Division of Applied BioScience and Biotechnology, and Environmental-Friendly Agriculture Research Center, Chonnam National University) ;
  • Kim, Yong-Woong (Division of Applied BioScience and Biotechnology, and Environmental-Friendly Agriculture Research Center, Chonnam National University) ;
  • Lee, Hyang-Burm (Division of Applied BioScience and Biotechnology, and Environmental-Friendly Agriculture Research Center, Chonnam National University) ;
  • Kim, Kil-Yong (Division of Applied BioScience and Biotechnology, and Environmental-Friendly Agriculture Research Center, Chonnam National University)
  • 류지연 (전남대학교 응용생물공학부, 친환경농업연구사업단) ;
  • 김영덕 (전남대학교 응용생물공학부, 친환경농업연구사업단) ;
  • 김용웅 (전남대학교 응용생물공학부, 친환경농업연구사업단) ;
  • 이향범 (전남대학교 응용생물공학부, 친환경농업연구사업단) ;
  • 김길용 (전남대학교 응용생물공학부, 친환경농업연구사업단)
  • Received : 2006.07.19
  • Accepted : 2006.08.03
  • Published : 2006.08.28
Download PDF
⟨ Previous Next ⟩

Abstract

A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperellum and named as Trichoderma asperellum T-5 (TaT-5). The fungus released lytic enzymes such as chitinase and ${\beta}$-1, 3-glucanse, and produced six antifungal substances in chitin broth medium. To demonstrate the protective effect of TaT-5 against damping-off in cucumber plant caused by Rhizoctonia solani, TaT-5 culture broth (TA), chitin medium (CM) and distilled water (DW) were applied to each pot at 10 days after sowing, respectively. Then, the homogenized hyphae of R. solani were infected to each pot at 1 week after TaT-5 inoculation. During experimental period, fresh weight of shoot and root in cucumber plant more increased at TA treatment compared to other treatments. PR-proteins (${\beta}$-1, 3-glucanase and chitinase) activities in cucumber leaves markedly increased at CM and DW treatments, but the activity slightly increased and then decreased at TA treatment at 3 days after infection of R. solani. The activity of PR-proteins activities in cucumber roots at all treatments decreased with time where the degree of decrement was more alleviated at TA treatment than CM and DW. These results suggest that the lytic enzymes (chitinase and ${\beta}$-1, 3-glucanse) and antifungal substances produced by TaT-5 can reduce the pathogenic attack by R. solani in cucumber plants.

몇 년동안 게껍질이 풍부하게 있었던 밭토양에서 강한 키틴분해능력을 가진 Trichoderma 곰팡이를 분리하였다. 분리된 곰팡이의 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence 분석과 형태학적 특징을 살펴본 결과 Trichoderma asperellum으로 동정되었고, 이를 Trichoderma asperellum T-5 (TaT-5)로 명명하였다. 이 곰팡이는 chitianse와 ${\beta}$-1,3-glucanase같은 lytic enzyme을 분비하며, 키틴배지 상에서 6가지의 항 곰팡이성 물질을 생산했다. R. solani가 원인인 오이의 모잘록병에 대해 TaT 5의 방제효과를 보기 위해서 TaT-5 배양액(TA), chitin medium(CM), 증류수(DW)를 씨를 심은 후 10일 째에 각 pot에 관주했다. 그리고 관주 1주일 후에 R. solani의 균사를 갈아서 각 pot에 주었다. 실험기간 동안에 오이의 지상부와 지하부 생체중은 다른 처리구에 비하여 TA 처리구가 더 많이 증가하였다. 오이 잎에서 PR-protein (chitianse, ${\beta}$-1,3-glucanase) 활성은 R. solani 감염 후 CM과 DW에서 증가를 보였고, TA 처리구에서는 증가하다가 감소하는 경향을 보였다. 뿌리에서는 모든 처리구가 감소하는 경향을 보였지만 TA 처리구가 CM과 DW 처리구보다 감소하는 정도가 적었다. 오이의 잎과 뿌리에서 lignification related enzyme(POD, PPO, PAL)활성은 R. solani 감염 초기에는 증가하다가 점점 감소하는 경향을 보였다. 이러한 결과들은 TaT-5에 의하여 생산된 lytic enzymes와 항 곰팡이성 물질들이 오이에 R. solani의 공격을 줄여준다고 생각된다.

Keywords

References

  1. Benitez, T., Rincon, A.M., Limon, M.C. and Codon, A.C. 2004. Biocontrol mechanisms of Trichoderma strains. Int. Microbial. 7, 249-260
  2. Bara, M.T.F., Lima, A.L. and Ulhoa, C.J. 2003. Purification and characterization of an exo-$\beta$-1, 3-glucanase produced by Trichoderma asperellum. FEMS MicrobioL Lett. 219, 81-85 https://doi.org/10.1016/S0378-1097(02)01191-6
  3. Chen, C, Belanger, R.R., Benhamou, N., and Paulitz, T.C. 2000. Defense enzymes induced in cucumber roots by treatment with plant growth promoting rhizobacteria (PGPR) and Pythium aphanidennatum. Physiol. Mol. Plant Pathol. 56, 13-23 https://doi.org/10.1006/pmpp.1999.0243
  4. Caruso, C, Chilosi, G., Caporale, C. Leonardi, L., Bertini, L., Magro, P., and Buonocore, V. 1999. Induction of pathogenesis related proteins in germinating wheat seeds infected with Fusarium culmorum. Plant Sci. 140, 107-120
  5. Claydon, N., Allan, M., Hanson, J.R., and Avent, A.G. 1987. Antifungal alkyl pyrones of Trichoderma harzianum. Transactions of the British Mycological Soc. 88, 503-513 https://doi.org/10.1016/S0007-1536(87)80034-7
  6. Dennis, C., and Webster, J. 1971. Antagonistic properties of species groups of Trichoderma. II. Production of volatile antibiotics. Transactions of the British Mycological Soc. 57, 41-48 https://doi.org/10.1016/S0007-1536(71)80078-5
  7. EI Shora, H.M. 2002. Properties of phenylalanine ammonia lyase from marrow cotyledons. Plant Sci. 162, 1-7 https://doi.org/10.1016/S0168-9452(01)00471-X
  8. Enkerli, J., Felix, G., and Boller, T. 1999. Elicitor activity of fungal xylanase does not depend on enzymatic activity. Plant Physiol. 121,391-398 https://doi.org/10.1104/pp.121.2.391
  9. Endo, A., Hasumi, K., Sakai, K., and Kanbe, T. 1985. Specific inhibition of glyceraldehydes-3-phosphate dehydrogenase by koningic acid (heptelidic acid). J. Antibiot. 38, 920-925 https://doi.org/10.7164/antibiotics.38.920
  10. Ghisalberti, E.L., and Sivasithamparam, K. 1991. Antifungal antibiotics produced by Trichoderma spp. Soil Biol, Biochem. 23, 1011-1020 https://doi.org/10.1016/0038-0717(91)90036-J
  11. Howell, C.R. 1998. The role of antibiosis in biocontroL In: Harman GE, Kubicek C.P. (eds) Trichoderma & Gliocladium, vol. 2. Taylor & Francis, Padstow, pp 173-184
  12. Howell, C.R. 2003. Mechanisms Employed by Trichoderma species in the Biological Control of Plant Diseases: The History and Evolution of Current Concepts. Plant dis. 87, 4-10 https://doi.org/10.1094/PDIS.2003.87.1.4
  13. Hoffland, E. Hakulinen, J., and Pelt, J. 1996. Comparison of systemic resistance induced by avirulent and nonpathogenic Pseudomonas species. Phytopathology 86, 757-762 https://doi.org/10.1094/Phyto-86-757
  14. Inbar, J. Abramski, M. Coen, D., and Chet, l. 1994. Plant growth enhancement and disease control by Trichoderma harzianum in vegetable seedlings grown under commercial conditions. Eur. J. Plant Pathol. 100, 337-346 https://doi.org/10.1007/BF01876444
  15. Inbar, J., and Chet, I. 1995. The role of recognition in the induction of specific chitinases during mycoparasitism by Trichoderma harzianum. Microbiology 141,2823-.2829 https://doi.org/10.1099/13500872-141-11-2823
  16. Jung, W.J., An, K.N., Jin, Y.L., Park, R.D., Lim, K.T., Kim, K.Y., and Kim, T.H. 2003. Biological control of damping-off caused by Rhizoctonia solani using chitinase producing PA.Cnibacillus illinoisensis KJA 424. Soil BioI. Biochem. 35, 1261-1264 https://doi.org/10.1016/S0038-0717(03)00187-1
  17. Jung, W.J., Jin, Y.L., Kim, Y.C., Kim, K.Y, Park, R.D., and Kim, T.H. 2004. Inoculation of PA.Cnibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici. BioI. Control. 30, 645-652 https://doi.org/10.1016/j.biocontrol.2004.03.006
  18. Kim, S.J. 2006. Biocontrol of lite Blight (Phytophthora capsici) in pepper by chitin broth containing multitude of chitinolytic bacteria. A Master's Thesis, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Republic of Korea
  19. Kleifeld, O., and Chet, I. 1992. Trichoderma harzianum interaction with plants and effect on growth response. Plant Soil 144, 267-272 https://doi.org/10.1007/BF00012884
  20. Lee, H.J., Park, K.H., Shim, J.H., Park, R.D., Kim, Y.W., Cho, J.Y., Hwangbo, H., Kim, Y.C, Cha, G.S., Krishnan, H.B., and Kim, K.Y. 2005. Quantitative Changes of plant defense enzymes in biocontrol of pepper (Capsicium annuum L.) late blight by antagonistic Bacillus subtillus HJ927. J. Microbiol. Biotechnol. 15, 1073-1079
  21. Lingappa, Y., and Lockwood, J.L. 1962. Chitin media for selectived culture of actinomycetes. Phytopathology 52, 317-323
  22. Lorito, M. 1998. Chitinolytic enzymes and their genes. In Trichoderma and Gliocladium (Kubicek CP & Harman GE, eds), pp. 73-99. Taylor & Francis, London
  23. Lawrence, C.B., Joosten, M.H.A.J., and Tuzun, S. 1996. Differential induction of athogenesis-related proteins in tomato by Alternaria solani and the association of a basic chitinase isozyme with resistance. Physiol. Mol. Plant P. 48, 361-377 https://doi.org/10.1006/pmpp.1996.0029
  24. Lieckfeldt, E., Samuels, G.J., Nirenberg, H.I., and Petrini, O. 1999. A morphological and Mol.ecular perspective of Trichoderma wiride : Is it one or two species? Appl. Environ. Microb. 65, 2418-2428
  25. Mohammadi, M., and Kazemi, H. 2002. Change in peroxidase and polyphenol oxidase acrivities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance. Plant Sci. 162, 491-498 https://doi.org/10.1016/S0168-9452(01)00538-6
  26. Mitchell, R, and Alexander, M. 1961. The mycolytic phenomenon and biological control of Fusarium in soil. Nature 190,109-110 https://doi.org/10.1038/190109a0
  27. Monte, E. 2001. Understanding Trichoderma : between biotechnology and microbial ecology. Int. Microbiol. 4, 1-4
  28. Nampoothiri, K.M., Baiju, T.V., Sandhya, C, Sabu, A., Szakacs, G., and Pandey, A. 2004. Process optimization for antifungal chitinase production by Trichoderma harzianum. Process Biochem. 39, 1583-1590 https://doi.org/10.1016/S0032-9592(03)00282-6
  29. Nandakumar, R., Babu, S., Wiswanathan, R., Raguchander, T., and Samiyappan, R. 2001. Induction of systemic resistance in rice against sheath blight disease by Pseudomonas fluorescens. Soil BioI. Biochem. 33, 603-612 https://doi.org/10.1016/S0038-0717(00)00202-9
  30. Okuda, T., Fujiwara, A., and Fujiwara, M. 1982. Correlation between species of Trichoderma and production patterns of isonitrile antibiotics. Agr. Biol. Chem. 46, 1811-1822 https://doi.org/10.1271/bbb1961.46.1811
  31. Ordentlich, A., Elad, Y, and Chet, I. 1988. The role of chitinase of Serratia marcescens in biocontrol of Sclerotium rolfsii. Phytopathology 78, 84-88
  32. Paulitz, T., and Belanger, R. 2001. Biological control in greenhouse systems. Annu. Rev. Phytopathol. 39, 103-133 https://doi.org/10.1146/annurev.phyto.39.1.103
  33. Pow, M.J., Cordier, C, Dumas Gaudot, E., Gianinazzi, S., Barea, J.M., and Azcon Aguilar, C 2002. Localized versus systemic effect of arbscular mycorrhizal fungi on defense responses to Phytophthora infection in tomato plants. J. Exp. Bot. 53, 525-534 https://doi.org/10.1093/jexbot/53.368.525
  34. Rey, M., Delgado jarana, J., and Benitez, T. 2001. Improved antifungal activity of a mutant of Trichoderma harzianum CECT 2413 which produces more extracellular proteins. Appl. Microbiol. Biot. 55, 604-608 https://doi.org/10.1007/s002530000551
  35. Russell, J., Tweddell, Suha, H., Jabaji Hare, and Pierre, M. Charest. 1994. Production of chitinases and $\beta$-1. 3-glucanses by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani. Appl. Environ. Microb. 60, 489-495
  36. Sanz, L., Montero, M., Redondo, J., Liobell, A., and Monte, E. 2005. Expression of an $\beta$-1, 3-glucanase during mycoparasitic interaction of Trichoderma asperellum. FEBS J. 272, 493-499 https://doi.org/10.1111/j.1742-4658.2004.04491.x
  37. Singh, P.P., Shin, Y.C., Park, C.S., and Chung, Y.R. 1999. Biological control of Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology 89.92-99 https://doi.org/10.1094/PHYTO.1999.89.1.92
  38. Silva, H.S.A., Romeiro, R.S., Macagnan, D., Halfeld Vieira, B.A., Pereira, M.C.B., and Mounteer, A. 2004. Rhizobacterial induction of systemic resistance in tomato plants : non specific protection and increase in enzyme activities. Biol. control 29, 288-295 https://doi.org/10.1016/S1049-9644(03)00163-4
  39. Suarez, B., Rey, M., Castillo, P., Monte, E., and Liobell, A. 2004. Isolation and characterization of PRA I. a trypsin like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Appl. Microbiol. Biot. 65, 46-55
  40. Tuzun, S. 2001. The relationship between pathogen-induced systemic resistance (ISR) and multigenic (horizontal) resistance in plants. Eur. J. of Plant Pathol. 107, 85-93 https://doi.org/10.1023/A:1008784417222
  41. Viterbo, A., Haran, S., Friesem, Ramot, O., and Chet, I. 2001. Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol. Lett. 200, 169-174 https://doi.org/10.1111/j.1574-6968.2001.tb10710.x
  42. Viterbo, A., Montero, M., Ramot, O., Friesem, D., Monte, E., Llobell, A., and Chet, I. 2002. Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203). Curr. Genet. 42, 114-122 https://doi.org/10.1007/s00294-002-0345-4
  43. Wang, S.L., Shih, I.L., Wang, C.H., Tseng, K.C, Chang, W.T., Ywu, Y.K., Ro, J.J., and Wang, C.L. 2002. Production of antifungal compounds from chitin by Bacillus substilits. Enzyme Microb. Tech. 31, 321-328 https://doi.org/10.1016/S0141-0229(02)00130-8
  44. Yedidia, I., Benhamou, N., and Chet, I. 1999. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl. Environ. Microb. 65,1061-1070
  45. Yedidia, I., Benhamou, N., Kapulnik, Y., and Chet, I. 2000. Induction and accumulation of PR proteins activity during early stages of root colonization by the mycoparasite Trichoderma harzianum strain T-203. Plant Physiol. Biochem. 38, 863-873 https://doi.org/10.1016/S0981-9428(00)01198-0
  46. Zhang, W., Dick, W.A., and Hoitink, H.A.J. 1996. Compostinduced systemic acquired resistance in cucumber to Pythium root rot and anthracnose. Phytopathology 86, 1066-1070 https://doi.org/10.1094/Phyto-86-1066
  47. Zhao, H.C., Zhao, H., Wang, B.C., and Wang, J.B. 2005. Effect of local stress induction on resistance-related enzymes in cucumber seeding. Colloids and Surfaces B: Biointerfaces 43,37-42 https://doi.org/10.1016/j.colsurfb.2005.01.017
  48. Zheng, H.Z., Kim, Y.W., Lee, H.J., Park, R.D., Jung, W.J., Kim, Y.C., Lee, S.H., Kim, T.H., and Kim, K.Y. 2004. Quantitative changes in PR proteins and antioxidative enzymes in response to Glomus intra radices and Phytophthora capsid in pepper. J. Microbiol. Biotechn. 14, 553