In Vitro and Enzymatic Studies of Inhibitory Activities of Three Trichoderma Species against Pythium Aphanidermatum in Infected Sweet Pepper

Author(s): Oluyemi Olawumi Idowu, Abiodun Olusola Salami, Ayodeji Christopher Oni

This study investigated the antifungal activities of Trichoderma species against Pythium aphanidermatum; determined and measured the induction of defense enzymes (such as peroxidase, polyphenol oxidase and phenylalanine ammonia lyase) by Trichoderma species in seedlings inoculated with Pythium aphanidermatum. This was with a view to obtaining the best antagonist among the Trichoderma species. Inhibition percentages of the three Trichoderma species on Pythium aphanidermatum were determined via dual inoculation method and via production of volatile compounds. Fifteen day old seedlings of treated and untreated seeds were inoculated with 5 X 10⁵ spores/ml of Pythium aphanidermatum. Extraction of enzymes was carried out at different time interval of 1, 3, 5, 7, 9 and 11 days after pathogenic inoculation and they were assayed. The phenolic content for each of the treatment was also determined. The in vitro results suggested that, Trichoderma harzianum and T. koningii inhibited the growth of Pythium aphanidermatum mainly through their higher competitive abilities for space (on the plate) and nutrients at the expense of P. aphanidermatum, while T. atroviride inhibited the growth of P. aphanidermatum on the third day before occupying the space and absorbing nutrients from the substrates. The peroxidase activities were higher in T. atroviride plus pathogen in comparison with its activities in T. atroviride alone, T. harzianum alone and pathogen alone, while higher accumulation of phenolic contents was recorded in dual inoculations with T. atroviride than with T. harzianum. The study confirmed the ability of T. atroviride to inhibit the infection of P. aphanidermatum through the induction of activities of oxidative enzymes, competition and mycoparasitism, which was found to be high in interactions with the pathogen at all levels.

Trichoderma harzianum, Trichoderma atroviride, Trichoderma koningii, Pythium aphanidermatum, Inhibition percentages and Induction of defense enzymes

1. Abo-Elyousr, K.A.M., Hashem, M., and Ali, E.H. (2009). Integrated control of cotton root rot disease by mixing fungal biocontrol agents and resistance inducers. Crop Protection, 28: 295 – 301.
2. Ajith, P. S. and Lakshmidevi, N. (2010). Effect of volatile and non-volatile compounds from Trichoderma spp. against Colletotrichum capsici incitant of anthracnose on bell pepper. Nature and Science, 8 (9):265-269.
3. Avdiushko, S.A., Ye ,X. S. and Kuć, J. (1993). Detection of several enzymatic activities in leaf prints cucumber plant. Physiological and Molecular Plant Pathology. 42: 441-454.
4. Brunner, K., Peterbauer, C. K., Mach, R. L., Lorito, M., Zeilinger, S. and Kubicek, C.P. (2003). The N-acetylglucosaminidase of Trichoderma atroviride is essential for chitinase induction by chitin of and major relevance to bio-control. Current Opinion in. Genetics. 43: 289-295.
5. Chittoor, J. M., Leach, J. E. and White, F. F. (1997). Differential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. oryzae. Molecular Plant to Microbe Interactions.10: 861 to 871.
6. Das, K. K., Panda, D., Nagaraju, M., Sharma, S. G. and Sarkar, R. K. (2004). Antioxidant enzymes and aldehyde releasing capacity of rice cultivars (Oryzae sativa L.) and determinants of anaerobic seedling establishment capacity. Bulgarian Journal Plant Physiology. 30:34-44.
7. Doka, O., Bicanic, D. D., Dicko, M. H. and Slingerland, M. A. (2004). Photoacoustic approach to direct determination of the total phenolic content in red sorghum flours. Journal of Agricultural Food Chemistry. 52: 2133-2136.
8. Goyal, S. P., Jandaik, C. L. and Sharma, V. P. (1994). Effect of weed fungi metabolites on the mycelial growth of Agaricus bisporus (Lang) Imbach. Mushroom Research. 3: 69-74.
9. Hammerschmidt, R. (1999). Phytoalexins: what have we learned after 60 years? Annual Reviews of Phytopathology 37: 285-306.
10. Hammerschmidt, R. (2005). Phenols and plant pathogen interactions: The saga continues. Physiological and Molecular Plant Pathology. 66: 77-78.
11. Harman, G. E., Howell, C. R., Viterbo, A., Chet, I. and Lorito, M. (2004). Trichoderma species opportunistic, avirulent plant symbionts. Nature Reviews Microbiology., 2:43-56.
12. Harman, G.E., (2006). Overview of mechanisms and uses of Trichoderma spp. Phytopathology, 96: 190-194.
13. Hiraga, S., Sasaki, K., Ito, H., Ohashi, Y. and Matsui, H. (2001).A large family of class III plant peroxidases. Plant and Cell Physiology42: 462-468.
14. Kerkeni, A., Daami-Remadi, M., Tarchhoun ,N. and Khedher, B.N. (2007) In vitro and in vivo suppression of Fusarium oxysporium f.sp. radicis-lycopersici the causal agent of Fusarium crown and root rot of tomato by some compost fungi. International Journal of Agricultural Research. 2, 111-115.
15. Kullnig, C., Mach, R.L., Lorito, M., and Kubicek, C. P. (2000). Enzyme diffusion from Trichoderma atroviride (T. Harzianum P1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42gene expression before mycoparasitic contact. Applied Environmental Microbiology, 66: 2232–2234.
16. Li, L. and Steffens, J. C. (2002). Over expression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247.
17. Melo, G.A., Shimizu, M.M., and Mazzafera, M. (2006). Polyphenol oxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust. Phytochemistry., 67:277-285.
18. Mishra, V. K. (2010). In vitro antagonism of Trichoderma species against Pythium aphanidermatum. Journal of Phytology, 2(9): 28-35. www.journal-phytology.com.
19. Morsy, M., Ebtsam, K. A., Abdel-Kawi, Khalil, M. N. A. (2009). Efficiency of Trichoderma viride and Bacillus subtilis as bio-control agents against Fusarium solani on tomato plants. Egyptian Journal of Phytopathology. 37:47-57.
20. Narasimha Murthy, K., Nirmala Devi, D., and Srinivas, C. (2013). Efficacy of Trichoderma asperellum against Ralstonia solanacearum under greenhouse conditions. Annuals of Plant Sciences. 02 (09):342-350.
21. Nawrocka, J., Snochowska, M., Gajewska, E., Pietrowska, E., Szczech, M., and Małolepsza, U. (2011). Activation of defense responses in cucumber and tomato plants by selected polish Trichoderma strains. Vegetable Crops Research Bulletin. 75:105–116.
22. Nicholson, R.L. and Hammerschmidt, R. (1992) Phenolic compound and their role in disease resistance. Annual Reviews of Phytopathology. 30:369–389
23. Padmodaya, B. and Reddy, H.R., (1996). Screening of Trichoderma spp. against Fusarium oxysporium f.sp. lypersici causing wilt in tomato. Indian Journal of Mycology and Plant Pathology 26,266-270.
24. Quiroga, M., Guerrero, C., Botella, M.A., Barcelo´, A., Amaya, I., Medina, M.I., Alonso, F.J., de Forchetti, S.M. , Tigier, T. and Valpuesta, V. (2000). A tomato peroxidase involved in the synthesis of lignin and suberin, Plant Physiology. 122:1119–1128.
25. Reddy, K. J., Wang, L. and Gloss, S. P. (1995). Solubility and mobility of copper, zinc and lead in acidic environments. Plant and Soil. 171, 53-58.
26. Salami, A.O. (1998). Biochemical interactions of mycorrhiza and soil microorganisms on growth of pepper (Capsicum annum. Linn.) seedlings. Ph.D Thesis. Pp: 126-127.
27. Salami, A. O., Olawole, O. I. and Oni, A. C. (2011). Effect of interactions between Glomus mosseae and Pythium aphanidermatum on the growth performance of okra plant seedlings. Journal of Science Research. Nigeria.
28. Shashidhara, S., Lokesh, M.S., Lingaraju, S., Palakshappa, M.G. (2008). In vitro evaluation of microbial antagonists, botanicals and fungicides against Phyptophthora capsici Leon. The causal agent of foot rot of black pepper. Karnataka Journal of Agricultural Sciences 21, 527-531.
29. Stevens, L. H., Davelaar, E., Kolb, R. M., Pennings, E. J. M. and Smit, N. P. M. (1998). Tyrosine and cysteine are substrates for black spot synthesis in potato. Phytochemistry 49:703–707
30. Svetlana, Z., Stojanovic, S., Ivanovic, Z., Gavrilovic, V. Tatjana, P. and Jelica-balaz. (2010). Screening of antagonistic activity of microorganisms against Colletotrichum acutatum and Colletotrichum gloeosporioides, Archives of Biological Science., Belgrade, 62(3), pp611-623.
31. Thangavelu, R. A., Palaniswami, B. and Velazhahan, R. (2004). Mass production of Trichoderma harzianum for managing Fusarium wilt of banana. Agriculture Ecosystem Environment, Vol. 103, pp. 259–263.
32. Thomashow, S.L. (1996). Biological control of plant root pathogens. Current Opinion in Biotechnology. 7: 343-347.
33. Van Wees, S.C.M., van der Ent, S., and Pieterse, C.M.J. (2008). Plant immune responses triggered by beneficial microbes. Current Opinion in Plant Biology. 11: 443–448.
34. Vanitha, S. C., Niranjana, S. R. and Umesha, S. (2009). Role of phenylalanine ammonia lyase and polyphenol oxidase in host resistance to bacterial wilt of Tomato. Journal of Phytopathology. 157: 552-557.
35. Verma M., Brar S.K., Tyagi R.D., Surampalli R.Y. and Valero J.R.(2007). Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochemical Engineering Journal. 37, 1-20.
36. Watanabe, T. (2002). Pictorial Atlas of Soil and Seed Fungi: Morphologies of Cultured Fungi and Key to species. 2nd Edn. CRC Press.
37. Yedidia, I., Benhamou, N., and Chet, I. (1999). Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Applied Environmental Microbiology. 65: 1061-1070.
38. Zieslin, N. and Ben-Zaken, R. (1993). Peroxidase activity and presence of phenolic substances in peduncles of rose flowers. Plant Physiology and Biochemistry. 31:333-339.