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Title:	Efficiency of trichoderma isolates and bacillus subtilis UKM1 as biocontrol agents against magnaporthe grisea, rhizoctonia solani and fusarium solani in rice
Authors:	Hamdia Z. Ali (P57901)
Supervisor:	Kalaivani Nadarajah, Assoc. Prof. Dr.
Keywords:	Biological pest control agents Trichoderma Fungi as biological pest control agents Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	13-Mar-2014
Description:	The aim of this study is to determine the effectiveness of Malaysian Trichoderma isolates in combination with Bacillus subtilis UKM1 as biocontrol agents against Rhizoctonia solani, Fusarium solani and Magnaporthe grisea in rice plant. The pathogenicity of one isolates of M. grisea, two isolates of R. solani and three isolates of F. solani were studied on rice plant under greenhouse conditions. Data from this experiment showed that the isolates varied in their pathogenic ability, one isolate from each pathogen that was most virulent was chosen for use in all further assays. In vitro tests conducted with 22 isolates of Trichoderma and one isolate of B. subtilis UKM1 against R. solani F. solani and M. grisea via the dual culture technique showed that isolates exhibited high antagonistic activity and reduced radial growth for each pathogen. In addition the plant microbe interaction at the cellular level was observed under a SEM where the level of inter and intracellular proliferation of the above three microbes in three rice varieties; cv MR219, Pongsu seribu and Maswangi were tested. Reactive oxygen intermediate species (ROIs) production was also observed as being expressed at the location of pathogen proliferation through 3,3'-Diaminobenzidine (DAB) assays. As ROIs are known to trigger defense response, the expression levels of Pathogenesis related (PR) gene was monitored in all varieties used in this study. Pongsu Seribu showed the highest level (40.33%) of PR gene expression. Greenhouse studies were conducted to determine the effectiveness of Trichoderma isolates to suppress M. grisea, R. solani R2 and F. solani F2 growth in autoclaved soil where four parameters were tested; pre-emergence seedlings, post-emergence seedlings, disease incidence and disease severity for each pathogen. The studies show Trichoderma isolates exhibited greater efficiency of reducing disease severity in M. grisea isolates T2, T7 and T8 (11, 8.7 and 11% respectively), R. solani isolates T2 and T7 (15.3 and 17.7%), and F. solani F2 isolates T2, T7, T8 and T9 (8.67, 6.7, 4 .3 and 11% respectively) after 60 days post transplanting. Six Trichoderma isolates T2, T7, T8, T9, T11 and T21 were chosen among 22 Trichoderma isolates tested in nonautoclaved. When tested in combination with B. subtilis UKM1, the highest suppression for disease severity on M. grisea was shown by isolates T2 and T9 (4.5, 8.9% respectively), isolates T2, T7, T8 and T21 (6.7-8.9%) in R. solani R2 and F. solani F2. All isolates gave significant reduction (p<0.05) of (6.7 to 11.1% respectively) after 60 days post transplanting. The greenhouse experiments with the above six isolates showed that the suppression levels were greater in non-autoclaved soil compared to autoclaved soil. These six isolates were pre-screened on chitin plates to determine its ability to digest chitin therefore indicating the presence of chitinase which is a good disease resistance candidate gene. As a consequence of dual culture assays, the greenhouse experiments and chitinolytic assays, Trichoderma T2 was determined as the most effective isolate and was used as template for the isolation of endochitinase gene using Trichoderma asperellum chitinase (ChiB) gene sequence as template (NCBI).The product (ChitT2 gene) which is 1200 bp long was sequenced and the sequence data was analyzed in silico and the protein was classified as an endochitinase Class V belonging to the family 18 glycosyl hydrolase. The 3D structure was also resolved implicating the possible mode of interaction of this enzyme. A transformation construct was generated with this amplified product and used to transform rice calli via the Agrobacterium mediated transformation method.,Ph.D.,Certification of Master's / Doctoral Thesis" is not available"
Pages:	238
Call Number:	SB976.F85A438 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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