Biochemical characterization Transformation, Mass Production and Formulation of Beauveria Bassiana and Metarhizium Anisooliae Isolates

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The overall objective of the work done in this dissertation was to investigate thirty one isolates of entomopathogenic fungi, including 17 B. bassiana and 14 M. anisopliae isolates for their biochemical characteristics at the enzyme and molecular level and transformation, mass production and formulation aspects were studied as well. The first aspect of the thesis describes the biochemical characterization of the thirty one isolates and selection of isolates based on virulence candidate enzymes. The extracellular enzyme activity of cuticle hydrolyzing enzymes such as protease, chitinase and lipase was investigated. The induction and repression mechanisms of these enzymes were studied using different medium constituents. Pr1 activity was induced by supplemented protein in the media whereas chitinase activity was repressed by glucose. In-Gel enzyme activity was studied and a predominant chitinase of 58 & 14.3 kDa was observed in activity gels for B. bassiana and M. anisopliae isolates respectively whereas a 97 and 66 kDa protease was frequent for both B. bassiana and M. anisopliae. Two isolates were finally screened out (B. bassiana (UB9) and M. anisopliae (UM10) isolates) and used for further studies. The protease and chitinase enzymes from these two isolates were partially purified and characterized. The purified fraction was characterized on the basis of temperature, pH and effect of inhibitors. Molecular weight of the enzymes was detected on SDS-PAGE. A conventional 33 kDa chitinase was purified from B. bassiana and 23 kDa chitinase was purified from M. anisopliae. 47 & 43 kDa protease was purified from B. bassiana and M. anisopliae respectively. The genes of virulence determinant enzymes viz. protease and chitinase were studied as well. Chitinase and protease specific primers (two primer pairs for each gene and each isolate) were designed based on the conserved sequences. PCR amplified products were sequenced (Xcelris Labs) and sequences were analyzed by BLAST (BAST N & BLAST X) and conserved domains in the amplified sequence were detected by CD finder. 351 & 312 bp and 434 & 438 bp amplified fragments were observed for B.bassiana and M. anisopliae chitinase gene respectively. A 504 & 517 bp and 535 & 551 bp amplified fragments were observed for B. bassiana and M. anisopliae protease gene respectively. Sequence alignments showed conserved sequences are present in the amplifications. Pathogenicity potential of B. bassiana (UB9) and M. anisopliae (UM10) isolates against cottonbollworm Helicoverpa armigera was investigated. Bioassay was performed using Diet Surface Technique and 5 replicates were taken for each treatment. Fungus was reisolated from the mycosed cadaver of H. armigera and several virulent factors of indigenous conidia and insect passaged conidia were investigated. M. anisopliae (UM10) was more effective against 1st and 2nd instar larvae of H. armigera than B. bassiana (UB9). The second aspect deals with the studies optimizing protoplast yield from M. anisopliae (UM10) and B. bassiana (UB9) and transformation of these strains to herbicide resistance. Several factors were investigated influencing protoplast release from mycelium (B. bassiana (UB9) and M. anisopliae (UM10)) including effective cell wall lysing enzyme, osmotic stabilizer, mycelium age and enzyme incubation time. 10 mg/....
Supervisor: G. K. Saini