Studies On Insect Pathogenesis Related Protein From Beauveria Bassiana Including Six Fungal Strains Virulence Comparison Against Galteria Mellonella And Myzus Persicae

In this work we compare six entomopathogenic fungal strains for virulence or toxicity againstMyzuz persicae and Galleria mellonella, isolate and purify insect toxin protein from EPF, Beauveriabassiana70. Similarly we isolate a cuticle degrading protease gene from Beauveria bassiana70by PCRtechnique, using specific primers. The main results are summarized as follows;1-Five bioassays, two dermal and three intra-hemocelic were performed for dermal andintra-hemocelic virulence or toxicity comparison of six fungal isolates (three of each, Beauveriabassiana and Verticillium lecanii), using Galleria mellonella as a model. Dermal bioassay consists ofconidial application virulence bioassays, with and without wetting agent while other three were conidial,filtrate and crud proteins intra-hemocelic injection bioassays. An isolate Beauveria bassiana252(Bb252) had highest virulence against Galleria mellonella, either of conidial shower or in the presence ofwetting agent (tween80) or intra-hemocoel injection of spore's suspension. The median lethal time(LT50) for Bb252, both in conidial shower and in the presence of wetting agent were14.87and12.07days, with cumulative mortality96.67and90.0%respectively at concentration of190±30conidia/mm2and1x108conidia/ml respectively after19days of inoculation. Similarly the cumulative mortality ofintra-hemocelic conidial injection of Bb252was (96.67%after48H), with the lowest LT50(10.2H)value at concentration1x108conidia/ml while with the intra-hemocelic filtrate and crud proteinsinjection, V3(100%after48H) and Verticillium lecanii2(V2)(56.67%after60H) were found to havehighest cumulative mortalities and low LT50values (9.6and89.6H) respectively.2-Two bioassay, conidial shower and filtrate application, of six EPF strains, three each of Beauveriabassiana and Verticillium lecanii, were screened for pathogenicity test against the Myzus persicae toselect high virulent isolate with the most suitable application and to determine the role of individualenzyme in its virulence. The percent mortality rate of filtrate was found higher as compared to percentmortality of conidial showering. Verticillium lecanii3(V3) showed highest virulence or toxicity againstthe target pest treated either with conidial shower (80.70%) or filtrate (88.36%) application whileBeauveria bassiana70(Bb70) and Beauveria bassiana76(Bb76) showed high toxicity (77.14%and80.86%) in filtrate application at6thday of incubation. Correlating the aphidicial activities to theenzymes production (Chitinase, protease and lipase), lipase was assumed to participate more in the totalvirulence or pathogenicity as compared to protease and chitinase.3-A novel insect toxic protein, Bb70p was purified from entomopathogenic fungi, Beauveriabassiana70(Bb70) with a molecular weight of35.5kDa, and pI~4.5. Bb70p posesses high affinity foranion exchangers, no affinity for hydrophobic group (phenyl) exchanger, active between the pH rang of4-10, better in slightly acidic pH, and remains active from4-60oC for10m, high activity from37-40oCwhile no activity was observed at100oC. The toxin protein activates the PO cascade and the protein ispure having no glycogenic side residue. The protein caused high mortality by intra-hemocelic injectionto Galleria mellonella with LD50of334.4μg/g body weight. The partial amino acid (denovo) peptidessequences of Bb70p sequence was compared for homology with the already described toxin proteins, using NCBI (BLAST) database, show no homology to the other toxins proteins of entomopathogenicfungi already described while100%homology was found to three hypothetical proteins fromClostridium sp.(bacteria).4-A cuticle degrading protease gene (1166bp) was isolated from Beauveria bassiana70by PCRtechnique using specific primers, protease1and protease2(P1and P2) designed on conservedsequences of seven Beauveria bassiana cuticle degrading genes (DNA or mRNA) sequences (NCBI).The amplified PCR product was sequenced and the nucleotides sequence was checked for homology tothe already described sequences on NCBI (BLAST) database. The BLAST result of the protease genepossesses98%homology to sequences (top9sequences) of cuticle degrading proteases from Beauveriabassiana. The gene was cloned into pMD-18-T vector, transformed into E.coli DH5α and was verifiedby identification PCR. The transformed E.coli DH5α sequence result possesses highest homology to thesequences of the cuticle degrading protease (DNA or mRNA) from Beauveria bassiana on NCBIdatabase (BLAST), verifying that gene is cuticle degrading protease.