Effect Of Simulated Insect Midgut Environment On The Spores Of Entomopathogenic Fungus Beauveria Bassiana

Infective spores(conidia and blastospore)of Beauveria bassiana hardly infects host insects via ingestion route,but the mechanism involved is poorly understood.For elucidating the mechanism involved in survival of spore in insect midgut,this thesis sought to 1)screen a transgenic strain that steadily expressed green fluorescence protein,2)determine variation of the fluorescence intensity(FI)of spore after treating with different simulated insect midgut digestive fluids through flow cytometry,3) observe structural changes of cell wall surface of digested spore under scanning electron microscope,and 4)establish variation of the viability of digested spore. Detailed results were summarized as follows:Screening the transgenic strain of B.bassiana.Several biotic tests were carried out to screen the most appropriate strain for the following research from 6 transgenic strains of B.bassiana.The FI of conidia was examined through flow cytometer,with a result that the isolate Bb2860G6 had the highest value.Conidial thermotolerance were assayed by exposing conidia to the thermal stress under 48℃(in water bath)for 45 min,and the viabilities of Bb2860G6,Bb2860G12 and Bb2860G4 after stress were still more than 55%.Analysis of the growth area showed that strain Bb2860G0,Bb2860G6 and Bb2860G8 had similar growth activity and grew significantly faster than Bb2860G1 at 7d.Bioassay results indicated that strain Bb2860G6,Bb2860G8 and Bb2860G0 have a similar virulence against M.persicae. Thus,the isolate Bb2860G6 was chosen as ideal representive of the B.bassiana transgenic strain with familiar physiological activity of wide type strain.Effect of simulated insect midgut environment on the conidia.The conidia of Bb2860G6 were suspended in the solutions of 0.05,0.1,0.5 and 1%enzymes similar to those present in different insect midguts and then incubated at 25 or 35℃for 4h, followed by examining the FI of conidia through flow cytometry and observing the structural changes of conidial wall under scanning electron microscope.As a result, the FI of the treated conidia was significantly enhanced firstly by proteinase K, pronase E,cellulose,and trpsin respectively and then reduced at higher enzyme concentrations.However,all the four enzymes(α-amylase,laminarinase,glucosidase and snailase)only caused slight FI variations of the treated conidia at tested enzyme concentrations.Both mixture of proteinase K/cellulase and trpsin/cellulase decreased the FI more significantly than any single enzyme in mixture and the decreases varied significantly at pH 5-11(proteinase K/cellulose:3-23%and trpsin/cellulose:28-47%). Conspicuous changes of the conidial wall(i.e.,illegibility of protein rodlet on the conidial surface)were also observed after 4-h treatment with the digestive enzymes. Viabilities of the conidia after treating by different stimulated insect digestive fluids were determined.The results suggested that the viabilities reduced after treating with 8 single enzymes,and the trpsin resulted in the most significant decrease of conidial viability(31%).The two sorts of enzyme mixtures reduced the viabilities more than any single enzyme in mixture,and the decreases varied significantly at pH 5-11 (proteinase K/cellulose:30-98%and trpsin/cellulose:21-91%).Effect of simulated insect midgut environment on the blastospore.The blastospores of Bb2860G6 were assayed by the same methods for the conidia.The results suggested that the FI of the treated blastospores was significantly enhanced by treatments with proteinase K,pronase E,cellulase and trpsin respectively,but reduced slightly by glucosidase and snailase.Both laminarinase andα-amylase enhanced the conidial FI at low concentration and reduced significantly at high concentration.The mixture of trpsin/cellulase enhanced the FI of the blastospore at all the tested concentrations and resulted in the highest increase of fluorescence intensity(48%)at 0.5%.While the mixture of proteinase K/cellulase resulted in the highest increase (10%)at 0.05%and caused decrease of 15%at 1.0%.The mixture of proteinase K/cellulase and trpsin/cellulase resulted in the FI increase of 1-36%and 35-214%at pH 5-11,respectively.Observed under SEM,the surface of blastospore became rough after treating with stimulated digestive fluids.Viabilities of the blastospores also reduced by 8 single enzymes,and laminarinase resulted in the most significant decrease in blastospore germination.The two sorts of mixed enzymes reduced the viabilities more than any single enzyme in mixture,and the decreases varied significantly at pH 5-11(proteinase K/cellulose:49-78%and trpsin/cellulose: 43-67%).In summary,this study has established the model system for evaluation the influences of insect midgut environment on fungal spore as biocontrol agents.All variations of the fluorescence intensity,structure of spore surface and spore viability suggested that the stimulated insect midgut digestive fluids have some negative impact on structure and activity of the conidia and blastospores.The results provide new insight into the survival mechanism of spore under insect midgut environment and suggest the promising ideas to enhance the fungal virulence against host insects via ingestion route.