Construction Of Engineering Bacteria Strain Of Entomogenous Fungus Regulating Immunity System Of Bemisia Tabaci By RNAi

Isaria fumosorosea is a species of common soil microorganism, and has widegeographical distribution, strong adaptability, and extensive host range, includingHomoptera, Lepidoptera, Diptera, Coleoptera and Hymenoptera, etc. According toincomplete statistics, insects in more than25families are infected by Isaria fumosorosea.Isaria fumosorosea is an important biological control fungus, and its preparations havecome into the market overseas. Present preparations of Isaria fumosorosea are usuallydeveloped by wild strains. since pathogenic processes of wild strains are long, insecticidalefficacy is slow, the field use dose is great, and the cost is high, these features constrain itswide spread use of Isaria fumosorosea. In consequence, the insecticidal speed of fungalpesticides is urgently needed to improve. At present time, employing genetic engineeringtechnology to select high virulence strains has become the hot spot of research. Thepurpose of this study is: constructing a strain of entomogenous fungus which could rapidlykill Bemisia tabaci by transgenic technology. This studies mainly include as follows:Construction of silent vector P-Z-F; protoplast preparation of Isaria fumosorosea strainIfB01and optimum condition of transformation with PEG4000; according to preliminaryscreening and the final selection to confirm recombination strain, and in vitro synthesis ofdsRNA; observing the biological characteristics of recombination strain, detecting toxicityof Isaria fumosorosea and in vitro synthesis of dsRNA to second instar nymphy of Bemisiatabaci. The results of this study are:（1）RNAi vector of immunogene T7L of Bemisia tabaci was constructed by silentvector pSilent-1plasmid of mycelial fungus. Interference vector was transferred intoprotoplast of Isaria fumosorosea strain IfB01by PEG4000mediating. One recombinationstrain, which could have stable inheritance, was gained by Hygromycin B preliminaryscreening and PCR final selection. We observed that the biological character ofrecombination strain and wild strains were similar. The primary bacterial colony was white,flesh red after producing spores, and the shape was irregular, upheaval. The sporulationquantity was great, and has no aerial mycelium. A large number of aerial mycelium weregenerated after5~6generations of cultivation, and the spores was rarely. The reverse sidewas faint yellow or yellowish-brown. Observed under microscope, the mycelium was longand thin, colorless and transparent with coremium. The conidiophore were verticillate, anda large number of colorless and transparent spores were produced. The spores werecolumnar to oval.（2）The best condition of protoplast formation of IfB01was enzymatic hydrolysate:1%snailase+2%cellulose,30℃,8h, PH6.0, osmotic stabilizer:0.7mol/L NaCl solution,the rotating speed100rpm, and the yield of protoplast was6.72×106pcs/mL. The colony numbers induced by40%PEG4000were the most,141, and the regeneration rate was7.05%.（3） Impregnation method was carried out to determine the toxicity of therecombination strain and dsRNA to the second instar nymphy of Bemisia tabaci. Theresusts showed that the virulence of recombination strain was better than wild strain IfB01.The maximum of cumulative mortality rate of recombination strain was86.67±2.31%, andwild strains76.00±2.00%,when the concentration was2.0×107/mL; mortality rate of thesecond instar nymphy of Bemisia tabaci were not over50%when the concentration ofspores was1.25×106/mL; insecticidal efficacy of dsRNA to the second instar nymphy ofBemisia tabaci was weak, cumulative corrected mortality rate of2000mg/L and4000mg/L was39.00±1.73%and57.67±1.53%, respectively.