Screening And Characteristic Of Bacillus Thuringiensis With Nematicidal Activity Against Ditylenchus Destructor

D.destructor, which mainly parasitize in sweet potato, could cause tremendous loss in agricultural production, and the disease caused by this worm has become more and more serious in these years. Traditional control against D.destructor includes rotation, application of chemical pesticides and breeding resistant varieties, which has exposed significant limitations because of the low efficiency, high investment and environmental problems. Bacillus thuringiensis(Bt), as its specifically high toxicity, harmless to mammals, and safe to environment, has been successfully applicated as microbial insecticides on control of plant parasitic nematodes. But so far, the research on the nematicidal of Bt against D.destructor has rarely been reported. In our study, we tested the nematicidal activities of 12 Bt strains against D. destructor, of which YBT-008 showed highest toxicity against D.destructor. The four insecticidal genes--cyt2Ba, cryllAa, cry4Aa and cy4Ba—encoded by YBT-008 have been analysed and verified using SDS-PAGE, LC-MS and PCR. Then we cloned their CDS, constructed expression vectors and finally the proteins were expressed in the acrystalliferous Bt strain BMB 171. The nematicidal activity of expressed proteins was tested, by which we aimed to acquire new gene and protein resources from Bt strains with high virulence against D.destructor.1. Screened out a Bt isolate YBT-008 with high virulence against D.destructor. The nematicidal activities of 12 Bt strains against D. destructor were tested by extracting the insecticidal crystal proteins (ICPs), incubated with dissolved ICPs, and the mortalities of the D. destructor were counted after 3 and 7 days. The activities of primarily screened strains were then assayed through diluting their bioassay protein samples. After primary and secondary screening, the YBT-008 strain showed the highest toxicity against D. destructor. The result of bioassay indicated the LC50 of YBT-008 against D, destructor was 203.76μg/mL.2. Analyzed and identified the four insecticidal genes encoded by YBT-008. The parasporal crystals formed during sporulation could be observed by microscope. By means of SDS-PAGE and LC-MS analysis, the ICPs—Cry4Aa, Cry4Ba, CryllAa and Cyt2Ba-- produced by YBT-008 were identified. And we further validated the fact that YBT-008 harbored cyt2Ba, cry11Aa, cry4Aa and cry4Ba, by means of PCR amplification.3. Cloned and expressed the above four genes. We designed the specific primer pairs with restriction sites to clone their full-length coding sequence, connected them to the shuttle vector pHT304 and then transformed into BMB171. The proteins could be observed by microscope and detected by SDS-PAGE, which meant that they all expressed successfully.4. Tested the nemaicidal activities of Cyt2Ba and Cry4Ba against D.destructor. We extracted the expressed ICPs, and tested their activities using the same bioassay procedure. The results showed that nematicidal activity of Cyt2Ba was more effective than Cry4Ba after 3 and 7 days. As the low expression amount of Cry11Aa and Cry4Ba, we need to optimize their expression amount and then detected their nematicidal activities.