Research On The Nematicidal Activity Of Bacillus Thuringiensis Against Bursaphelenchus Xylophilus And Cryoperservation Of The Nematode

Pine wood nematode disease (also called pine wilt disease) caused by Bursaphelenchus xylophilus(Steiner et Buhrer), is a destructive disease of pine trees. In present, Bursaphelenchus xylophilus is a threat to China forestry, and it is one of the quarantine pests in the world. Bacteria were shown nematicidal activity to other nematodes, like Caenorhabditis elegans. Screening nematicidal bacteria against B. xylophilus were performed. For the purpose of convenient culture of B. xylophilus, cryoperservation of this nematode at -80℃was established.In the first place, 210 samples were isolated from the soil, dead pine tissue and healthy pine plant tissue from Zhejiang forest. After assay for nematicidal activities in 96 wells, 8 strains among them were shown the significant nematicidal activities against B. xylophilus. All of the 8 strains produce the endospores. For further, Among these 8 strians, 4 Bt strains'crystal proteins tested on Bursaphelenchus xylophilus, were found to had higher nematicidal activity. The bioassay result showed that the mortality after 48h of zjfc85, zjfc14, zjfc65 and zjfc67 were 90.91%,85.58%, 75.80% and 60.00%.During 2008 to 2010, 120 exzamples including soil, diseased and healthy trunks of P. massoniana, Black pinus and Monochamus alternatus, were collected from the areas that pine wood nematode disease occurrenced in Zhejiang province. 210 isolates were obtained, 70 isolates that had spores were included. 96 well plate were used to test the nematicidal activity of all isolates. The results showed that 8 strains had nematicidal activity, 4 of the 8 strains showed higher activity, named zjfc14, zjfc65, zjfc67, zjfc85. Crystal proteins were extracted and their nematicidal activity against Bursaphelenchus xylophilus were tested. The bioassay result showed that the mortality of zjfc14, zjfc65, zjfc67, zjfc85 after 48h, were92.6%, 82.3%, 77.8% and 95.3%. Strain zjfc85 had the highest nematicidal activity.The colony morphology, physiologic and biochemical analysis results and the result of 16srDNA proved that the 4 strains were Bacillus thuringiensis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the crystal proteins of the four Bt strains were different from each other.In this paper, cryoperservation of Bursaphelenchus xylophilus at -80℃was estabilished. Followed by investigation of different cryoprotectants affecting the survival and propagation, cryoperservation with 15% (V/V) glycerol as the protectant was found as the best for cryoperservation, followed by 10% (V/V), 20% (V/V) and 25%(V/V). The last was 1% DMSO. Under the circumstance of 15% (V/V) glycerol, the survival rate maintained as high as 36.5% after defrosting, and nematodes remained normal at propagation. There was no significant difference between 3 days and 30 days later after defrosting, which indicated that cryoperservation with 15% (V/V) glycerol was suitable for B. xylophilus at -80℃, substantially.In this study 4 different Bt strains that had high nematicidal activity were obtained. They can be basic materials in theresearch of biocontrol of pine wood nematode disease. And the cryoperservation method of Bursaphelenchus xylophilus were primarily constructed, which can solve the problems in the research of Bursaphelenchus xylophilus.