Study On Toxicity Of Bacillus Thuringiensis And Esteya Vermicola Against Pine Wood Nematode(Bursaphelenchus Xylophilus)

Pine Wood Nematode?PWN,Bursaphelenchus xylophilus?,the main pathogen of pine wood nematode disease?PWND?,not only causes devastating damage to Pinus species,but also becomes a global quarantine pest.Due to the strong concealment of pine wood nematodes,it is extremely difficult to directly use PWN as a target for biological control.Bacillus thuringiensis?Bt?is one of the most widely used microbial insecticide all over the world.Some strains'fermentation broth of Bt has toxic activity against PWN.Esteya vermicola?Ev?,an endophytic fungus discovered for the first reported in PWN,can produce crescent-shaped spores that have invasive activity and lethality against PWN.In this study,the screening of nematicidal Bt strains was started to study the virulence stability of the virulent nematicidal Bt fermentation broth and the combined virulence of Bt and Ev against PWN.The purpose is to provide certain theoretical basis and technical support for joint use of Ev and Bt to control PWN.Direct contact killing method was used to determine the nematicidal activity and stability of Bt fermentation broth in order to screen highly toxic Bt strain.The results showed that the nematicidal activity of strain D fermentation broth was the highest among four strains.The mortality of pine wood nematode treated by strain D fermentation broth was over 70%for 8 h,and the mortality rate reached 100%at 24 h.The results of toxicity stability test showed that strain D fermentation broth was stable to proteinase K,resistant to alkali,insensitive to strong acid and temperature,and did not decrease obviously at the temperature of 4?for 50 d.It was initially determined that the nematicidal active substance in the strain D fermentation broth was?-exotoxin.Polymerase Chain Reaction amplification results showed that the 16S rDNA fragment of strain D was 100%similar to Bacillus thuringiensis KNU-07.The combined toxicity of Ev and Bt was determined by morphological changes of PWN,mortality and mortality when indoor treatment of PWN with Ev spore suspension and Bt fermentation broth.The results showed that the nematodes treated by Ev spore suspension could cause leakage of body contents and shrinkage,bending and fracture of body cavity.The combination of high concentration of Ev spore suspension and Bt fermentation solution could significantly increase the mortality of nematodes,with a maximum of 100%when combined with Ev spore suspension at a concentration of 2.5×10⁸ cfu·mL^-1 and three-fold diluted Bt fermentation broth.The combined treatment of pine wood nematodes died faster,and the mortality rate would continue to rise with the extension of treatment time.Test results of combined toxicity of Ev spore suspension and Bt fermentation broth in Greenhouse showed that the survival rate of Chinese pine?Pinus tabuliformis Carrière?seedlings,which infected with PWN,treated with combination of Ev spore suspension at high concentration and three-fold diluted Bt fermentation liquid was higher than the single use of two treatment.And there was no significant difference in the effect between the prevention group and the control group.The survival rate of the seedlings in the prevention group was higher than the seedlings in the control group when the high concentration Ev spore suspension was used alone.The result of using three-fold diluted Bt fermentation broth alone was in the opposite.