Determination Of The Control Effect Of 2% Emamectin Benzoate Emulsifiable Concentrate On Pine Wood Nematode Disease And Preliminary Screening Of Fungi For Biocontrol Of Pine Wood Nematodes

Pine wood nematode disease,a worldwide quarantine disease,has emerged on a large scale in China and is currently one of the most severe forest diseases in the country.Bursaphelenchus xylophilus,the causative agent of pine wilt disease,is a migratory endoparasitic nematode that can cause rapid wilting of the host pine tree by damaging the water transport system.In the search for effective control measures against B.xylophilus,this study investigated two approaches:trunk injection treatment and screening of biocontrol fungi for pine wilt disease.The following results were obtained:1、Indoor impregnation with 2%emamectin benzoate emulsifiable concentrate（trade name:Songgaofei）showed higher nematicidal activity compared to 2%emamectin benzoate microemulsion（trade name:Linyuansongyun）,with an LC₅₀ of 41.808 mg/L,significantly lower than the LC₅₀ of 75.959 mg/L for 2%emamectin benzoate microemulsion（P<0.05）,indicating stronger nematicidal activity.The results of the greenhouse prevention group showed that the prevention effect of 2%emamectin benzoate emulsifiable concentrate was significant,and pine trees did not die after 2 years.The results of the greenhouse treatment group showed that the earlier the treatment time with 2%emamectin benzoate emulsifiable concentrate,the better the treatment effect;2 years later,after being inoculated with nematodes,the pine trees did not die after 7 days of injection treatment,while after 30 days of injection treatment,Pinus bungeana did not die,P.tabuliformis had a mortality rate of 20%,and P.armandii had a mortality rate of 40%.Field experiments showed that 2%emamectin benzoate emulsifiable concentrate had a significant preventive effect against massoniana pine wilt disease in the forest,with a cumulative mortality rate of 2%and 5%for the treated pine trees after 2 years,much lower than the mortality rate of 20%and 25%for the control group.2%emamectin benzoate emulsifiable concentrate exhibited good translocation in P.bungeana,P.tabulaeformis,and P.armandii,with lower residual levels in P.tabulaeformis than in the other two pine species 30 days after treatment.Over time,there are differences in changes in the levels of emamectin benzoate in different pine varieties.15 days ago,the content of emamectin benzoate in all pine varieties increased;After 15 days,the content of emamectin benzoate in the bodies of P.bungeana and P.armandii gradually decreased,while there was no significant decrease in the content of emamectin benzoate in the bodies of P.tabulaeformis.The residual levels of emamectin benzoate gradually increased in different pine species 15 days after treatment and then leveled off or decreased.2%emamectin benzoate emulsifiable concentrate showed significant nematicidal activity against B.xylophilus,with significant preventive and curative effects in greenhouse and field conditions,good translocation in different pine species,and thus has the potential for further promotion and use.2、During the process of cultivating B.xylophilus with Botrytis cinerea,a strain of Clonostachys pseudochroleuca,a hyperparasitic fungus with nematicidal activity,was discovered.Through morphological and molecular identification using ITS1/ITS4 and Tb2a/Tb2b primers,the strain was preliminarily identified as C.pseudochroleuca.When 2000B.xylophilus were inoculated onto PDA plates covered with C.pseudochroleuca mycelium,only 46.67±52.49 nematodes per dish were recovered after 8 days,with 6.67±9.43 nematodes per dish being viable.The fermentation broth and spore suspension of C.pseudochroleuca showed nematicidal activity against B.xylophilus,with corrected mortality rates of 88%for the fermentation broth after 48 hours and 88.33%for the spore suspension at a concentration of 1×10⁸ cells/m L after 24 hours.C.pseudochroleuca also exhibited a significant inhibitory effect on egg hatching,with a 100%inhibition rate observed when exposed to a 2×fermentation broth.Furthermore,C.pseudochroleuca has a hyperparasitic effect on B.cinerea and can enclosed the host hyphae,growing from the host hyphae by uptaking the host plasma as a source of nutrients.3.The effects of 30 edible fungi on the population reproduction of B.xylophilus were compared.The results showed that the number and total number of alive B.xylophilus isolated from all edible fungi plates were significantly lower than the negative control,B.cinerea（P<0.05）.Lentinula edodes showed the best control effect,with no nematodes isolated from the culture medium after eight days;fewer than 200 lived nematodes isolated from the 19 edible fungi culture media per dish,the control effects were mostly good;while the inhibition effect of Flammulina velutipes and Morchella esculenta on the population reproduction ability of B.xylophilus was the lowest.The corrected lethal rate of B.xylophilus treated with fermentation broth of 19 edible fungi was from 34.00～86.67%after 24 hours,and from 68.67～96.33%after48 h,indicating significant nematicidal activity.The corrected nematicidal activities of Ganoderma sinense fermentation broth at 24 hours and 48 hours were significantly higher than that of other edible fungi（P<0.05）,with 86.67±2.05%and 96.33±1.70%,respectively.Additionally,,the mycelium of Pleurotus ostreatus and P.cornucopiae displayed trapping and digestion effects on B.xylophilus under optical microscope.