A Preliminary Study On The Culture Of Esteya Vermicola And Its Application In Pine Wilt Disease

Pine wilt disease（PWD）is a global quarantine and destructive epidemic disease that can cause rapidly wither and die to pine forests,serious damage to forest resources,and great losses to the national economy.The pathogenic factor of PWD is Bursaphelenchus xylophilus（Aphelenchida:Aphelenchoididae）,which is spread by human and beetles.At present,biocontrol microorganisms and plant nematicidal substances have been widely studied in biological control of PWD.Esteya vermicola,the first recorded parasitic fungus of B.xylophilus,exhibits high infestation and parasitism rate against B.xylophilus and has the prospect and potential as a biocontrol agent against PWD,which obtained patents in the United States and South Korea.South Korea’s Chungnam National University has provided our laboratory with the potential parasitic fungi.Based on previous studies,this study focuses on five new aspects of solid culture conditions of E.vermicola,the effect of water on fungal growth,the difference in infection concentration,the prevention and control of trunk injection,and the related research with Monochamus alternatus（Coleoptera:Cerambycidae）.A relative study on the growth conditions and infective vigor of this fungus was carried out in order to lay a foundation for the use of the fungus to control PWD and provide relevant theoretical proof.The main findings are as follows:（1）Effects of different rice mediums on the sporulation of E.vermicola:Using rice medium to culture,E.vermicola began to grow obvious hyphae on the third day,and on the fifth day,the fungal colonies grew over the surface of the rice.The highest total spore concentration was 6.4×10⁷CFU/m L,which belongs 50g rice+50 m L pure water+0.5g yeast powder+0.05g KH₂PO₄after culturing E.vermicola 7 days.The highest lunar spores concentration was 8.8×10⁶CFU/m L,which belongs 50g rice+50 m L pure water+0.05g KH₂PO₄after culturing E.vermicola 10 days.（2）Effect of different water content rice medium on the sporulation of E.vermicola:Different water content（14.00%、42.67%、57.00%、65.60%and 71.33%）rice medium significantly affected the sporulation of the fungus.the fungus has the weakest sporulation when the water volume is 14.00%and 71.33%.When the water content was 42.67%,it was significantly higher than other water treatment,and the highest in the ninth day was 6.0×10⁷CFU/m L.The highest concentration of lunar spores in each treatment occurred in rice medium with a water content of 57.00%and 65.60%.（3）Determining the infectivity of different concentrations of E.vermicola spore suspension against B.xylophilus:Experiments on the infection rates of different spore concentrations of 1×10⁵,1×10⁶,1×10⁷and 1×10⁸/m L showed that the spore killing rate of1×10⁸/m L（58.68%）was significantly higher than that of other cultures after 1d;after 3days,the mortality of spore-treated nematodes reached more than 90%,and there was no significant difference between the treatments.（4）Trunk injection E.vermicola to control PWD:There were no significant difference between the survival rate of pine after injecting E.vermicola and that in the control group after 9 months.Artificial infection of nematodes after two months of injection of E.vermicola,the survival rate of P.massoniana was 61.76%in E.vermicola group,which was significantly higher than that of the avermectin group 54.84%and the nematode group27.96%.Artificial infection of nematodes after three months of injection of E.vermicola,the survival rate of P.massoniana was 65.32%in E.vermicola group,which was significantly higher than that of the nematode group 49.29%.The survival rate of P.massoniana increased with the increase in the concentration of E.vermicola spore suspension.Under the treatment of 1×10⁷and 1×10⁸CFU/m L,the pine survival rate was60%and 57%,which was significantly higher than the injection of avermectin 13%.However,the survival rate of pine with injecting 1×10⁶CFU/m L spore suspension was48.0%,and it was 43%in the injected water group.（5）Effect of E.vermicola on the transmission efficiency of M.alternatus:The rate of nematode carried by 152 M.alternatus trapped in Conghua Hot Spring Forest in May and June 2018 was 44.08%.The amount of nematodes contained in the single M.alternatus is between 1 and 66,000.The number of nematodes in 1-10 is 49.25%.Under the three treatments,M.alternatus had the weakest ability to directly infect the pine branches which were sprayed E.vermicola spore suspension,at 22.22%lower than the infection rate of M.alternatus on pine branches without any treatment（44.44%）and M.alternatus soaked with spore suspension to pine branches（69.23%）.