Response Of Pine Endophytic Bacteria Flora To Bursaphelenchus Xylophilus Infection And Construction Of Insecticidal Gene Engineering Bacteria With JK-SH007

Bursaphelenchus Xylophilus,which has the characteristics of being easy to spread,rapid onset and high mortality,is known as "AIDS" to pine.In the early stage of Bursaphelenchus Xylophilus infection,the pesticide application of the vaccine tree can greatly increase the cure rate.Therefore,through the study on the structure changes of pine endophytic bacteria flora during the early stage of B.xylophilus infection,it is possible to provide evidence for the key scientific problems to be solved urgently,including the early infection diagnosis and control of B.xylophilus.Microbial bio-agents have advantages in environment-friendly protection,disease and pest co-treatment,and plant-growth promoting,especially suitable for early ecological control of plant pests and diseases.Cry proteins,secreted by Bacillus thuringiensis,have high insecticidal activities.Introduction of genes into endophytic bacteria with disease-preventing and growth-promoting functions to achieve effectively heterologous expression is a promising strategy to biocontrol pine wilt disease.In this regard,microbial community structure analysis,homologous recombination,and other molecular biological techniques were used to analyze the response of endophytic bacteria in pinus massoniana(P.massoniana)to the early stage of infection of B.xylophilus after nematode inoculation in the field.We also studied the heterologous expression of cry genes and their insecticidal activities in the biocontrol bacterium Burkholderia pyrrocinia JK-SH007.The data we got laid the foundation for the construction of early diagnosis and ecological control system of B.xylophilus.The main results are as follows:(1)In the same forest area,flora map of healthy wild P.massoniana with different trunk diameter had no significant difference.The bacterial structure of non-inoculated and the inoculated water on the endophytic bacteria of P.massoniana was also no significant difference.These results show that there was no evident influencefrom mechanical damages and changes in field condition during the experiment.(2)With the increasing of infection time of B.xylophilus,the species and abundance of endophytic bacteria were increased over time.Endophyte Mucilaginibacter sp.M68C4 A and uncultured bacterium OTU2265 had a positive correlation to the increase of infecti on time of B.xylophilus.Endophyte uncultured bacterium MA13809b03 and Klebsiella sp.Arv-22-2.8 had a negative correlation to the increase of infection time.(3)We observed that the P.massoniana received inoculation of nematodes had a very high coincidence degree in the same time and space,which suggested that the flora of Masson Pine had a consistent response to the early infection of B.xylophilus.By using NMDS to analyze the data of endophytic flora from P.massoniana in the first 30 days and in the 40 th day ofinoculation,we found that the two layers were markedly separated,and the population structures of endophytic bacteria were significantly different.These results laid a theoretical basis for the technology development of early diagnosis of pine wilt disease.(4)JK-SH007 genetic manipulation system was successfully established,and the expression of recombinant Cry218 and Cry3 a protein in JK-SH007 was at a relatively high level.The expression vector was p HKT2.Cry218 gene promoter was Pr P1,nevertheless the promoter of Cry3 a gene was replaced to T7,which indicate that cry series of genes in the same expression system for promoters were different.Through replacement of different promoters,we enable to achieve high expression of cry gene in other expression system.(5)The optimal expression of Cry218 protein condition was as follows: JK-SH007(p H KT2-cry218)was used to inoculate LB medium containing trimethoprim(50 ?g/m L)to grow till to a serials cell density at OD600=0.8.The cultures were then kept in a 42°C shaking incubator with vigorous shaking for 12 hours,and the maximum yield of Cry218 ICP was about 0.25g/L.(6)The biological test results show that the engineered bacterial endophyte JK-SH007(p HKT2-Cry218)was toxic to the second instar silkworm,and JK-SH007(p HKT2-Cry3a)was toxic to the P.aenescens(Fairmaire)larvae.LC50=0.77(0.57-1.04)g/L and 0.63(0.48-0.82)g/L.At the same time,the engineered bacterial endophyte JK-SH007(p HKT2-Cr y218)and JK-SH007(p HKT2-Cry3a)had a Toxic activity to the larvae.But the control host bacteria JK-SH007 also showed considerable toxicity,so the action mechanisms o f engineering bacteria on pine wood nematode remains to be further studied.