The Population Structure Dynamics And Molecular Responses Of Pine Wood Nematode Under The Stress Of ?-pinene

Pine wilt disease?PWD?,an intricate disease system caused by the pine wood nematode?Bursaphelenchus xylophilus?,is one of the major forest disease.After the pine wood nematode?PWN?invading pine trees,it will multiply in a short time and cause the defensing response of pine trees.The infected pines begin to release a large amount of terpenes which enter the tracheid after vaporization and lead to cavity in the xylem and last cause pine trees wilt.Terpenes as an important substance of pine trees to against the PWN,it play important roles of the infecting process.Reproducing,migrating and pathogenesis of PWN in the whole process of infection,there is an interaction relationship with pine terpene.Therefore,this study selected?-pinene?High concentration of?-pinene?214.5mg/ml?and low concentration of?-pinene?56.33mg/ml??,which is the largest proportion in the terpenes produced by pines,as stimulator to explore its stress on the populations,population structure and sex ratio of pine wood nematode in order to found the propagate strategies of PWN under the stress of?-pinene.Besides,based on the transcriptome data,reproduction-related genes BX-clec and BX-F58genes were found responding to?-pinene stress,their sequences were cloned and functions on propagate strategies of PWN were tested by RNAi to elucidate the responding mechanism of PWN at physiological and molecular levels under?-pinene stress conditions.Our study provides a theoretical basis for the research on the pathogenic mechanism and breeding rules of PWN.The main conclusions of this thesis are as follows:1)We established the mating system of PWN?3?:1??to research the population structure and sex ratio.The results showed that populations of PWN had a significant growth trend after they were inoculated on PDA,the maximum population appeared on the 10^thday after inoculated,and then the populations began to decline from the 11^thday.The population and sex ratio grew fastest within the first 7d,the number of males began to increase and sex ratio to decline after 7d,it is because the hyphae in the PDA plate is completely fed by the PWN at this time and the pine wood nematode is caused by a lack of food in an unfavorable environment.During the whole cultivated period which larvae were the largest proportion in the population structures,the maximum number of larvae and populations were appeared on the 10^thday after inoculated and both of them have the same changing rules,the results showed that there was a positive correlation between the larvae number and the total population number of PWN.According to the dynamics of population,we got 7 key breeding time points:4^th,6^th,7^th,9^th,10^th,11^thand 12^thfor the following study.2)The population,sex ratio of PWN under the high concentration of?-pinene?214.5mg/ml?was higher than the control and low concentration of?-pinene?56.33mg/ml?.We found that PWN could produce more female nematodes and regulate population structure under the high concentration of?-pinene?proportion of larvae and eggs in the population were relatively stable?,but PWN produced more male nematodes and regulated population structure?proportion of larvae and eggs were reduced?under the low concentration of?-pinene.Our results suggested that PWN changed their propagate strategies by sex ratio and population structure to maintain the population growth under the stress of?-pinene.3)Base on transcriptome data,we found two reproduction related genes responding to?-pinene stress which named as BX-clec?BUX.s00713.777?and BX-F58?BUX.s01254.227?.Functional annotations in the PWN genome-wide website indicated that two genes are involved in the regulation of the development of the reproductive system,reproduction,larval development,embryonic development and egg hatching.We cloned the two genes and got CDS sequence homology to PWN genome-wide website with identification 100%,the length of BX-clec gene was 543 bp and BX-F58 gene was 486bp.The BX-clec belonged to CLECT class family genes and homology of clec-1 gene with Caenorhabditis elegans is 55%,BX-F58belonged to F1-ATPase-delta class family genes and homology of F58F12.1 gene with Caenorhabditis elegans is 70%by compared on NCBI.Both genes have only one proteins domain and proteins encoded were stable,the gene function were relatively conservative.Real-time quantitative results indicated that BX-clec and BX-F58 genes responded to?-pinene stress under high or low concentrations of?-pinene treatment and there was a significant difference in the amount of expression.4)BX-clec,BX-F58 and the negative control GFP of RNAi vector which to construct and the ds RNA was successfully synthesized in vitro,to soaked PWN by ds RNA solution.The real-time quantitative PCR results showed that BX-clec and BX-F58 were successfully interrupted,which was 73%and 46%respectively decreased under the low concentration of?-pinene.Under the high concentration of?-pinene treatment,the population of the interference group were lower than those of the control group,under the low concentration of?-pinene treatment,the proportion of larvae in the BX-clec gene interference group was higher than those of control group from the 7^thday after inoculated,the proportion of larvae in BX-F58 gene interference group and control group was not shown significant difference.Under the high concentration of?-pinene,the proportion of larvae in the BX-clec gene interference group was lower than control group from the 7^th,while the proportion of larvae in the BX-F58gene interference group was lower than control group from the 10^thday.The sex ratio of interference group was lower than control group of two genes,under the high concentration of?-pinene.The sex ratio of interference group and control group were not obvious,under the low concentration of?-pinene.When the gene of BX-clec and BX-F58 were interference,the proportion of larvae and sex ratio were began to decline and lower than control group under the high concentration of?-pinene,and the population was to decline.The result suggested that BX-clec and BX-F58 genes were involved in the interaction between PWN and pine terpene,it also regulates the population,population structure and sex ratio of PWN at the same time.About all,PWN used different propagate strategies under the high and low concentrations of?-pinene to maintain its population growth.The BX-clec and BX-F58 genes were involved in these changes of propagate strategies of PWN,thereby affecting the change of population,it is one of the key breeding-related genes which the process of infestation of pine trees by pine wood nematode.