Study On The Occurrence And Viral Infection Of Euproctis Pseudoconspersa

The tea caterpillar,Euproctis pseudoconspersa is an important tea pest.In recent years,the application of chemical pesticide has been gradually reduced in tea gardens while the occurrence of E.pseudoconspersa is increasing.The larvae of E.pseudoconspersa not only damage the yield of tea plants by direct feeding,but also cause swelling and itching of human body.This brings a lot of inconvenience to the production and management of tea garden.The occurrences of E.pseudoconspersa are different due to regional environment conditions,therefore,the forecast and prediction can guide timely control,which relied on the national E.pseudoconspersa monitoring.In addition,E.pseudoconspersa nucleopolyhedrovirus is an important natural pathogen to control this pest.Higher population density of E.pseudoconspersa easily causes the epidemic of the virus disease,which therby can reduce the population of this tea pest.Normally,the virulence among viral isolates exists difference to the tea caterpillar,thus the continuous collection of viral resources is important for reserving prevention and control means to E.pseudoconspersa in different production areas.With the development of high-throughput sequencing technology,more and more RNA viruses have been discovered.In the view of the new cultivation and management of tea garden,the unknown occurrence of E.pseudoconspersa and the insufficience of virus excavation,this thesis focuses on the pest niche,occurrence and prediction of E.pseudoconspersa in tea garden,as well as the excavation of DNA virus and RNA virus,in order to provide theoretical support for the research and development of green prevention and control technology of E.pseudoconspersa.The main results are as follows:1.The occurrence and genetic diversity of E.pseudoconspersaBased on the investigation and analysis of the interspecific competition relationship of the main lepidopteran pests in the tea garden(Yongchuan District in Chongqing Municipality),the results showed that the spatial distribution between E.pseudoconspersa and Caloptilia theivora and the temporal distribution between E.pseudoconspersa and Mahasena colon were low.Therefore,they had little influence among above populations.While E.pseudoconspersa coincide with Darna trima and Iragoides fasciata in the occurrence period and canopy level,the competition between E.pseudoconspersa and D.trima,as well as E.pseudoconspersa and I.fasciata at the time and space niche level existed.The field monitoring of four tea areas in China(Qingyuan City in Guangdong Province,Wuxi City in Jiangsu Province,Xinyang City in Henan Province,and Yongchuan District in Chongqing Municipality)showed that E.pseudoconspersa occurred 3?4 generations per year,and generations occurred in Qingyuan(4 generations)are the most.Based on the monitoring data of the above four tea areas,and using the discriminant analysis,the key factors that can affect the occurrence of E.pseudoconspersa were the maximum temperature in March,the average temperature in the first ten days of February,the precipitation in January and the average humidity in the last ten days of March.While there are two key meteorological factors affected the occurrence peak period of E.pseudoconspersa,such as the precipitation in the middle ten days of January and the first ten days of February.The Fisher's discriminant function was constructed,and the accuracy of discrimination were 33.3% and 66.7%,respectively.The mitochondrial COI and ND5 genes of E.pseudoconspersa from 7districts in China were analyzed to investigate the genetic diversity of different geographical populations.The results showed that the genetic differentiation within populations played a key role in the evolution of E.pseudoconspersa,but the gene exchange among populations was weak.2.Identification of DNA viruses and their virulence assay in E.pseudoconspersa and D.trimaThe samples of the tea pests(E.pseudoconspersa and D.trima)were collected in the field,and the viruses were identified by molecular methods and virus particle morphology.Three isolates of E.pseudoconspersa nucleopolyhedrovirus(Ep NPV)and one isolate of D.trima granulovirus(Datr GV)were identified,and the genome of Datr GV was unknown to date.The total length of Datr GV genome was 103,846 bp and concluded 118 ORFs accounting for 90.5% of the whole genome.Based on the phylogenetic tree constructed with 38 core proteins of insect baculovirus and collinearity analysis,the highest similarity was found between Datr GV and Pr GV.The Datr GV genome contains 10 unique ORFs and 18 direct repeat homologous region sequences,and the same sequence has different repeat combination.The virulence bioassay of two Ep NPV strains to different populations of E.pseudoconspersa was perforemed.It was found that the virulence of different isolates to different populations of E.pseudoconspersa was different.None of isolaes had significantly higher virulence to all populations of E.pseudoconspersa than the other isolates.Datr GV had strong virulence to the host itself,and also had certain virulence to Thosea sinensis and I.fasciata.It is worth noted that Datr GV could enhance the virulence of Ep NPV to E.pseudoconspersa.3.Discovery of RNA virus in E.pseudoconspersa and infection frequency and genetic differentiation of the RNA virusBased on high-throughput sequencing technology and bioinformatics analysis,a negative-stranded RNA virus(Bunyavirus,Ep BYV)was identified in E.pseudoconspersa.The small RNA derived from viruses in E.pseudoconspersa was analyzed.It was found that the small RNA derived from Ep BYV had strong signal,and appeared the highest peak at 20 nt,and symmetrically distributed in the whole viral genome.The above results suggested that the virus can replicate in the host and trigger host's RNAi antiviral immune response.Based on the transcriptome sequencing data and the detection of viral frequency,Ep BYV had a high infection frequency in E.pseudoconspersa.Based on Rd Rp gene sequence analysis,it is found that Ep BYV virus has a high degree of differentiation and less gene exchange in the six populations of E.pseudoconspersa.The genetic differentiation mainly arised from the variation between the populations,followed by the variation within the populations.4.Immune responses genes identification to Ep NPV and Ep BYV infection in the E.pseudoconspersaBased on the transcriptome databases of 6 lepidopteran pests including E.pseudoconspersa,Ectropis grisescens,Buzura suppressaria,Caloptilia theivora,M.colona and I.fasciata,the key genes of each immune pathway were identified and annotated.There were 18,74,30 and 122 genes in the RNAi,Imd,Jak-Stat and Toll pathways,respectively.Except for the partial Toll gene and SPZ gene were expanded or contracted,the types and quantities of the other genes in other pathways and Toll pathway were the same as those in Bombyx mori.Based on the transcriptome data of E.pseudoconspersa larvae inoculated with Ep NPV and E.pseudoconspersa larvae with high titer and low titer Ep BYV,168 and 235 DEGs were obtained respectively.Further analysis of E.pseudoconspersa against Ep NPV,it was found that the immune associtated genes such as heat shock protein and some epidermal proteins were up-regulated,while the differential expressions of E3-ul-Ufd4,diapausin A4,Car E-6,yellow,Ten-X and cath D-H were found in high titer and low titer Ep BYV samples.In summary,the occurrences of E.pseudoconspersa in the field were monitored,and it is found that there are 3 ? 4 generations a year in the four tea areas within the monitoring region.The Fisher's discriminant function had constructed in predicting the occurrence degree and peak of the first generation of E.pseudoconspersa;At the same time,it was found that the niche competition between E.pseudoconspersa and D.trima was the strongest in tea garden.The DNA viruses of the two pests were collected and identified,and their virulence to their own hosts was strong.D.trima granulovirus had synergistic effect on the virulence of E.pseudoconspersa nucleopolyhedrovirus to E.pseudoconspersa;Based on the transcriptome data,E.pseudoconspersa Bunyavirus was identified,and the infection frequency was high in the field population,and there was differentiation among the populations;The differential genes of E.pseudoconspersa in response to E.pseudoconspersa nucleopolyhedrovirus and Bunyavirus were analyzed.These results could be helpful in the development of the green control method of E.pseudoconspersa,as well as the efficient control of E.pseudoconspersa in together with the reduction of chemical pesticides.