Identification Of A Novel Rhabdovirus Rice Stripe Mosaic Virus And It Vector Transmission Characters

Rice is one of the important staple crops in our country and the world,while the virus disease poses a serious threat to the rice production in Southeast Asia.Especially in the rice area of South China,most viruses often occurred together,and a serious economic loss caused by the outbreaks of large area.The detection of viruses is the technological basis of disease prediction and prevention.In this study,a multiplex RT-PCR method was established to monitor the occurrence of four common rice viral diseases in South China.Furthermore,a new rice viral disease was found during the field investigation,and focused on the transmission characteristics of transmission vector,Recilia dorsalis.At the same time,the RNA silencing suppressor encoded by RSMV was preliminarily identified.The major results are as follows.1.The distribution characteristics and occurrence of four major rice viruses(SRBSDV,RRSV,RGDV and RBSDV)in south China were clarified.Based on the characteristics of four viral genomes,we designed and screened the specific primers of four viruses,and established a multiplex RT-PCR method.Based on this method,582 potentially infected rice samples collected from Southern China were analyzed.The results showed that the above four viruses were distributed in south China,among which RGDV was most common virus,and the detection rate was 36.08%,followed by RRSV,SRBSDV and RBSDV,detection rate were 34.02%,30.07% and 0.515%,respectively.Further analysis found that there were significant geographical differences in the distribution of the four viruses in southern China,RRSV mostly distributed in southern Hainan,RGDV mainly occurred in the southwest and northeast of Guangdong,SRBSDV occurred across south China,and RBSDV only occurred in the northern parts of Guangxi.2.A novel rice virus,rice stripe mosaic virus(RSMV),was discoveried and identified.Field investigation and artificial inoculation revealed that diseased plants exhibited slight dwarfing,with leaves showing yellow stripes or mosaic and occasional twisting,inferior heads tended to halfway emerged and grains were often unfilled.Electron microscopic observation revealed that enveloped bacilliform matured virons were distribution in the cytoplasm of infected leaves tissues,the viral particles were 300 to 3775 nm long and 45 to 55 nm in wide(n = 50).The complete genome of the virus was obtained by small-RNA deep sequencing and reverse transcription-PCR product sequencing.The anti-genome contains seven non-overlapping genes in the order 3'-N-P-P3-M-G-P6-L-5'.In addition,we confirmed that RSMV encoded N,P,M G and L are viral structural proteins by purified protein sequencing.Phylogenetic analysis based on protein N,G and L amino acid sequences indicated that RSMV is a new member of the genus Cytorhabdovirus of the family Rhabdoviridae,and and is most closely related to CBDa V.RSMV can be transmisit by leafhopper Recilia dorsalis(Hemiptera:Cicadellidae)and naturelly infect field weeds,Digitaria sanguinalis and Monochoria vaginalis.RSMV is now mainly distributed in southwestern Guangdong Province of China.In some fields,the incidence was higher than 70% and seriously harming rice production.3.The transmission characteristics of RSMV by vector Recilia dorsalis were determined.The results of this study showed that adults and nymphs can be acquired for RSMV after feeding 3 min on the diseased plants,and the viruliferous adults and nymphs were 19.2%(10/52)and 24.4%(10/41),respectively.The virulifierous adults were 28%(14/50),25.9%(14/54)and 29.3%(12/41)after feeding for 5min,10 min and 30 min,respectively.When the nymphs were fed for 5min and 10 min,the virulifierous nymphs were 36.4%(12/33)and 32.7%(17/52),respectively.However,the frequency of virulifierous nymphs significantly increased to 45%(18/40)after feeding 30 min on diseased plants.After feeding for 1 h,the virulifierous adults and nymphs were reaching 40.4%(21/52)and 50%(15/30),respectively.After feeding for 3 hours,the rate of infection was significantly increased,and the infection rate of nymphs(66.7%)was higher than that of adults(58.9%).On the other hand,neither virulifierous adults nor nymphs could transmit RSMV to health rice seedlings after feeding for 3min,5min and 10 min on the diseased plants.The transmission rate of virulifierous adults and nymphs were 5%(1/20)and 15%(3/20)after feeding for 30 min.However,after feeding for 1h,the transmission rate of adults and nymphs were significantly increased to 50%(12/24)and 57.1%(16/28),respectively.In addition,the shortest circulative transmission periods of RSMV in the vector was 6 days,the longest was 18 days,the majority ranged from 8 to 16 days,and there were significant difference among individuals.Part of tested vectors throughout the circulative transmission periods,can continuously transmit RSMV before the end of life,some of vectors exist intermittent period,the shortest and longest time were 2 days and 6 days,respectively.A small number of viruliferous vectors can not be transmitted the virus before death and few viruliferous individuals can not be transmitted the virus during their lifespan.4.P protein encoded by RSMV was preliminarily identified as a RNA silencing supressor.In this study,the full length of the six genes encoded by RSMV(except L gene)were cloned and ligated to the plant expressed vector,respectively.These plasmids were transformed into Agrobacterium tumefaciens EHA105 and mixed with Agrobacterium containin 35S-GFP construct,followed by co-inflitration of transgenic Nicotiana benthamiana line 16 c.The preliminarily results showed that RSMV-encoded P protein had a relatively weak local RNA siliencing suppressor proteins activity and delayed systemic silencing.Our study began with the field investigation,clarified the occurences and distribution characteristics of the mainly four rice viruses in southern China,which was provide a basic data to deep understanding of the occurrences regularity and optimization of disease mornitoring or control system.Furthermore,the discovery and identification of RSMV as well as research of the transmission characteristics of vector not only reveals the diversity of plant viruses in nature,but also the timely discovery of RSMV lays the foundation for the development of prevention and control measures to ensure the satety of grain production.