Identification Of Viruses Infecting Cucurbit Crops In Guangdong Province And Analysis Of Important Virus Characteristics

Cucurbit crops are important cash crops in Guangdong,and virus diseases are one of the main diseases in their production.However,the virus diseases on cucurbit crops in Guangdong hasn’t been systematically investigated and identified.This paper investigated the occurrence of virus diseases on cucurbit crops in Foshan,Guangzhou etc13 administrative cities of Guangdong.Viruses infecting cucurbit crops in Guangdong were identified using small RNA deep sequencing and RT-PCR detection method.The complete genome sequences of Cucumber green mottled mosaic virus(CGMMV)、 Guangdong isolates of Watermelon virus A(WVA)and Zucchini tigre mosaic virus(ZTMV)were cloned and sequenced.The pathogenicity of CGMMV and the resistance of cucumber varieties were determined.The results of the study were as follows:1.Cucubit crop virus diseases occurred and damaged to varying degrees in every cucurbit crop-growing areas in Guangdong.Through general survey combined with systematic investigation,it was found that cucurbit crop virus diseases occurred and damaged at 5% to 30% of disease incidence,even 100% in 13 administrative cities including Foshan,Guangzhou,Huizhou,Maoming,Qingyuan,Shaoguan,Zhanjiang,Zhaoqing,Chaozhou,Meizhou,Jieyang,Shantou,and Yangjiang.The diseased plants exhibited mosaic,yellowing,shrinking,mottle,blistering and bubbling symptoms,but mainly mosaic symptom.2.There exist sixteen viruses infecting cucurbit crops in Guangdong.Using small RNA deep sequencing and sequence comparativ analysis,16 viruses were detected in the mixed diseased samples of cucurbit crops from Guangzhou,Huizhou,Zhanjiang etc.including Bottle gourd mosaic virus(BGMo V),Cucurbit aphid-borne yellows virus(CABYV),Cucurbit chlorotic yellow virus(CCYV),CGMMV,Cucumber moasic virus(CMV),Lagenaria siceria endorna virus(Ls EV),Melon aphid-borne yellows virus(MABYV),Melon yellow spot virus(MYSV),Papaya ringspot virus(PRSV),Squash leaf curl China virus(SLCCNV),Tobacco stripe virus(TSV),Watermelon mosaic virus(WMV),Watermelon silver mottle virus(WSMo V),WVA,ZTMV and Zucchini yellow mosaic virus(ZYMV).It was first found that BGMo V infected cucurbit crops in Chinese mainland,and TSV,WVA,ZTMVdistributed in Guangdong.3.The viruses and predominant viruses infecting 9 cucurbit crops in Guangdong were made clear.Using RT-PCR primers selected for 16 viruses,10 viruses were detected in 116 pumpkin diseased samples collected from various areas of Guangdong,and 4 viruses including PRSV(28.45%)、SLCCNV(21.55%)、 ZTMV(22.41%)and ZYMV(22.41%)were predominant viruses.Seven viruses were detected in 88 cucumber diseased samples,and MYSV(32.95%)was predominant virus.Eleven viruses were detected in 72 winter melon and winter gourd diseased samples,and WSMo V(48.61%)was predominant virus.Ten viruses were detected in 43 gourd diseased samples,and CGMMV(55.81%)and WVA(48.84%)were predominant viruses.Five viruses were detected in 13 melon diseased samples,and ZTMV(41.67%)and ZYMV(66.67%)were high.Four viruses were detected in 8 loofah diseased samples,and ZYMV(50 %)was high.Five viruses were detected in 7white melon diseased samples,CCYV(71.43%)and PRSV(71.43%)were high.ZTMV and ZYMV were detected in 8 bitter gourd diseased samples and only ZYMV was detected in 4 watermelon diseased samples.4.The complex infection of two or more viruses on cucurbit crops in Guangdong was common.Out of 357 suspected diseased samples of cucurbit crops collected from 13 cities including Foshan and Guangzhou etc,105 samples(29.42%)were negative and 256samples(70.58%)were positive by RT-PCR with specific primers for the 16 viruses.The samples in 256 diseased samples infected by one,two,three,four,and five viruses accounted for 33.61%,22.41%,8.96%,5.04%,and 0.56%,respectively,and the complex infection rate was as high as 52.38%.5.The complete genome sequences of three important viruses were cloned and sequenced.Through segmented RT-PCR amplification,the full-length genome nucleotide sequences of Guangdong isolate of CGMMV,WVA and ZTMVwere determined.The genome of CGMMV-GDLZ was 6423 nt(MK933286),containing 4 ORFs to encode replication-related protein,Rep,MP and CP.Phylogenetic analysis showed that CGMMV-GDLZ clustered with CGMMV isolate Shandong(KJ754195).The genome of WVA isolate HZHL was 8369 nt(MK292710.1)containing 4 ORFs to encode replication-associated polyprotein(RP),movement protein(MP),24.8k Da protein and coat protein(CP).The identity of WVA-HZHL and WVA isolate KF was 93.23%.The genome of ZTMV isolate XW was 10320 nt(MN267689),encoding ployprotein.The ployprotein was futher cleaved into P1,HC-Pro,P3,6K1,CI,6K2,VPg,NIa-Pro,NIb and CP.Phylogenetic analysis showed that ZTMV-XW clustered with France isolate(KC345608).6.CGMMV has pathogenicity to eight crops and the resistence levels of cucumber varieties existed significantly different.It was proved that CGMMV could infect 8 crops by agroinfiltration inoculation with CGMMV infectious clones,p CB301-CGMMV,including pumpkin,cucumber,winter melon,calabash,loofah,balsam pear,watermelon,and cabbage.The resistance levels of 10 cucumber varieties to CGMMV were evaluated using agroinfiltration inoculation with p CB301-CGMMV.The results showed that Jinyou 611 and Jinyou 409 cucumbers were resistance to CGMMV,Shantou cyan cucumber and Minile cucumber were susceptible to CGMMV,and the other 6 cucumber varieties were tolerant to CGMMV.