Study On Genetic Diversity And Long-Distance Movement Determinants Of Watermelon Mosaic Virus

WMV(Watermelon mosaic virus,genus Potyvirus,Family Potyviridae),transmitted by aphids in a non-persistent manner,is one of the most important viruses infecting Cucurbitaceae,Aceraceae and Leguminosae crops.It caused huge yield and quality losses on crops.Here,we prepared WMV CP antiserum,and characterized the biological and serological properties of two WMV isolates including ailanthus isolate CN:AIL-TA:17 and zucchini isolate CN:ZUC-JN:17,and analyzed the genetic diversity of those two WMV isolates,and obtained the WMV full-length infectious clone based on CN:ZUC-JN:17,and screened the resistance of 81 varieties to WMV,and analyzed the role of WMV P1 in determining the host range.The main results are shown as below:(1)WMV CP antiserum with high titer and specificity was obtained.WMV CP was expressed in Escherichia coli,and used to immune rabbit to produce antiserum.The WMV CP antiserum can specifically recognize WMV CP,but neither cross reaction to closely related Papaya ringspot virus(PRSV),Potato virus Y(PVY),Zucchini yellow mosaic virus(ZYMV)from the genus Potyvirus,nor to Cucumber green mottle mosaic virus(CGMMV)from the genus Tobamovirus.The titer of WMV CP antiserum determined by ELISA was 1:8192.(2)The biological propertities of two WMV isolates including zucchini isolate CN:ZUC-JN:17 and ailanthus isolate CN:AIL-TA:17 were analyzed.Western blotting analysis revealed that the two WMV isolates from different hosts were serologically related.CN:ZUC-JN:17 and CN:AIL-TA:17 isolates were mechanically inoculated into Nicotiana benthamiana and zucchini.Results showed that CN:AIL-TA:17 can infect N.benthamiana plants,but not the zucchini plants,in contrast,CN:ZUC-JN:17 can systematically infect zucchini plants,but not the N.benthamiana plants.CN:ZUC-JN:17 can only infect the inoculated leaves of N.benthamiana plants.(3)The genetic diversity of CN:ZUC-JN:17 and CN:AIL-TA:17 based on complete genome sequences was analyzed.The complete genome sequences of CN:ZUCJN:17 and CN:AIL-TA:17 were obtained by RT-PCR and 5' RACE(Rapid amplification of cDNA ends).Sequence analysis revealed that the complete genome of CN:ZUC-JN:17 and CN:AIL-TA:17 was 10028 and 10045 nucleotides(nt),respectively.CN:ZUC-JN:17 had a big open reading frame of 9645 nt,encoding a polyprotein of 3215 amino acids;CN:AILTA:17 has a big open reading frame of 9657 nt,encoding a polyprotein of 3219 amino acids.Comparison with the WMV complete genome sequences obtained from NCBI database showed that CN:ZUC-JN:17 and CN:AIL-TA:17 had the highest sequence identity of 93.9% and 89.7% at nucleotide level with isolate CN:SQU-TA:16 and CN:AIL-BJ,respectively.Phylogenetic analysis based on WMV complete genome sequences indicated that isolate CN:ZUC-JN:17 was clustered into groups III;CN:AIL-TA:17 and CN:AIL-BJ were clustered into groups VI.Recombination events were detected in CN:ZUC-JN:17,which is a daughter of CN:WMV-WS:16,CN:WMV-CHN:05 and FR:FBR04-37:04.No recombination was detected in isolate CN:AIL-TA:17.Sequence similarity analysis revealed that P1-encoding region is the most variable area between CN:AIL-TA:17 and CN:ZUC-JN:17.(4)WMV infectious clone pCamWMV and pCamWMV-GFP was obtained.The WMV full-length infectious clone based on isolate CN:ZUC-JN:17 was constructed.It can infect all the agro-infiltrated zucchini,melon and watermelon plants.GFP was inserted between NIb and CP in the infectious clone pCamWMV to obtain pCamWMV-GFP.(5)Resistance of 81 varieties to WMV were determined.To know the resistane of the main Cucurbitaceae crops growed in Shandong province,pCamWMV-GFP was agroinfiltrated on 2-3 weeks seedings of different Cucurbitaceae crops.Results showed that among the identified 14 zucchini varieties,two varieties of Wansheng Fengbao and Shengfeng Jinzhu were resistant to WMV,and the remaining varieties were susceptible or highly susceptible to WMV;among the 13 watermelon varieties,two varieties of lvbao Xinxiu and Langchao Yihao were medium-resistant to WMV,and the rest ones were susceptible or highly susceptible to WMV;among the 24 melon varieties,only one variety of Mianpi Huanggua was resistant to WMV,and the rest ones were susceptible or highly susceptible to WMV;among the 16 cucumber varieties,Xingjun Beibei is medium-resistant to WMV,and the rest ones were highly resistant to WMV;among the nine pumpkin varieties,Aiwei 80 Nangua was high resistant to WMV,and the rest ones were resistant or medium resistant to WMV;among the five Bottle gourd varieties,Zhepu Liuhao and Hu Zigua were susceptible to WMV,and the rest three varieties are highly susceptible to WMV.(6)Analysis of WMV P1 determines long-distance movement in N.benthamiana.To get natural mutant of WMV,the infectious clone pCamWMV-GFP was inoculated into N.benthamiana,subsequently,the inoculated leaves were used as inoculum to infect the healthy N.benthamiana plants.This process was repeated until natural mutants which can infect N.benthamiana systemically were observed.Comparison between the natural mutants and pCamWMV-GFP revealed mutations in P1-encoding region.Replacing P1-encoding sequencing of pCamWMV-GFP by that of CN:AIL-TA:17 to produce WMV chimeras pCaZUC-JN-AIL-TA-P1.Results showed that pCaZUC-JN-AIL-TA-P1 can infect N.benthamiana systemically.These results indicate confirmed the essential role of P1-encoding sequence in determing the long-distance movement of WMV.