The Molecular Diversities Analysis Of Melon Chlorotic Yellows Pathogen And Investigation Of The Disease Occurrence Rule

Melon chlorotic yellows is a new emerging disease caused by Cucurbit chlorotic yellows virus (CCYV), which is a member of the genus Crinivirus of Closteroviridae family. This virus is transmitted by Bemisia tabaci semi-persistently and can’t be inoculated by mechanical way or other manners. This virus infects watermelon, melon, cucumber in nature. When the plants are infected by this virus, initial chlorosis appears at the base and middle portions of the older leaves, and the whole plants become yellows in the end. The disease caused serious hazards for melon crops in autumn.CCYV was first reported in Japan in2004and discovered in Shanghai and Ningbo in China in2007. Now, this disease distributes in Hainan, Guangxi, Taiwan, Zhejiang, Shanghai, Jiangsu, Henan, Shangdong and Hebei in China. Only very few studies on CCYV were documented up to now. Cloning and analyzing the full genome of the virus, investigating the occurrence pattern of the disease can help us to ascertain the molecular characteristics of the pathogen, analyze the pathogenic mechanism, do the antiviral research by modern genetic engineering means and understand the occurrence rule of CCYV in China.To reveal the molecular characteristics, ten pairs of primers were designed based on the CCYV genome sequence. The RNA extracted from samples was used as template for Reverse Transcription Polymerase Chain Reaction (RT-PCR) to amplify the sequences of the whole genome. The whole genome sequences of Henan and Hainan isolates were alignmented with the published sequences in GenBank. The results showed that the genome of this virus is composed by two segments, RNA1and RNA2. RNA1is8607nt in length while RNA2is8041nt. RNA1shared99.85%nucleotide acid identities with that of all the published sequences and RNA2shares99.90%nucleotide acid identities with that of all the published sequences. To further analyze the molecular diversities of the Coat Protein (cp) gene, the specific primers of cp gene were designed to amplify the nucleotide sequences of cp genes of13isolates from Shanghai, Ningbo, Sanya and Zhengzhou. After the sequences were compared with the published sequences in GenBank, the results showed that the nucleotide sequences of cp gene of all the isolates have very high identities reaching up to99.75%. There was only14-site varied in all the isolates. So the nucleotide sequences of cp gene did not have molecular diversities. All those results indicated that no remarkable diversity was found among the CCYV isolates and suggested that all CCYV might share the same origin.To reveal the occurrence rule of CCYV, the greenhouse melons in autumn were investigated in Ningbo, including the occurrence of CCYV, Bemisia tabaci, the variation of fruit character and the level of susceptibilities to CCYV during the whole growing season. The results showed that the occurrence of Bemisia tabaci had two peaks during the whole melon growing season. The first peak was one week after fertilization and the second peak was two weeks before mature. The symptom of CCYV began to appear after the first peak and the rise rate of the disease was fastest after the second peak. The appearance, weight, shape and taste of fruits from infected plants were changed. Different cultivar showed varied incidences of the disease. The incidence of the disease of09A-71was lowest. To reveal the biotype of Bemisia tabaci transmitting CCYV in China, the DNA extracted from Bemisia tabaci collected from Zhengzhou and Ningbo was used as template for PCR with the specific primers of biotype Q and B Bemisia tabaci. The results revealed that they were biotype Q in these two areas. To ascertain the Bemisia tabaci carrying virus or not, the RNA extracted from Bemisia tabaci was used as template for RT-PCR with the cp gene specific primer. And result showed that three whiteflies as one sample were sufficient to be detected for carrying virus.