Identification And Detection Of Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus (CGMMV) is an important seed-borne virus, which is responsible for devastating losses of cucurbitaceous crops. The virus is one of the potential hazardous pests in China. At present, it has been intercepted and captured frequently by port plant quarantine department, and it has been found at several regions recently. It's long distance spread is transmitted through international trade or domestic trade of seeds. Therefore, it is impendency to develop detection progresses of CGMMV. In this study, methods of identification and molecular detection of CGMMV were developed. These methods can help to intercept and capture the virus more efficiently. Major results are described as follows:(1) CGMMV was detected by mechanical inoculation, transmission electron microscopy, double antibody sandwich ELISA ( DAS-ELISA ) and reverse transcription polymerase chain reaction (RT-PCR) in a cucumber sample which was collected from a greenhouse in Nanning city, Guangxi province.(2) Immuno-capture RT-PCR ( IC-RT-PCR ) method was established by optimizing the RT-PCR reaction system and amplification procedures. A coat protein(CP) encoding gene was amplified by IC-RT-PCR, and the PCR product was cloned into pMD18-T vector. Sequences analysis showed that CP gene of this isolate had 486 nucleotides, encoding 161 amino acids with theoretical molecular weights of 17．3 kDa. Sequence alignment showed it shared 91．4 %～99．4 % nucleotide acid identities and 98．8 %～100 % amino acid identities with the published sequences, respectively.(3) Six pairs of primers were designed to amplify the coding region of CGMMV which were isolated from Lagenaria siceraria leaves with inoculated by single ring spot on Chenopodium amaranticolor. Six nucleotide sequence fragments were cloned, sequenced and then reconstructed. Sequence analysis showed that this isolate had 6188 nucleotides in length, encoding 1144 amino acids. Comparison showed it shared 60．1 %～98．2 % nucleotide acid identities and 58．4 %～99．0 % amino acid identities with the published sequences, respectively.(4) The IC-RT-PCR was applied directly to detect the virus infested seeds. The results showed that IC-RT-PCR could directly detect CGMMV in infected seeds, which not only simplifies the RNA extraction, but also decline the demand of samples. IC-RT-PCR is specific, simple and convenient, and could be used as a rapid and efficient detection method for CGMMV diagnosis. The IC-RT-PCR is a hopeful method in practical application.