Sequencing Of Cucumber Green Mottle Mosaic Virus And Detection Of Disease Using The Prepared Antiserum

Cucumber green mottle mosaic virus (CGMMV) causes the serious loss in Cucurbitaceae plants. At present, CGMMV had been reported in a number of provinces in China.Here, To reveal the molecular character of CGMMV occurred in Zhejiang province, three CGMMV isolates from melon, watermelon and bottle gourd samples showing mottle, mosaic and distort symptoms on leaf were identified. The complete genomic RNA sequences of the three CGMMV isolates were determined by assemble two fragments amplified from these samples by RT-PCR which overlapped the whole CGMMV genome. All of the three CGMMV isolates were consisted of 6423 nucleotides with the typical character genome of genus Tobamovirus. Multiple sequences alignment comparison showed that the complete nucleotide identities between the three CGMMV isolates were 99.11-99.67%, while the coat protein amino acid sequences were absolutely identical. Phylogenetic tree generated from all CGMMV complete nucleotide sequences available suggested that CGMMV could be separated into two subgroups. The three Zhejiang isolates determined in this work belonged to subgroup I, and the genetic relationship of these Zhejiang isolates was close to that of the Korean CGMMV isolates.To reveal the occurrence and distribution of CGMMV in Zhejiang and Shanghai, the coat protein of CGMMV was used for preparation of antiserum against the virus. Using the antiserum, CGMMV on the cucurbit samples in the fields was investigated. Results showed CGMMV infection on cucurbit crops was common in the fields in Zhejiang province and Shanghai city. In Zhejiang Province, CGMMV was detected in Jiashan, Haining, Shaoxing, Lin’an, Cixi regions and the detection rate above 87%. In Shanghai, CGMMV was detected in Fengxian, Jinshan, Qingpu, Jiading area.We also detected the feasibility of amiRNA on controling CGMMV. An amiRNA named amiR-CGMMV targeting CGMMV was constructed, and was then transferred into Agrobacterium tumefaciens. Subsequently, the amiR-CGMMV solution was been infiltrated into N. benthamiana using Agrobacterium transient expression system, followed by the inoculation of CGMMV at twelve hours later. Northern blot showed that the amount of virus accumulation in the experimental group was significantly lower than that of the control group at three days past inoculation, which sugguests that the amiR-CGMMV could inhibit the infection of CGMMV.