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Studies on the Molecular Identification and Detection Methods of Cucumber Green Mottle Mosaic Virus (CGMMV)

Author: LiHaiMing
Tutor: ChenQiJian；WuZuJian
School: Fujian Agriculture and Forestry University
Course: Plant Pathology
Keywords: Cucumber green mottle mosaic virus RT-PCR DAS-ELISA NASH Detection and identification
CLC: S432.41
Type: Master's thesis
Year: 2009
Downloads: 93
Quote: 2
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Abstract


Cucumber green mottle mosaic virus (CGMMV) is a quarantine virus that is harmful to Cucurbitaceae plants, such as watermelon, muskmelon and cucumber et al..It severely threatens agriculture in many countries and regions over the world. In this study mechanical inoculation test was applied using cucumber leaves infected by CGMMV and reverse transcription polymerase chain reaction (RT-PCR) was applied to detect and identify. The 6188bp coding region of the virus were successfully amplified by RT-PCR,and the sequence homologous was analysed. The detection sensitivity of DAS-ELISA, RT-PCR and NASH (Nucleic acid spot hybridization) were compared.The main results are described as follows:1. A pair of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein (CP) gene of CGMMV and comfirmed that the cucumber leaves were CGMMV. Typical systemic symptoms were developed on those hosts of cucumber, watermelon, muskmelon, pumpkin and bottle gourd after inoculated with CGMMV, and local symptoms on Chenopodium amaranitcolor, No symptom on Nicotiana tabacum var. K326 and N. glutinosar.2. Coding region sequences of the isolate CGMMV-GX had over 96% similarity to the CGMMV isolates from different countries and regions deposited in GenBank. And the 97.3% similarity with CGMMV-W in Japan was the highest.The CP gene sequences were compared with the sequences of the homologous genes of other isolates of CGMMV.The result showed that the highest homology could reach 100% at the nucleic acid level with CGMMV-GX-G (DQ647384). The differences between CGMMV-GX、CGMMV-GX-G with CGMMV-France , CGMMV-W-Japan were lied in 3 base sites of nucleic acid only three differeent nucleic acid bases could be detected between CGMMV-GX, CGMMV-GX-G, CGMMV-France and CGMMV-W-Japan. Phylogenic trees were constructed based on the sequenses of the entire coding regions and CP gene of CGMMV isolates from different regions of the world. Certain relation between different CGMMV isolates and geological distribution was found.3. The results of comparison of detecting sensitivity showed: RT-PCR was more sensitive than DIG-labeled NASH; DAS-ELISA was lowest sensitive.4. It was found NASH was suitable to be applied in CGMMV detection after evaluating the three detecting methods.Considering the fact that the quarantine detection standard has not been unveil.This study explored the possibility of using the combination of DIG-Label NASH and differential host test to detect CGMMV.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful > Virus
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