Identification Of Four Etiological Viruses Causing Grapevine Leafroll By RT-PCR In China

Grapevine leafroll disease is one of the most important virus diseases of grapevines.It occurs in all grape-growing regions in the world. To date, ten different filamentousviruses identified as Grapevine leafroll-associated viruses (GLRaVs) have been isolatedand characterized from leafroll infected grapevines. These ten serologically differentviruses are numbered from 1 to 10 (GLRaV-1 to GLRaV-10) based on their discoveryorder, which all belong to the family Closteroviridae. One or more GLRaVs can causegrapevine leafroll. In this study, we used RT-PCR to identify GLRaVs species,preliminary clear the infection condition of GLRaVs, and analysis the sequences of somegenes of GLRaV-1 and GLRaV-7 Chinese isolates. The purpose is to lay the theoreticalfoundation for GLRaVs detection and anti-virus breeding grapevine in China. The resultsof this study are as follows:1.57 grapevine samples with typical leafroll symptoms were collecting fromNational grapevine germplasm center at Zhengzhou Fruit Institute, Chinese Academy ofAgricultural Sciences. The samples were detected for GLRaV-1, GLRaV-2, GLRaV-3,and GLRaV-7 by RT-PCR. The results showed that a total of 48 samples were positive.The positive samples rate of four GLRaVs was 68.42ï¼…, 21.05ï¼…, 7ï¼…and 5.26ï¼…respectively. It is a relatively common phenomenon of mixed infection of GLRaVs, 11grapevine varieties were mixed infection with two to three GLRaVs. 21 samples werepositive of GLRaVs in 26 wine varieties, and 24 in 27 table varieties, the positive ratewas 80.77ï¼…and 87.10ï¼…respectively. 24 were positive of GLRaVs in 27 red -fruitedvarieties, and 25 in 30 white-fruited ciltivas, the positive rate was 88.89ï¼…and 83.33ï¼…respectively. The results showed that there was no significant difference of positivesamples among different types of ciltivas.2. IC-RT-PCR method for GLRaVs detection was established preliminary. Somepositive samples detected by RT-PCR were detected for GLRaVs by IC-RT-PCR. Theresults showed that both phloem and petiole of grapevine were infected of GLRaV-1,GLRaV-2, GLRaV-3 and GLRaV-7. The detection results used by RT-PCR andIC-RT-PCR were Consistent. It is a rapid and sensitive method for GLRaVs detection.3. Fragment of GLRaV-7 HSP70 gene was cloned from Semilong in NationalIindoor Conservation Center of Virus-free Germplasm of Fruit Crops. The fragment ofGLRaV-7 HSP70 gene is 590bp in size, which putative encodes 196 amino acids. Thesequence was registered in GenBank and the registered number is EF093187. This is thesecond GLRaV-7 nucleotide sequence on NCBI. There is a little difference betweennucleotide sequences of fragment of GLRaV-7 China isolates S1 HSP70 gene (EF093187)and fragment of another reported GLRaV-7 HSP70 gene (Y15987). The similarity of the two nucleotide sequence is 91ï¼…, and the similarity of amino acid sequence is 93ï¼…. Thespcical 293bp fragment of GLRaV-1 CPd2 gene was obtained From Cabernet Franc. Thenucleotide sequence was registered on GenBank as EF583823. The sequence similaritiesof EF583823 with other nucleotide sequences of reported GLRaV-1 isolates are less than88ï¼….