Study On Molecular Detection Techniques Of Grapevine Leafroll Disease Etiological Viruses In China

Grapevine leaf roll disease is one of the most important diseases of grapevines worldwide. At present, eleven serologically distinct viruses belonging to the family Closteroviridae have been found associated with grapevine leafroll disease (GLD). These eleven viruses are named GLRaV-1, -2, -3, -4, -5, -6, -7, -8, -9, -Pr and -De respectively, which can cause grapevine leaf roll disease alone or together.. In this paper, we used ELISA and RT-PCR to identify GLRaVs species, preliminary analyzed the infection condition of GLRaVs in China. And based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. The sequences of part genes of GLRaV-4 and GLRaV-5 Chinese isolates were analyzed. The results as follows:1. In order to identify the GLD Etiological Viruses in China, the dormant grapevine cane samples from 58 individual vines which had shown severe leafroll symptoms were collected from a germplasm collection pot at Research Institute of Pomology, Chinese Academy of Agricultural Sciences. The samples were tested by ELISA and RT-PCR. The results showed that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, -4, -5 and -7 are present, a total of 81.0% samples were infected and the rate of six GLRaVs was 20.7%, 17.2%, 62.1%, 3.4%, 5.2%, and 15.5% respectively. To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. It is a common phenomenon of mixed infection with two or three GLRaVs. The mixed infection rate was 39.6%.2. In order to improve detection efficiency and decrease cost, based on the detection of GLRaVs by single RT-PCR, a multiplex RT-PCR was applied to detect 4 grapevine leafroll-associated viruses, GLRaV-1, -3, -4 and -5. In this paper, we studied the mainly factors affecting RT-PCR reaction. The results showed that template concentration, primer concentration, Taq DNA polymerase concentration, annealing temperature and reaction cycles make more influences on the result in the system than that of extension time and dNTP concentration.3. The HSP70 and CP gene of GLRaV-4 were cloned from"Autumn Royal". The CP gene of GLRaV-5 were cloned from"Malaga Rose".Sequence analysis showed that the HSP70 gene consists of 442 nucleotides and encodes a polypeptide consisting of 146 amino acids. The registered number in GenBank is GQ849394. The sequence showed 99% identical nucleotides with the corresponding region of one GLRaV-4 isolate from Italy (GenBank Accession No. AF039553). There are two distinct nucleotides.The CP gene consists of 300 nucleotides and encodes a polypeptide consisting of 100 amino acids. The registered number in GenBank is GQ479041. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-4 isolate from Chile (GenBank Accession No. EU746621) showed 99â„… identity . There is one distinct nucleotide.The CP gene consists of 690 nucleotides and encodes a polypeptide consisting of 227 amino acids. The registered number in GenBank is GQ246625. A comparison of the CP gene sequences with the corresponding nucleotide sequences of another GLRaV-5 isolate from Argentina (GenBank Accession No. EU815935) showed 95â„… identity . There are 33 distinct nucleotides.