Genetic Variability Analysis And Molecular Detection Of Grapevine Fanleaf Virus

Grapevine fanleaf virus(GFLV) is responsible for fanleaf degeneration, which is one of the most severe virus diseases of grapevines worldwide. GFLV causes substantial crop losses, reduces fruit quality and shortens the longevity of grapevines in the vineyard. Study on about grapevine fanleaf disease was late in China, and the complete genome sequence of GFLV has not been gotten since it was found in Xingjiang in 1980. In order to establish a stable, reliable, fast and sensitive molecular detection methods for GFLV, and investigate the distribution of GFLV in China as well as the characteristics of molecular variation. In this study, we explored the nested RT-PCR, and real time quantitative RT-PCR detection for GFLV, and nested RT-PCR were used to detect GFLV in grapevine samples collected from 19 regions of China. Movement protein and coat protein sequences were cloned and sequenced, and the gene variations were further analyzed. The mainly results are as follows:This study established a nested RT-PCR method for GFLV detection; it is based on the FLMP1, FLMPn1 and FLCP1, FLCPn1 as primers. The detection results of the method are accurate and reliable, and the FLMPn1 detection results are better than FLCPn1. The real time quantitative RT-PCR method of FL-HP1 for GFLV was established by using primer selection and system optimization. Sensitivity of real-time quantitative RT-PCR method was at least 10 times higher than that with nested RT-PCR. Real-time RT-PCR method was used to determine the content of GFLV in different grape plants and to detect 58 grape samples.There was noticeable differences among the content of GFLV in field samples,and real-time fluorescent quantitative RT-PCR method can detect more GFLV isolates than DAS-ELASA and RT-nPCR.In this study, GFLV in grapevine samples from 19 provinces and regions of China were detected by DAS-ELISA, RT-PCR, and nested RT-PCR. Among them, 376 samples for nested RT-PCR detection, 279 samples(belonging to 376 samples) for ELISA test. Of the samples, 11.4% tested positive by RT-PCR and nested RT-PCR, and 25.8% tested positive for GFLV by DAS-ELISA.Theseresultsshowed that 81 of 376 samples(21.5%) tested positive for GFLV using one or more of the above detection methods.We found that GFLV was distributed widely in ten major grapevine-growing areas in China, the detection rate of 6.1%-54.2%. GFLV was present in 32 of the 102 cultivars(31.4%), including cultivars thatCabernet Franc(22.2%), Cabernet Gernischt(15.8%), Italian Riesling(12.5%), Red globe(12.5%), Muscat Kyoho(11.1%), Cabernet Sauvignon(10.0%).Movement protein(MP) and coat protein(CP) gene were cloned and sequenced. The sequence data showed conservation of the 2BMP and 2CCP gene sequences among the GFLV isolates from distinct geographical regions of China, but the China isolates appeared to be distinct from the previously published GFLV isolates. We aligned sequences from our isolates and GFLV isolates downloaded from GenBank and found that the 2BMP nucleotide and amino acid sequences shared 78.3–86.6% and 89.3–96.4% identities, respectively, and the 2CCP nucleotide and amino acid shared 74.8–83.3% and 81.4–91.4% identities, respectively. The phylogenetic analysis showed a distinct evolutionary relationship between the Chinese and Italian, France, and United States populations. The sequenced GFLV isolates from China were monophyletic, and the evolution was not affected by other geographical origins.The complete sequences of RNA1 and RNA2 have been determined for a China isolate of GFLV(GFLV-SDHN). The two RNAs are, respectively, 7 367 and 3 788 nucleotides in length, excluding the poly(A) tails. The RNA1 of GFLV-SDHN has the longest genome reported to date. The 5’-UTR of GFLV-SDHN RNA1 is 22–24nt longer compared to other full-length GFLV isolates. The phylogenetic tree based on full-length RNA1 sequences showed that GFLV-SDHN and GDefV grouped together, but was distinct from the all full-length GFLV and ArMV isolates. A phylogenetic tree based on the full-length RNA2 sequences revealed that GFLV-SDHN wasdistinct from other GFLV and GDefV isolates. One recombination event in the 2AHP of RNA2 were found(nts 342-632), with GFLV isolates Shir-Amin and Takestan as putative parents. The results shown that the GFLV-SDHN isolate have distinct originswith other full-length GFLV isolates and were is a new variant of grapevine fanleaf virus. The complete sequences of RNA1 and RNA2 have been deposited in the GenBank database with the accession numbers KU522584 and KU522585.