Identification and sequence analysis of major viroids of grapevine in Shandong

Abstract:【Objective】Grape (Vitis vinifera L.) is one of the most susceptible fruit trees that may be infected by various phytopathogens. The wide distribution of these phytopathogens and serious damage on grape has aroused wide public concern. Viroid infection on grape results in yellow leaves, plant dwarfism, dysplasia, and even plant death. Viroid disease occurred in Xinjiang, Ningxia, Gansu, Liaoning and other grape growing areas, and the incidence rate could be as high as 100% in some cases. Shandong province is one of the major production region of fresh grape in China. In recent years, the viroid transmission increased due to the continuous expansion of planting area and the introduction of grapevine from other production regions. In this research, 149 grape leaf samples from the major production region in Shandong province were collected and detected by RT-PCR to identify the viroids.【Methods】About 0.1 g of each grape leaf sample was weighed and placed into 2 mL centrifuge tube; the sterilized steel balls and liquid nitrogen were added and the tissues were ground quickly in the grinder until the sample was pulverized. The total RNA was extracted according to the instructions to the kit. Then c DNA of each sample is synthesized by using Hi Script®II 1 st Stand c DNA Synthesis Kit. The symptomatic samples were detected using RT-PCR and the specific primers GYSVd1, GYSVd2, AGVd, HSVd and CEVd. The amplification system was as follows: 2×Es Taq Master Mix 15 µL, upstream and down-stream primers 0.5 μ L respectively, c DNA 1.0 µL, making up the total volume to 30 mL by dd H2 O.PCR amplification products were detected by 1% agarose gel electrophoresis and the indicated fragments were purified by reference to the kit. The purified target fragments were constructed into cloning vector p MD18-T. The recombinant vector was transformed into E. coli competent cells DH5α for DNA sequencing. The sequences were compared in Gen Bank. We downloaded the sequence of viroid isolates from NCBI and constructed the phylogenetic tree by using MEGA 5 Neighbor-Joining (NJ) . The tree of the branch confidence (Bootstrap) was analyzed repeatedly for 1 000 times.【Results】The specific bands of about 367 bp, 374 bp, 375 bp and 306 bp were amplified by primers of GYSVd1, GYSVd2, AGVd and HSVd, respectively. No specific band was amplified by CEVd primers. Compared with the reported isolates in Gen Bank, the coincidence of GYSVd1, GYSVd2, AGVd and HSVd was above 90%according to DNA sequencing and BLAST. None of the five viroids was detected in grape samples from Pingdu, Laiyang and Linshu; more than four viroids were detected in the samples collected from other sampling sites and complex infection of these viroids occured in most cases. In order to clarify the phylogenetic relationship of four kinds of viroids in the major producing regions of Shandong, we used GYSVd1, GYSVd2, AGVd, HSVd isolates from Qingdao, Yantai, Rizhao, Linyi and the isolates of different regions from Gen Bank to construct a phylogenetic tree. The results revealed that the GYSVd1 isolates of Shandong were mostly similar to the isolates of Brazil, South Korea, and the United States;the GYSVd2 isolates of Shandong were mostly similar to those of Beijing and Iran; AGVd isolates of Shandong were mostly similar to those of Iran, Chile and the United States; HSVd isolates of Shandong were mostly similar to those of Japanese and American.【Conclusion】Grapes in Shandong province were infected by GYSVd1, GYSVd2, AGVd and HSVd and the complex infection normally occured under natural conditions. GYSVd2 isolate was firstly reported in Shandong.