Virulence Identification Of PLMVd Isolates And Sequence Analysis Of Their Progenies

Peach latent mosaic viroid (PLMVd) is an important pathogen which can infect stone trees such as peach. It seriously affects the yield, fruit quality and growth. PLMVd can induce mosaic, chlorosis, and yellowing of peach. Previously, occurrence of PLMVd was confirmed in many peach orchards of China. Most of infected peach trees showed chlorosis along the veins and margin of leaves, and some showed yellowing of leaves. Previously, no one had demonstrated directly that these three kinds of symptoms were caused by PLMVd. In this study, PLMVd was isolated from peach, and the infectious RNA was constructed in vitro, and inoculated to GF305seedlings. The aim of this research was to establish a relationship between PLMVd and these three kinds of symptoms. The main results obtained are as follows:1. Five peach samples which showed chlorosis along the veins and leaf margin, and yellow mosaic in the field were selected. Dominant molecular variants (DXH-2-1, DXH-14-1, DXH-12-1, DXH-5-1and WH-5-3) from these five samples were used as materials. The dimerized cDNA of PLMVd was ligated into pGEM-18T vector, and the peach GF305seedlings were inoculated with dimeric RNA obtained through in vitro transcription. The viroid was detected by RT-PCR and symptoms were observed after inoculation. The results showed that the five isolates were inoculated successfully after15days post inoculation. After150d, the plants inoculated with DXH-14-1and DXH-12-1showed chlorosis along the leaf margin, the plants induced with DXH-5-1displayed slight yellowing, and the plants inoculated with isolate WH-5-3showed significant yellowing, which later changed into calico, all of the symptoms were similar to the field isolates, we can sure chlorosis along the leaf margin and yellowing were induced by PLMVd in China peach. The isolate DXH-2-1did not induce the symptoms in plants. we speculated that vein chlorosis symptoms were not induced by PLMVd2. We cloned the PLMVd progeny from inoculated GF305seedlings, and selected10clones from each isolate for sequencing. Results showed that progeny sequences shared97.9～98.8%,97.9～98.5%,97.6～98.8%,99.4～100%and97.6～99.7%identities with the DXH-2-1, DXH-14-1, DXH-12-1, DXH-5-1and WH-5-3original sequences, respectively. In the sequences obtained from isolates DXH-5-1and WH-5-3, two to four sequences were found to be same or highly similar (less than two mutations) with parental sequence, while for progenies of variants DXH-2-1, DXH-14-1and DXH-12-1, there existed four to seven mutations compared with the parental sequences. The results showed that all of the progeny sequences mutated after inoculation, which might provide a reasonable explanation for not all the plants inoculation with DXH-14-1and DXH-12-1showed the same symptoms as in the field. Further study could be done to explain the reasons for different degree of the descendants sequence variations of progeny sequences compared to the parental ones.Recombination was found in some progeny sequences (1-7). All the substitution sites located in the A loop of PLMVd secondary structure (203-208nt), six nucleotide sequences of the A loop were replaced with17nucleotide sequences, and formed a stable neck ring structure in original position. To eliminate the possibility of the variation and recombination caused by construction of in vitro infectious RNA, we selected isolate DXH-14-1and DXH-12-1to transcribe in vitro and analyze transcript sequences, the results showed that, after in vitro transcription and RT-PCR, the degree of variation in progeny sequence was much lower than the variation in vivo.3. To better analyze the PLMVd molecular variation, we constructed the monomer cDNA with cohesive ends of the PLMVd molecular variants (DXH-3-1), DXH-2-1, DXH-14-1, DXH-12-1, DXH-5-1and WH-5-3), then inoculated it onto the in vitro cultures of P. pyrifolia cv. Jinshui no.2. Sequence analysis of the progenies showed that there existe6(DXH-3-1),0(DXH-2-1),11(DXH-14-1).1(DXH-12-1),1(DXH-5-1) and19(WH-5-3) progeny sequences is same with parental sequence, and existed1(DXH-3-1),18(DXH-2-1),8(DXH-14-1),0(DXH-12-1),8(DXH-5-1) and0(WH-5-3) in the variant sequences shared high similarities with parental sequences (≥99%) with no more than three mutations. The progeny sequences of each isolates shared intra-isolate identities of97.9～100.0%,97.9～100.0%,96.9～100.0%,97.3～100.0%,98.6～100.0%,98.6～100.0%with DXH-3-1, DXH-2-1, DXH-14-1, DXH-12-1, DXH-5-1and WH-5-3, respectively. The mutation sites were mainly located in Loop A, the PSTⅠ arm and150th.