The Detection Of Main Apple Viruses And The Molecular Variability Analysis Of Apple Chlorotic Leaf Spot Virus In China

Apple virus diseases are widely distributed throughout the world, spreading fast and difficult to cure. Virus interfere the normal physiological function of the apple tree, affect the fruit quality and cause great economic lose.RT-PCR technique was used to detect four apple viruses, including Apple chlorotic leafspot virus （ACLSV）, Apple stem pitting virus （ASPV）, Apple stem grooving virus （ASGV） and Apple mosaic virus （ApMV） in this study.The main results are as follows:1. The RT-PCR method for test of four apple viruses （ApMV, ASPV, ASGV and ACLSV） in field was estebalished: At first, the best extraction method for apple total RNA extraction-improved RNA-extraction method was determined; The amplification effects were compared between published primers and self-designed primers. The optimal primers were selected; At last, the RT-PCR method was optimized. The method could detect the four viruses during the whole year, the detection sensitivity of which could reach as less as 2,000×dilution 5,000×dilution 10,000×dilution and 200×dilution of total RNA extracted from fresh sample for ASPV, ASGV, ACLSV and ApMV.2. The occurrence and the distribution of four apple viruses in China were made clear by detecting 327 samples collected from 13 provinces （municipalities/ autonomous regions）,: The four apple viruses occur widely in all the producing areas in China. The positive rate of ApMV, ASPV, ASGV and ACLSV was 80.1%, 65.1%, 73.7% and 69.7%. The mixed infection phenomenon of two or more kinds of viruses exists generally. The revalence of two apple viruses is 16.5%, the revalence of three apple viruses is 51.1% and the revalence of four apple viruses is 26.9%. The revalence of four apple viruses in China was the highest, which is followed by ApMV+ASGV+ACLSV, 16.8%, the revalence of ASPV+ACLSV was the lowest, 0.3%.3. An multiplex RT-PCR detection system to diagnose and detect the four viruses simultaneously was established: The different primer combination of the four viruses was test using the apple tissue cultures infected by the three latent viruses and apple tissues infected by ApMV as the material. Then the primers of ApMV and apple internal gene were added. On these bases, the multiplex RT-PCR detection method was optimized. The result showed that the combination of ASGVs-5’/ASGVs-3’, 2002ASPV-5’/2002ASPV-3’, ACLSV₄-5’/ACLSV₄-3’and EF-1α-5’/EF-1α-3’had the highest specificity in simultaneous detection of the four viruses. The sensitivity of the mehtod could reach as less as 2,000×, 1,000×, 1,000×and 100×dilutions of ASPV, ASGV , ACLSV and ApMV. At last, we verified the applicability of the method for field testing of the four viruses.4. Tne molecular variability of ACLSV in China was studied preliminary: The part coat protein （CP） sequence of 54 ACLSV isolates obtained from different region and different varieties were determined. The similarity and phylogenetic analysis were done between all the 54 isolates and 66 published isolates. The result showed that the isolates were separated into two major clusters. Cluster I contained 38 Chinese isolates and the‘B6 type’isolates. The variation site of 20 isolates was very distinctive, which were gathered in different sub-cluster distinguished from B6 type’. Cluster II contained 16 Chinese isolates classified one group to‘P205 type’. Phylogenetic analysis of all the isolates from different host, region and cultivar showed that there is certain correlation between the host type and molecular variation of ACLSV, but no correlation between the region and molecular variation of ACLSV and the correlation maybe exsit between host cultivar and the variation.