The Identification Of Pathogens Causing Apple Virus Disease In Shandong And Shaanxi Province And The Establishment Of Multiplex RT-PCR Detection System

Apple is major fruit and one of the advantages of agricultural products in China at present. The apple production areas involve in25provinces. Cultivated area and production ranks first in the world. Apple virus disease widely distribute in China, most viruses cause chronic damage which leads to weak tree, decline in yiled, lower quality, and become an important obstacle of apple production.In this study,54and50samples of apple leaves were collected in April and July in2012respectively from Shandong and Shaanxi provinces. The specific detection primers from screening literatures for five apple viral pathogens, Apple Mosaic virus (ApMV), Apple scar skin viroid (ASSVd), Apple green crinkle virus (AGrCV). Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), were used to test the partial randomly selected samples from Shandong and Shaanxi. The results showed that, except the ApMV, the rest four positive bands were detected by RT-PCR. In order to correctly determine the types of the virus, the clones were sequenced and analyzed. According to the test results of a single RT-PCR and sequencing, the primers of4kinds of viral pathogen were designed, combining with the sequences of related viruses which had been reported in the GenBank. After the system and condition of amplification were optimized, a multiplex RT-PCR system for simultaneous detection of ASSVd. ASPV, ASGV and ACLSV was finally established. Specificity and sensitivity testing were performed for the multiplex RT-PCR system. The results showed the specificity of multiplex RT-PCR amplification was strong, and it could simultaneously detect the four viral pathogens at the minimum detection limit level of10-2ng. With the established multiple RT-PCR detection, a total of100apple leaf samples from Shandong and Shaanxi were detected. the tests showed the four viral pathogens were detected in samples from Qingdao, Yantai, Weinan and Xianyang. The dominant virus in Shandong was ASPV and the detection rate was88.9%and84.4%respectively in samples of Qingdao and Yantai. The dominant virus in Shaanxi are ASGV and ASPV. The detection rate of ASGV is81.0%and79.3%respectively in Weinan and Xianyang. The detection rate of ASPV is81.0%and79.3%, respectively. In these four districts, the detection rate of ACLSV is lowest among the four viral pathogens. The mixed infection of viruses is serious, and the rate of mixed infection in the samples from Qingdao, Yantai, Weinan and Xianyang, is66.7%,75%,85.7%and89.7%respectively. The The largest proportion of mixed infection type in Qingdao, Yantai, Weinan and Xianyang is ACLSV+ASPV+ASSVd, ASGV+ASPV, ASGV+ASPV+ASSVd and ASGV+ASPV+ASSVd respectively.In order to study the variation of ACLSV, the full genome of ACLSV was amplified after being divided into6fragments which is2435bp,1435bp,861bp,1605bp,1095bp and749bp respectively and was affirmed after sequence analysis and was spliced by DNAStar SeqMan. The full genome of ACLSV is7557bp (GenBank no. KJ522693) and contains3ORFs coding RNA polymerase, movement protein and coat protein respectively.5’UTR and3’UTR contain a151nt and195nt non-coding region respectively. By phylogenetic tree analysis, the full genome of ACLSV has85.9%nucleotide similarity with the apple isolate B6from Japan (GenBank no. AB326224).The prokaryotic expression of coat protain (CP) gene of ASGV was studied. The clone recombinant vector of ASGV CP and expression vector pET-28a(+) were digested by restriction enzyme BamH I and Xho I in37℃overnight. After purifying and recycling the enzyme-digested products, T4DNA ligase was then added to the products in4℃overnight. The products were transformed into the cells of E.coli DH5a to breed. After bacterium solution was digested and detected by PCR, the positive bacteria liquid was sent for sequencing. The sequencing result showed that the target fragment of ASGV CP was coloned to the expression vector pET-28a(+) correctly, which indicated that the construction of prokaryotic expression vector was succeed. The prokaryotic expression vector plasmid pET-28a-ASGV-CP was extracted and transformed into the expression bacterium cell of BL21(DE3) pLysS. Culturing the bacterial solution in37℃till the OD value is0.6-0.8,IPTG was added till the final concentration is1mmol/L, then the bacterial solution was cultured in27℃for3.5h. With the bacterium centrifugation and suspension,2×Loading buffer was added and then boiled for10min. SDS-PAGE analysis after cooling, and electrophoresis displayed the goal protein bands in33kDa, which showed that the fusion protein in BL21(DE3) pLysS was expressed successfully. Recycling the pure fusion protein with the tag protein purification kit, which establishes the foundation for the next step of antiserum preparation.