Molecular Variability And Genome Study Of Latent Viruses In Shaanxi

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV) and Applestem grooving virus (ASGV) are three most econcmically important viruses infecting appletrees worldwide. In our research, the occurrence of the three latent viruses in apple–producingareas in Shaanxi, China and their molecular variability were analyszed and reported for thefirst time. Also, we reported a ASGV and a ASPV genome sequence, respectively.A total of550apple leaf samples from12different major apple–producing areas inShaanxi, China, were tested for infection by DAS–ELISA and multiplex RT–PCR. The resultsshowed a wide occurrence of three latent viruses in the12major apple–producing areas. Norelationship was found between the occurrence rate and the apple–producing areas. As to thesingle–virus infection, analysis revealed an ASGV infection level of67.6%, followed byACLSV infection level of58.5%, higher than that of ASPV (44.0%). In addition, the mixedinfection was analysed and the most common co–infection was ACLSV, ASPV with ASGV(17.8%). Among apple samples of different tree age, the virus occurrence was positivelycorrelated with apple tree age.Sequences of504nt corresponding to87%of the CP gene of31isolates were acquiredby RT–PCR and deposited in GenBank with the accession numbers KJ544862–KJ544880，KF134387–KF134398. Comparisons of the partical CP gene sequences of these31isolatesrevealed the pairwise identities ranging from83–99%and86–100%at the nucleotide andamino acid level, respectively. Phylogenetic analysis based on these nucleotide sequencesshowed that the91isolates deposited in GenBank fell into three groups (Group1–P205,Group2–B6and Group3–Ta Tao5). Our31ACLSV isolates were separated into the P205and B6groups, respectively. Multiple alignment analysis of the amino acid sequences of CPrevealed that there was a combination of six amino acids at positions40,59,75,86,130and184in isolates from each group that could be used to distinguish among the three groups. Tworecombination events were identified from all isolates by recombination analysis, and threeACLSV isolates collected in this study participated in these two events. Our results show thatmolecular variation was present and obvious in isolates of ACLSV collected in Shaanxiprovince.In our research, the complete nucleotide sequence of an ASPV isolate (ASPV–YL)collected from apple in Shaanxi, China, was determined. The genome was found to contain five open reading frames (ORFs). Sequence analysis revealed that ASPV–YL had a highestidentity (79.2%) with an apple isolate from Germany (D21829), and a lowest identity (71.9%)with a pear isolate from China (EU095327). Phylogenetic analysis of the genome and coatprotein genes revealed that ASPV isolates had high variables and they were separated intothree groups regardless of the geographic origin, maybe related to hosts. Our isolate was inGroup1. Multiple alignment analysis of the amino acid sequences of CP revealed that therewas a variable region (1–198aa) and a conserved region (199–405) in the CP gene. Selectivepressure analysis revealed that the ASPV population was under negative selection.In addition, a complete genomic sequence (6,495nt) of a Chinese ASGV isolate fromapple (ASGV–CHN) and complete coat protein gene sequences of14other ASGV isolatesfrom Shaanxi, China were amplified, cloned and sequenced. The genomic sequence of theASGV–CHN isolate was found to share a highest identity (92.5%) with a pear isolate fromChina and a lowest identity (80.5%) with a pear isolate from South Korea. Phylogeneticanalysis revealed that these15CP gene nucleotide sequences were separated into two groupsregardless of the geographic origin. To our knowledge, this is the first report of molecularvariability analysis of ASGV in apple trees in China.