Survey of Apple mosaic virus in apple-growing provinces of East Anatolia (Malatya and Van) by RNA probe hybridization assay and RT-PCR

Abstract

Two main apple growing provinces (Van and Malatya) of East Anatolia were surveyed for the presence of the Apple mosaic virus (ApMV). Dot-blot hybridization and RT-PCR tests were implemented to investigate the incidence of ApMV, testing a total of 481 samples collected from commercial apple orchards. Digoxigenin-labeled RNA probes of the ApMV were synthesized from the cloned PCR products and applied in dot-blot hybridization to detect the virus in RNA extracts isolated from fresh leaf tissues. A validated RNA probe (dot-blot) hybridization method was adopted to investigate the presence of ApMV infections in the apple orchards of East Anatolia. In order to determine the most suitable mass extraction methods and to ascertain the presence of virus, 3 RNA preparation procedures (QIAGEN RNA extraction kit, silica capture, and citric buffer) were tested. Among the tested extraction methods, silica capture was determined as the most suitable extraction method for dot-blot hybridization assays. ApMV was found in the surveyed locations with an average incidence of 0.8%. The infected trees showed apparent disease symptoms on the leaves of apple trees. The coat protein (CP) genes of 2 viral isolates selected from Malatya (ApMV-G and ApMV-M) were cloned and their complete nucleotide sequences and deduced amino acids were determined (GenBank acc. nos. JX155668 and JX155669). The CP cistron of the ApMV-G and M isolates contained 224 amino acid residues. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor-joining and the unweighted pair-group mean arithmetic methods with 100 bootstrap replicates. The CP sequence of ApMV-G and ApMV-M varied among the 21 isolates, with overall identity ranging from 88% to 99% and ranging from 91% to 99% at the nucleotide level.