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


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Korkmaz G., Sıpahıoglu H. M. , Usta M.

Turkish Journal of Agriculture and Forestry, vol.37, no.6, pp.711-718, 2013 (Peer-Reviewed Journal) identifier identifier

  • Publication Type: Article / Article
  • Volume: 37 Issue: 6
  • Publication Date: 2013
  • Doi Number: 10.3906/tar-1212-5
  • Journal Name: Turkish Journal of Agriculture and Forestry
  • Journal Indexes: Science Citation Index Expanded
  • Page Numbers: pp.711-718

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%. Te infected trees showed apparent disease symptoms on the leaves of apple trees. Te
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). Te 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 neighborjoining and the unweighted pair-group mean arithmetic methods with 100 bootstrap replicates. Te 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.