Mikrobiol. Z. 2017; 79(3):115-124.
Typing of Plum Pox Virus Isolates in the Central Ukraine
Kyrychenko A.1, Shcherbatenko І.1, Antipov I.2, Hrynchuk K.2
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
2National University of Life and Environmental Sciences of Ukraine
15 Heroiv Oborony Str., Kyiv, 03041, Ukraine
Aim. During the spring of 2016 in three locations of Central Ukraine – Cherkasy, Kyiv and Yahotyn, we found plum and pear trees displaying foliar symptoms similar to those causing by an important quarantine pathogen Plum pox virus (PPV), and cherry trees showing symptoms typical for Apple mosaic virus (ApMV) – one of the most widespread viruses which can infect over 65 plants species of Rosaceae family. The aim of this study was to perform detection, identifcation and molecular analysis of viruses causing diseases symptoms found by us on fruit trees in Central Ukraine. Меthods. Total RNA extracted from leaves of infected and healthy plants was used to generate cDNA with known PCR primers as well as with own primers designed by «Primer3» software. The amplifed DNAs of Cp gene fragments of ukrainian PPV isolates were sequenced by Sanger dideoxy sequencing method and were compared with those available in GeneBank. The comparison and molecular analysis of virus isolates were performed using BLAST, MultAline and MEGA 6 softwares, and a set of own a simple computer programs (utilities), tightly specialized for the solution of a narrow objectives of sequence analysis. Results. The primers have been developed for Plum pox virus detection proved to be effective for revealing and molecular diagnostic of PPV in three regions of the Central Ukraine. Using these primers three PPV-D isolates, named seq1, seq2, seq3, have been selected from pears in Cherkasy (seq1) and from plums in Yahotyn (seq2) and Kyiv (seq3). According to sequence comparison of 181-base Cp gene fragments, the plum isolates seq2 and seq3 turned out to be identical among themselves and have 95.6% sequence identity to pear isolate (seq1). Ukrainian isolates obtained have shown high identity (95 - 99.4 %) with all of 33 PPV-D isolates from different countries and host plants tested in our study. The 181-base sequences of these isolates contain 30 nucleotide replacements concerning seq1 sequence. The vast majority of replacements (27 of 30) are synonymous and do not cause of amino acid substitutions in the viral coat proteins. PPV-D isolates form 6 groups of members with identical nucleotide replacements, which remind Vavilov’s homologous series of heritable variation of morphological traits. Conclusion. The primers have been developed were successfully used for detection of PPV-D isolates from pear and plum trees in three regions of Central Ukraine. It is the frst time the PPV has been reported in Cherkasy.Our studies provide information about spreading PPV isolates, that can be valuable in understanding the epidemiological role of this virus. Groups of identical nucleotide replacements, revealed by us in Cp gene sequence fragments of different PPV isolates, remind Vavilov’s homologous series of heritable morphological traits, and are of interest for clarifcation of the molecular basis of hereditary variation and parallel mutations.
Key words: Plum pox virus, sharka, ukrainian isolates of Plum pox virus, groups of virus isolates with identical nucleotide replacements.
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