PHYLOGENETIC ANALYSIS OF CYMBIDIUM MOSAIC AND ODONTOGLOSSUM RINGSPOT VIRUSES ISOLATED FROM PHALAENOPSIS SP

Cymbidium mosaic virus and Odontoglossum ringspot virus are the most common and widespread viruses in ornamental orchids. Infections caused by these viruses can lead to a decrease in the phenotypic diversity of orchid collections, and as a result – to their complete depletion. The aim of the study is to determine the origin of Ukrainian isolates of orchid viruses in the collection of protected soil from O.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv. The properties of nucleotide and amino acid sequences of the coat proteins (CP) of Cymbidium mosaic virus (CymMV) and of Odontoglossum ringspot virus (ORSV) were investigated. RNAs of CymMV and ORSV were isolated from leaves of Phalaenopsis sp. collected from A.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv, amplified through RT-PCR and sequenced. Obtained sequences were compared at nucleotide and amino acid levels with CymMV and ORSV isolates available in the GenBank. ORSV isolated in Ukraine shared 96-99 % and 93,4-98 % CP similarity to other known ORSV isolates at nucleotide and amino acid levels, respectively. CymMV isolated in Ukraine revealed approximately 77-97 % similarity for nucleotide sequences and 84-100 % for amino acid sequences to isolates from the GenBank. Phylogenetic analysis showed that studied ORSV and CymMV isolates may have common origin with some South Korean isolates.

The amplified products from Phalaenopsis sp. samples were purified using MinElute Gel Extraction Kit (Qiagen, Great Britain). Resulting PCR products were sequenced using Applied Biosystems 3730x1 DNA Analyzer using Big Dye terminators, version 3.1 (Applied Biosystems, USA).
Phylogenetic analysis was carried out using MEGA 9 software.
Results and Discussion. Orchid plants from collection of A.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv with symptoms of mosaic, spots, necrosis and chlorosis have been detected. Symptoms of mosaic on the leaves were an alternation of light and dark green areas, which formed a mosaic pattern. Symptoms of necrotic or annular spotting are the formation of small (0.5-2 mm) and medium-sized (2-4 mm) black necrosis of round and oval shape on the leaf blade, sometimes surrounded by a narrow border (Fig. 1). A fairly common symptom was yellowing of the leaf blade in combination with the formation of marginal leaf necrosis. Symptoms of mosaic, yellowing and ring necrosis can occur on plants under the influence of CymMV and ORSV [12,13,14].
CymMV and ORSV were detected by ELISA with appropriate antisera in all selected samples of orchid plants. It should be noted that mixed infection with CymMV + ORSV was detected in plants of Phalaenopsis sp.
Further leaves of Phalaenopsis sp. with the symptoms typical of CymMV and ORSV infection ( Fig. 1) confirmed by DAS-ELISA were used for total RNA extraction. The total RNA was amplified in RT-PCR. Agarose gel electrophoresis revealed the presence of expected amplified products with corresponding molecular weight (Fig. 2).
Gene sequence of Ukrainian ORSV isolate (ORSV-PHA-Ukr) were compared with another known isolates of ORSV available from the GenBank. For this study, we used published 32 full-genomic sequences of ORSV. Since full genomic sequences of ORSV isolates were used for the analysis, we were able to compare CP gene sequence of ORSV-PHA-Ukr with that of other isolates (Table 1). As shown in Table 1, the degree of CP gene similarity of ORSV-PHA-Ukr in comparison with other ORSV isolates from GenBank is quite high. This demonstrates high level of conservancy of ORSV genome (or at least its CP gene).

T a b l e 1. Comparison of partial nucleotide and amino acid sequences of ORSV isolated in
To determine possible origin of ORSV isolate sampled in Ukraine, phylogenetic analysis based on its amino acid sequence was conducted. Maximum Likelihood method was chosen as a discrete method, which was performed using Poisson model. As can be seen from the resulting phylogenetic tree (Fig. 3), ORSV-PHA-Ukr isolate and one of the isolates from the South Korea were clustered together suggesting their possible common origin. The obtained nucleotide sequence of CP gene of CymMV isolated in Ukraine (CymMV-PHA-Ukr) was pairwise compared with the other 33 CymMV isolates from the GenBank. Basing on the mutations in monomorphic sites, CymMV isolates can be divided into two monophyletic clusters at the nucleotide level -subgroup A and subgroup B [17]. According to our data and basing on the nucleotide sequence, CymMV-PHA-Ukr isolate belongs to subgroup A and shares 88-97 % similarity with other CymMV isolates from subgroup A and 77-87 % with the CymMV isolates from subgroup B. The deduced amino acid sequence of CymMV-PHA-Ukr shared 84-100 % similarity with other CymMV isolates.
For the further research of CymMV-PHA-Ukr phylogenetic tree of nucleotide sequence was constructed using ML method. As shown on Fig. 4, CymMV isolate sampled in Ukraine may have common origin with some isolates from the South Korea. In conclusion, it was established that ORSV isolate collected from Phalaenopsis sp. in Ukraine shared high homology at the nucleotide and amino acid levels with other known ORSV isolates basing on CP gene/protein sequence. CymMV isolate sampled from orchids in Ukraine showed 77-97 % similarity to the virus isolates available from the Gen-Bank. Isolates of both viruses (ORSV-PHA-Ukr and CymMV-PHA-Ukr) supposedly have common ancestors with the ORSV and CymMV isolates from the South Korea. Since the plants from tropical orchids' collections in botanical gardens in Ukraine were originally imported from eastern countries, we suggest that ORSV and CymMV may have invaded orchid collections with imported plant material, with subsequent spread in Ukraine.