Strain attribution of Ukrainian isolates of Zucchini yellow mosaic virus and their occurrence in Ukraine

V. O. Tsvigun, T. O. Rudneva, T. P. Shevchenko © 2016 V. O. Tsvigun et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 578.856 Strain attribution of Ukrainian isolates of Zucchini yellow mosaic virus and their occurrence in Ukraine


Introduction
Zucchini yellow mosaic virus (ZYMV) is one of the most economically dangerous viruses for all cucurbit crops.Thus, a biological, serological and molecular characterization of this virus is of great importance.ZYMV is widespread on all continents except Antarctica.
ZYMV belongs to Potyvirus genus of Potyviridae family.It was first described in Italy in 1973 [1].ZYMV infects 15 plant species from 7 different fam-ilies.However, only the members of Cucurbitaceae family develop severe symptoms [2].An occurrence of ZYMV was reported from more than 50 countries.It causes yield losses ranging from 25 to 50 % depending on the pathogenicity of the virus strain.The symptoms include mosaics and yellowing leaf blade [3].Knobs appear on fruits.Notably, in Ukraine this virus was first detected approximately 10 years ago [4].
The nucleotide sequences of approximately 400 strains and isolates of ZYMV were published in Viruses and Cell ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 3. P 235-241 doi: http://dx.doi.org/10.7124/bc.000925 GenBank.The nucleotide sequences of the coat protein (CP) and polymerase (NIb) genes as well as the amino acid sequences of their products are commonly used to establish a phylogenetic relationship between ZYMV strains.According to the literature data, the N-terminal part of CP is more variable than the C-terminal part of the NIb genome region [5].Moreover, the selection pressures were shown to be different: it was ten times higher for the C-terminal part of NIb than for the variable N-terminal part of CP.
According to the topology of phylogenetic tree built using the Nib/CP genome region, the ZYMV isolates form three distinct groups: A, B and C [5].Group A consists of three clusters supported on the phylogenetic tree by bootstrap of relatively high values .Cluster I includes the most frequently detected strains.Most of them belong to the serotype 1.The proposed referent strain of this subgroup is ZYMV-NAT.Cluster II is not supported by high bootstrap values.However, three different methods of phylogenetic analysis estimated the distinction of this cluster.The members of cluster II cannot be distinguished by serological tests, since all mutations in the coat protein gene sequences are synonymous; the amino acid sequences are identical.The strains of cluster III are presented by a small proportion of the ZYMV isolates, which were detected on four continents and connected with the strains of other clusters from Austria, Italy and Spain [5].
Group B includes one cluster consisted of five isolates from Reunion and neighboring islands, which differ from other isolates [5,6].Group C consists of several Chinese, Polish and Australian isolates.
Viral infection leads to an increase in the number of infected plants, decreases the quality of fruits and causes losses of yield.As ZYMV are widespread in the world, it is important to analyze phylogenetic relationships between Ukrainian isolates and the reported strains of this virus to propose possible ways of ZYMV dissemination from other countries to Ukraine as well as from Ukraine to other countries.
The purpose of current study was to establish the strain attribution of Ukrainian isolates of ZYMV and to examine their occurrence in Ukraine.

Materials and Methods
Plant samples were collected following the visual examination of virus symptoms.The equal number of samples was collected from each location.Plant material was homogenized in 0.1 phosphate buffered saline (PBS), pH 7.4, 1:2 (m/v).Plant components were removed by centrifugation at 5000 g for 20 min at 4 °C using centrifuge PC-6 [7].The supernatant was taken for diagnostic tests.Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was used for detection of viral antigens.DAS-ELISA was performed using commercial test system of Loewe (Germany) according to the manufacturer's recommendations.The ELISA results were read at 405/630 nm using microplate reader Termo Labsystems Opsis MR (USA) with Dynex Revelation Quicklink software.For obtaining statistically significant results, three times repeated testing of each sample was performed [8].Arithmetic mean was calculated for each sample [9].
Total RNA was extracted from plant samples using RNeasy Plant Minikit (Qiagen, Great Britain) [10].Reverse transcription polymerase chain reaction (RT-PCR) was carried out using primers specific to Nib/CP genome region of ZYMV [5]: Forward primer -5`-ATGTCGAGTATCACATTTCC-3`; Reverse primer -5` GGTTCATGTCCCACCAAGC-3T hese primers are complementary to 8200-8220 and 8800-8819 positions of genome and allow amplifing the fragments of 605 bp encompassing the C-terminal part of Nib coding region and the N-terminal part of the coat protein coding region.
RT-PCR was performed in the following parameters: 30 min at 50 °C, followed by 30 cycles -1 min at 95 °C, 30 s at 95 °C, 30 s at 55 °C, and final cycle -1 min at 72 °C.RT-PCR products were analyzed by electrophoresis in 1.5 % agarose gel using markers Gene Ruller 100 bp DNA Ladder plus (Fermentas, USA) [11].Obtained amplicons were extracted from agarose gel and purified using Thermo Scientific GeneJET Extraction Kit (USA).The purified amplicons were sequenced using Applied Biosystems 3730x1 DNA Analyzer with Big Dye terminators, version 3.1 (Applied Biosystems, USA).
Finally, we obtained Nib/CP sequences of the following Ukrainian isolates: ZYMV-10G (extracted from Cucurbita pepo in Poltava region), ZYMV-10P (extracted from Cucumis melo in Vinnytsia region), ZYMV-38/14 (extracted from Citrullus lantanus in Cherkasy region), ZYMV 5/13 (extracted from Cucurbita pepo in Poltava region) and ZYMV-B (extracted from Cucumis melo in Cherkasy region).The obtained nucleotide sequences were compared with their homologs from the strains reported from different countries using NCBI.The nucleotide sequences were analyzed using MEGA 6 software.The sequences were aligned by CLUSTAL W. Tree branches were bootstrapped with 1,000 replications [12].

Results and discussion
126 samples of plants from Cucurbitaceae family were screened for the presence of viral antigens.The plant samples were collected in agroecosystems of different regions of Ukraine: Vinnytsia, Zaporizhzhia, Kyiv, Kirovohrad, Odessa, Poltava, Cherkasy and Chernihiv.Cucurbit plants (cucumbers, squashes, pumpkins, and zucchini) showed the following symptoms: puckering, distortion, vein banding, yellowing, filamentary, yellow mosaic on leaf blade; dark green spots of different size, knobs and malformations on fruits (Fig. 1).
Further, DAS-ELISA was performed for detection of ZYMV.Antigens of ZYMV were detected in 51 plant samples out of 126.Zucchini yellow mosaic virus was detected in the mixed infections with Watermelon mosaic virus-2 and Cucumber mosaic virus.The survey conducted in 9 regions demonstrated that ZYMV is widespread in the central area of our country (Kyiv, Vinnytsia, Cherkasy and Poltava regions) as well as in Zaporizza region.ZYMV infection was not detected either in the northern (Chernigiv region) or in the southern (Kirovograd, Odessa regions) areas of Ukraine (Fig. 2).These finding allows a preliminary conclusion that the epidemiological situation with regard to ZYMV is likely more favorable in the latter regions, although more detailed studies on the ZYMV distribution are needed to confirm this suggestion.In any case, the detection of ZYMV infection in five of nine inspected regions points on a quite high prevalence of ZYMV in Ukraine.
The obtained sequences overlap the NIb/CP cleavage site at position 8542.This fragment is commonly used to study the variability of potyviruses, because the N-terminal part of the coat protein is known to be highly variable, whereas the polymerase is more conserved.RT-PCR resulted in amplification of a cDNA of expected 605 bp.
Further, the obtained amplicons were sequenced for establishing their phylogenetic relationships.The sequences were compared with the Nib/CP sequences of ZYMV strains and isolates previously reported in GenBank.The strains and isolates selected for phylogenetic analysis have different geographic origin, group attribution, biological and serological properties.The levels of identity between Ukrainian isolates and 49 isolates from GenBank ranged from 58 to 100 %.The phylogenetic tree was constructed using Neighbor-Joining method in MEGA 6 (Fig. 4).
The phylogenetic analysis revealed that Ukrainian isolates have low level of variability of the Nib/CP genome region: level of identity between Ukrainian isolates is 99,7 % .
According to the literature data, all isolates were proposed to be distinguished into three groups, and within group A four subgroups were separated by G. Romay (2014).The topology of resulted phylogenetic tree confirmed this grouping: three the most numerous clusters consisted of four subclusters [6].Ukrainian isolates were located in subgroup I within group A. Besides Ukrainian isolates, this subgroup also included isolates from Austria (AJ420017), Slovenia (AJ420018), Serbia (HM072432), Hungary (AJ459955), Slovakia (DQ124239) and Venezuela (JX310104 VE10-303, JX310118 VE10-279, JX310109 VE10-290, JX310108 VE10-292, JX310116 VE10-295).The levels of identity between the members of subgroup I were shown to vary from 94.3 to 100 %.The ZYMV isolates circulating in Ukraine have a monophyletic origin with European isolates.

DQ925448, DQ925451) and Poland (EF178505).
Ukrainian isolates showed 58 -77.8 % identity with members of this group.The lowest level of identity was shown with isolates from Vietnam (58 %).
Phylogenetic analysis of Ukrainian isolates points out that the nucleotide sequence of Nib/CP shared high level of identity between each other and previously published strains and isolates of ZYMV.They showed the level of identity ranging from 72.7 to 100 %.

Conclusions
Identification of infected plants in five of nine inspected agroecosystems suggests quite high prevalence of ZYMV infection in Ukraine.The topology of reconstructed phylogenetic tree confirmed the previously established clustering of the ZYMV isolates into three groups.Phylogenetic positioning of the Ukrainian ZYMV isolates on this tree demonstrated that they belong to subgroup I of group A. This group is the most numerous group, which consists of members of diffe rent geographic origin.The highest levels of identity were revealed between Ukrainian isolates and isolates from Austria (100 %), Slovenia (96 %), Serbia (100 %), Hungary (99.4 %), Slovakia (99.4 %).This fact clearly indicates that Ukrainian isolates have an European origin.ZYMV are transmitted by seeds.Contaminated seed material may be considered as a possible way of introduction of new strains in Ukraine as well as from Ukraine to other countries and continents.

Fig. 1 .
Fig. 1.Symptomatic plants from Cucurbitaceae family: dark green mosaic on leaf blade of Cucurbita pepo; filamentary of leaf blade of Cucurbita pepo; light green mosaic on leaves of Cucurbita pepo; knobs and ring spots on fruit of Cucurbita pepo.

Fig. 2 .
Fig. 2. Incidence of plants from family Cucurbitaceae infected by Zucchini yellow mosaic virus.