First Evidence of the Occurrence of Turnip Mosaic Virus in Ukraine and Molecular Characterization of Its Isolate

Received: 7 September 2017 | Accepted: 1 March 2018 DOI: 10.1111/jph.12703

ORIGINAL ARTICLE

First evidence of the occurrence of Turnip mosaic virus in Ukraine and molecular characterization of its isolate

Oleksiy Shevchenko1 | Ryosuke Yasaka2,3 | Olha Tymchyshyn1 | Tetiana Shevchenko1 | Kazusato Ohshima2,3

1Virology Department, ESC “Institute of Biology and Medicine”, Taras Shevchenko Abstract National University of Kyiv, Kyiv, Ukraine A total of 54 samples of Brassicaceae crops showing symptoms of mosaic, mottling, 2 Laboratory of Plant Virology, Department vein banding and/or leaf deformation were collected in Kyiv region (northern central of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga, Japan part of Ukraine) in 2014–2015. A half of collected samples was found to be infected 3The United Graduate School of Agricultural with Turnip mosaic virus (TuMV), and TuMV was detected in samples from Brassica Sciences, Kagoshima University, Kagoshima, oleracea var. capitata (cabbage), Raphanus sativus, Brassica juncea, Raphanus sp., Japan Sinapis alba, Camelina sativa and Bunias orientalis (weed). The full-length sequence of Correspondence the genomic RNA of a Ukrainian isolate (UKR9), which was isolated from cabbage, O. Shevchenko, Virology Department, ESC “Institute of Biology and Medicine”, Taras was determined. Recombination analysis of UKR9 isolate showed that this isolate Shevchenko National University of Kyiv, was an interlineage recombinant of world-Brassica and Asian-Brassica/Raphanus phy- Kyiv, Ukraine. Email: [email protected] logenetic groups. This study shows for the first time the occurrence of TuMV in Ukraine. Funding information Japan Society for the Promotion of Science KEYWORDS Brassicaceae, evolution, recombination, Turnip mosaic virus, Ukraine

1 | INTRODUCTION TuMV probably occurs worldwide and has been found in both temperate and subtropical regions of Africa, Asia, Europe, Oceania Turnip mosaic virus (TuMV) is a member of Potyvirus genus belong- and North and South America (Ohshima et al., 2002; Provvidenti, ing to the Potyviridae family of plant viruses. TuMV has flexible fil- 1996; Schwinghamer et al., 2014). In Europe, TuMV was reported amentous particles 700–750 nm long containing a single-stranded from the United Kingdom (Pallett et al., 2008), Spain (Segundo et al., positive-sense genomic RNA of about 10,000 nucleotides (King, 2003), Italy (Guglielmone et al., 2000; Ohshima et al., 2002), Greece Adams, Carstens, & Lefkowitz, 2012). TuMV genome has a single (Jenner & Walsh, 1996; Tomimura et al., 2004), Germany (Tomimura, open reading frame (ORF) translated into a large polyprotein which Gibbs, Jenner, Walsh, & Ohshima, 2003), the Netherlands (Ohshima is subsequently cleaved into 10 functional proteins by virus-encoded et al., 2002), Czech Republic (Petrzik & Lehmann, 1996), Hungary enzymes (Gibbs & Ohshima, 2010; Urcuqui-Inchima, Haenni, & (Horvath, Juretic, Besada, & Mamula, 1975), Bulgaria (Kovachevsky, Bernardi, 2001). Furthermore, an overlapping “pretty interesting 1975), Poland (Kozubek, Irzykowski, & Lehmann, 2007) and Russia Potyviridae ORF” (PIPO) exists in the +2 reading frame within the (Ohshima et al., 2002). region encoding protein 3 (P3) (Chung, Miller, Atkins, & Firth, 2008). Ukraine is one of the largest European countries between TuMV has an extremely wide host range but infects mostly plant the eastern EU states and Middle East region, where TuMV was species from the Brassicaceae family and induces persistent symp- detected in Turkey and Iran (Farzadfar et al., 2009; Korkmaz, toms (mosaic, mottling and chlorotic local lesions). TuMV is consid- Tomitaka, Onder, & Ohshima, 2008; Yasaka et al., 2017). Despite ered one of the most damaging and economically important viruses Ukraine’s geographical location and wide cultivation of differ- for domesticated Brassica plants. TuMV is transmitted by many aphid ent Brassica crops for centuries, TuMV has never been found on species non-persistently as well as mechanically from plant to plant field crops in this country. The only published record of TuMV but not by seed (Walsh & Jenner, 2002). occurrence in Ukraine describes a single virus-infected sample

Journal of Phytopathology. 2018;166:429–437. wileyonlinelibrary.com/journal/jph © 2018 Blackwell Verlag GmbH | 429 430 | SHEVCHENKO et al. of wild orchid Orchis purpurea from the Crimea peninsula back in 2 | MATERIALS AND METHODS 2004. However, the virus was not recovered from the host plant (Korotieieva & Polishchuk, 2004). 2.1 | Sample collection TuMV isolates are biologically classified into five host infect- Samples were collected in the crop-producing areas of Kyiv ing types. [OM] host infecting type isolates infect some plants (northern central part of Ukraine) where Brassicaceae plants were of Brassicaceae but not Brassica plants. (B) host infecting type cultivated during the growing seasons of 2014–2015. Sampling lo- isolates infect Brassica plants latently and occasionally and do cations included two botanical gardens (Botanical garden of Taras not infect Raphanus plants. B host infecting type isolates infect Shevchenko National University of Kyiv and Botanical garden of many species of Brassica plants systemically with mosaic on un- the National Academy of Sciences of Ukraine), the city centre, inoculated leaves but do not infect Raphanus plants. B(R) host Museum of Folk Architecture and Life of Ukraine (open-air loca- infecting type isolates infect many species of Brassica plants sys- tion w/o agricultural activity), and private gardens where differ- temically with mosaic symptoms but occasionally infect Raphanus ent brassica plants were regularly cultivated. Several large fields plants, and BR host infecting type isolates infect both Brassica in Luka and Gorenychi villages used for commercial cabbage cul- and Raphanus plants systemically with mosaic symptoms (Nguyen tivation were chosen for sampling in Kyiv region. Brassica plants et al., 2013). (cabbage, red radish, mustard, gold of pleasure, hill mustard), Phylogenetic relationships using the genomic sequences of dif- showing TuMV-like symptoms of mosaic, mottling, vein banding ferent TuMV isolates collected from around the world showed that and/or leaf deformation, were collected. Some symptomatic non- TuMV isolates fall into six major TuMV phylogenetic groups called Brassicaceae species were also collected (Ohshima et al., 2002). Orchis, basal-Brassica (basal-B), Iranian, basal-Brassica/Raphanus Several leaves were collected from each plant, placed in airtight (basal-BR), Asian-Brassica /Raphanus (Asian-BR) and world-Brassica plastic bags filled with silica gel with colour indicator (Wako Pure (world-B) (Nguyen et al., 2013; Ohshima et al., 2002; Yasaka et al., Chemical Industries, Ltd., Osaka, Japan). 2017). The basal-B group of (B) or B host infecting type isolates is most variable, not monophyletic and originating from both non- brassicas and brassicas from Eurasia. Closest to it is the monophy- 2.2 | Enzyme-linked immunosorbent assay letic basal-BR group of BR host infecting type Eurasian isolates. The least variable group is the Asian-BR group formed by BR host Collected samples were tested for TuMV infection by direct infecting type isolates, mostly from Raphanus plants from East double-antibody sandwich enzyme-linked immunosorbent assay Asia. The world-B group is composed of isolates from all conti- (DAS-ELISA), as described by Clark and Adams (1977), using nents, mostly from brassicas, and consists of the B host infecting TuMV-specific polyclonal antibodies (Ohshima et al., 2002). type isolates from Europe and BR host infecting type isolates from Briefly, 0.5 g leaf tissue was ground to a powder with a mortar Asia. and pestle in 10 ml phosphate-buffered saline, pH 7.4, contain- The studies indicate that recombination plays an important role ing 0.05% Tween 20, 2.0% polyvinylpyrrolidone (MW 40 000) and in TuMV evolution. The phylogeographic analyses showed that 0.2% bovine serum albumin. Nunc MaxiSorp™ microtitre plates TuMV probably originated from a virus of wild orchids in Germany (Thermo Fisher Scientific K.K., Yokohama, Japan) were coated and, while adapting to wild and domestic brassicas, spread via with TuMV-specific polyclonal antibodies (1:200) in carbonate Southern Europe to Asia Minor no more than 850 years ago. The buffer. Leaf extracts were then added to the plates in duplicate populations of Orchis and basal-B group in Europe and Asia Minor wells and incubated for overnight at 4°C. The presence of TuMV in are older than other populations of this virus (Nguyen et al., 2013; the samples was detected in 200 μl homogenate by TuMV-specific Yasaka et al., 2017). antibodies conjugated to alkaline phosphatase using p-nitrophenyl Ukraine is also one of the possible candidates for the emergence phosphate substrate (Wako Pure Chemical Industries, Ltd.). of TuMV, as there are many different orchid species growing natu- Absorbance values at 405 nm were measured using a ImmunoMini rally, especially in the west of the country (Carpathian region) and in NJ-2300 microtitre plate reader (BioTec Co. Ltd., Tokyo, Japan). its southern part (Crimea peninsula), but not exclusively. The exten- Absorbance values, measured 60 min after adding the substrate, sive list of Ukrainian orchid species includes Orchis militaris—one of greater than three times those of the negative controls were con- the tentative “hotbeds” of TuMV emergence. The discovery of TuMV sidered positive. TuMV-positive samples were tested by DAS- in wild-growing O. purpurea (Korotieieva & Polishchuk, 2004) may ELISA for Cucumber mosaic virus (CMV) infection using the specific support this view. polyclonal antibodies (Japan Plant Protection Association, Tokyo, In the study, TuMV was collected in Ukraine from naturally in- Japan) as described above. fected host plants, all from Brassicaceae family. The full genome sequence of one of the Ukrainian isolates (biologically classified into 2.3 | Host range tests BR host infecting type) was determined for the first time, and its phylogenetic relationships with worldwide isolates are discussed. TuMV UKR9 isolate from 27 ELISA-positive Brassicaceae samples The result showed the occurrence of TuMV in Ukraine. collected in Kyiv and Kyiv region in 2014–2015 was inoculated on SHEVCHENKO et al. | 431

TABLE 1 Host reaction of Turnip mosaic virus UKR9 isolate

Plant Common name Seed origin Symptoma

Asteraceae Calendula officinalis cv. Orange star Pot Marigold Chile LI/M (12/12) Lactuca sativa cv. Emrap 231 Lettuce USA −/− (12/12) L. sativa cv. Salinas 88 Lettuce USA −/− (12/12) Brassicaceae Brassica juncea cv. Hakarashina Mustard Italy LI/sM (6/6) Brassica napus cv. Norin-32 go Oilseed rape Japan LI/M (6/6) Brassica narinosa cv. Tatsuai Rosette pakchoi Australia LI/sM (5/6), −/− (1/6) Brassica oleracea var. botrytis cv. Snow crown Cauliflower Australia −/− (6/6) B. oleracea var. botrytis cv. Snow queen Cauliflower Chile −/− (6/6) B. oleracea var. capitata cv. Ryozan-2go Cabbage Japan LI/Mo, VC (6/6) B. oleracea var. capitata cv. Shinsei Cabbage Japan LI/Mo, VC (5/6), −/− (1/6) B. oleracea var. capitata cv. Sosyu Cabbage Japan (LI/Mo, VC)b (3/6), −/− (3/6) B. oleracea var. Grand duke Kohlrabi Italy LI/M, VC (6/6) B. oleracea var. italica cv. Challenger Broccoli Japan (LI/CS, Mo)b (2/6), −/− (4/6) B. oleracea var. italica cv. Endever Broccoli Japan (LI/Mo, VC)b (3/6), −/− (3/6) B. oleracea var. italica cv. Pixcel Broccoli Chile (LI/Mo, VC)b (3/6), −/− (3/6) Brassica pekinensis cv. Kyoto-3go Chinese cabbage Japan LI/M (6/6) B. pekinensis cv. Nozaki-1go Chinese cabbage Japan LI/M (6/6) B. pekinensis cv. Nozaki-2go Chinese cabbage Japan LI/M (5/6), −/− (1/6) Brassica rapa cv. Hakatasuwari Turnip Japan LI/sM (6/6) Camelina sativa cv. Calena Gold of pleasure Austria LI/M (6/6) Eruca sativa cv. Odyssey Rocket Italy LI/sM (6/6) Matthiola incana cv. Christmas rouge Stock Japan LI/M (6/6) Raphanus sativus cv. Everest Chinese radish USA LI/sM (6/6) R. sativus cv. Taibyosobutori Japanese radish New Zealand LI/sM (6/6) Solanaceae Nicotiana benthamiana Japan LI/M (6/6) Nicotiana clevelandii Japan NS/− (6/6) Nicotiana glutinosa Japan NS/Mo, VC (6/6) Petunia hybrid cv. F1 duo red Petunia USA LI/M (6/6) P. hybrid cv. F1 rond white imp Petunia Chile LI/M (6/6) Chenopodiaceae Chenopodium amaranticolor – Japan CS/CS (2/2) Chenopodium quinoa Quinoa Japan CS/CS (3/3)

LI, local infection; CS, chlorotic spots; M, mosaic; Mo, mottle; NS, necrotic spot; Ru, rugose; S, stunting; sM, severe mosaic; VC, vein clearing; –, not infected. aReaction of inoculated leaves/upper leaves. bSymptoms appear occasionally.

Chenopodium quinoa and serially cloned through single lesions at least species (Table 1). Inoculated plants were kept for at least 4 weeks in a three times. UKR9 isolate was subsequently propagated in Brassica glasshouse at 25°C and symptoms were recorded. rapa cv. Hakatasuwari (turnip) and Nicotiana benthamiana. UKR9 isolate was used for host range test. Leaves from systemically infected turnip 2.4 | Extraction of viral RNA and sequencing plants were homogenized in 0.01 m potassium phosphate buffer (pH 7.0), and the extract was used to mechanically inoculate young indica- Total RNA was extracted from TuMV-infected B. rapa using Isogen tor plants using the wide panel of Brassicaceae and non-Brassicaceae (Nippon Gene, Tokyo, Japan) and first-strand cDNA synthesis was 432 | SHEVCHENKO et al. performed using PrimeScript™ reverse transcriptase (Takara Bio (Martin, Murrell, Golden, Khoosal, & Muhire, 2015). These analyses Inc., Otsu, Japan), according to the manufacturer’s instructions. were performed using default settings for the different detection al- Polymerase chain reaction (PCR) amplifications were performed gorithms and a Bonferroni corrected p-value cut-off of .01 for linear using high-fidelity Platinum™ Pfx DNA polymerase (Invitrogen, sequences. All sequences that had been identified as likely recom-

Carlsbad, CA, USA). PCR products were separated by electropho- binants by rdp4 software, together with all those used in this study, resis in agarose gels. The expected fragments were excised from were rechecked again using the original software siscan version 2 (Gibbs the gels, cleaned using the QIAquick Gel Extraction Kit (Qiagen et al., 2000) with both 100 and 50 nt windows. These analyses also K.K., Tokyo, Japan). Nucleotide sequence from UKR9 isolate assessed which non-recombinant sequences had regions that were was determined using five independent RT-PCR products (5T- closest to regions of the recombinant sequences and hence indi- P3 region; 5′ end to nt 3230, HCpro-6K1 region; nt 1835–3732, cated the likely lineages that provided those regions of the recom- P3-VPg region; nt 3092–6099, CI-NIb region; nt 5609–7299, Nia- binant genomes. PolyA region; nt 7164 to 3′ end) to cover the full genome. The primers used for amplification were; Tu5T4P (nt 1–26, 5′-AAAA 2.6 | Phylogenetic analyses ATATAAAAACTCAACATAACAT-3′) and TuP3OP1M (nt 3209– 3230, 5′-CGCTGTATCTGCCGCCTAAATC-3′) for 5T-P3 region, The amino acid sequences of the polyproteins were aligned with TuKA1HC11P (nt 1835–1854, 5′-TTCATATGGGGTGAGAGAGG-3′) the outgroup sequences, described above, using CLUSTAL_X2 and Tu596K17M (nt 3713–3732, 5′-TCTGCGTCAAACATCATGAG-3′) (Larkin et al., 2007) with TRANSALIGN (kindly supplied by Georg for HCpro-6K1 region, TuP3OP1P (nt 3092–3113, 5′-CARAT Weiller) to maintain the degapped alignment of the encoded CTTGGACGAAGCATGGA-3′), TuVPG8M (nt 6077–6099, 5′-TCAA amino acids. Two sequences of Japanese yam mosaic virus (JYMV) ATCCATACATGTTGATGAA-3′) for P3-VPg region, Tu59CI9P (GenBank Accession codes: KJ701427 and AB016500), one of (nt 5609–5628, 5′-GTGCTTGARGGAGCRAAGTC-3′) and TuNI Scallion mosaic virus (ScaMV) (NC_003399), one of Wild onion B14M (nt 7280–7299, 5′-ACYGTGTGCTTYGTCACAAG-3′) for CI- symptomless virus (NC_030391), two of Narcissus late season yellows NIb region, and Tu59NIA3P (nt 7164–7184, 5′-GCAARCTAATMT virus (NLSYV) (NC_023628 and JQ326210) and two of Narcissus CAGACCTYG-3′) and Tu3T9M (nt 9835-polyA, 5′-GGGG yellow stripe virus (NYSV) (JQ911732 and JQ395042), were used

CGGCCGCT15-3′) for NIa-PolyA region. The sequences of adjacent as an outgroup, because these virus species are members of TuMV regions of the genome were overlapped by at least 400 bp to ensure phylogenetic group. These produced sequences of 8208 nt. The that they were from the same genome and were not from different phylogenetic relationships of the partial genomic sequences were components of a genome mixture. inferred using the Maximum Likelihood (ML) method in PhyML DNA sequencing was performed by primer walking in both di- v.3 (Guindon & Gascuel, 2003). The best-fit model of nucleotide rections using the BigDye Terminator v3.1 Cycle Sequencing Ready substitutions for each dataset was determined using jModeltest Reaction Kit and an Applied Biosystems Genetic Analyser DNA 0.1.1 (Posada, 2008). For the ML analysis, we used the general model 310 (Applied Biosystems, Foster City, CA, USA). Nucleotide time-reversible (GTR) model of nucleotide substitution, with rate sequence data were assembled using bio-edit Version 5.0.9 (Hall, variation among sites modelled using a gamma distribution and a 1999). proportion of invariable sites (GTR+I+r4). This model was selected

in r (Schliep, 2011) using the Bayesian information criterion, which has been shown to perform well in a variety of scenarios (Luo et al., 2.5 | Recombination analyses 2010). Branch support was evaluated by bootstrap analysis based The genomic sequences of the 238 isolates of TuMV available on 1,000 pseudoreplicates. The inferred trees were displayed from the international sequence databases and one Ukrainian iso- by TreeView (Page, 1996). Nucleotide and amino acid similarities late (UKR9) were used for recombination analyses. To add the gap were estimated using the Kimura two-parameter method (Kimura, sequences in the nucleotide sequences, the amino acid sequences 1980) and the Dayhoff PAM250 matrix (Dayhoff, Barker, & Hunt, corresponding to polyprotein regions were firstly aligned using 1983), respectively. CLUSTAL_X2 (Larkin et al., 2007) with TRANSALIGN (kindly supplied by Georg Weiller, Australian National University, Canberra, 3 | RESULTS Australia) to maintain the degapped alignment of the encoded amino acids. The aligned 5′ and 3′ untranslated region (UTR) sequences 3.1 | Surveys and detection of TuMV by DAS-ELISA were then reassembled to form complete sequences of 9082 nt. The aligned sequences were first checked for incongruent rela- A total of 54 plant samples showing virus-like symptoms, mosaic and tionships that might have resulted from recombination, using RDP mottling leaves were collected in different sites of the Kyiv region. (Martin & Rybicki, 2000), GENECONV (Sawyer, 1999), BOOTSCAN TuMV has been detected by DAS-ELISA in 27 plant samples (50%) (Salminen, Carr, Burke, & McCutchan, 1995), MAXCHI (Smith, 1992), including Brassica oleracea var. capitata, Raphanus sativus, Raphanus CHIMAERA (Posada & Crandall, 2001) and SISCAN algorithms sp., Sinapis alba, Brassica juncea, Camelina sativa, Brassica sp. and

(Gibbs, Armstrong, & Gibbs, 2000) implemented in rdp4 sof t war e Bunias orientalis (weed). SHEVCHENKO et al. | 433

TuMV generally showed mosaic, vein banding and leaf deforma- 3.4 | Recombination sites tion on cabbage plants, whereas mosaic and mottling were common for naturally infected radish and mustard plants. Six of 27 plant sam- The full genomic sequence of UKR9 isolate was assessed for evi- ples (22%) were mixed infected with CMV. dence of recombination using rdp4 software. A “clear” interlineage recombination site signal of UKR9 isolate (p values smaller than 1 × 10−6) was obtained using the RDP (3.68 × 10−21), BOOTSCAN 3.2 | Host range test (2.83 × 10−21), MAXCHI (3.12 × 10−11), CHIMAERA (2.14 × 10−13) −14 A TuMV isolate (UKR9) was collected in private garden and isolated and SISCAN (6.42 × 10 ) programs in rdp4 sof t war e, wher eas “t ent a- from B. oleracea var. capitata (cabbage) plant showing vein banding. tive” recombination site signal (p value of 3.76 × 10−2) was obtained

The cabbage plant infected with UKR9 isolate reacted strongly with using geneconv program. The site was further investigated by origi-

TuMV-specific antibody but not with CMV-specific antibody in DAS- nal software siscan version 2 (Figure 1). The UKR9 isolate sequence ELISA. This isolate was selected for the subsequent study of host showed significant affinities (Z values > 3.0) with the Rn98 sequence range using wide panel of plants including species of Brassicaceae, in the region corresponding to the 5′ end of the genome to the HC- Solanaceae, Chenopodiaceae and Asteraceae families (Table 1). Pro gene (nt 1–1374 corresponds to the UK1 genome). However, UKR9 isolate systemically infected both Brassica (cabbage, UKR9 isolate had affinity to TUR9 from the HC-Pro to the 3′-end of Chinese cabbage, kohlrabi, mustard, rape and turnip) and Raphanus the genome (nt 1375–9833). This indicated that UKR9 isolate was (Chinese and Japanese radish) plants. UKR9 isolate only occasionally a single recombinant and an interlineage recombinant of world-B infected different varieties of broccoli and did not infect cauliflower. (Rn98) × Asian-BR (TUR9) parents (detected here as minor and major Therefore, this isolate belongs to BR host infecting type. parents, respectively).

3.3 | Nucleotide sequence 3.5 | Phylogenetic relationships and sequence similarity The genomic sequence of UKR9 isolate, excluding the 5′ end primer sequences of 26 nt, was determined and deposited in DDBJ/EMBL/ ML phylogenetic trees were initially calculated from the genome se- GenBank databases (Accession number KY399991). The sequence quences of the 239 isolates, including the recombinants (data not of the isolate was 9833 nt long. The results indicated that these shown). However, there were inconsistencies in and poor bootstrap sequences encompass regions encoding P1, HC-Pro, third protein support for some lineages in the resulting trees, as found previously (P3), 6 kDa 1 protein (6K1), cylindrical inclusion protein (CI), 6 kDa (Nguyen et al., 2013; Ohshima et al., 2002, 2007). Therefore, the 2 protein (6K2), genome linked viral protein (VPg), nuclear inclusion trees were recalculated from the partial genomes (nt 1375–9833, proteinase protein (NIa-Pro), nuclear inclusion b protein (NIb) and corresponds to the UK1 genome) of 95 isolates, discarding the re- coat protein (CP) genes with 1086, 1374, 1065, 156, 1932, 159, 576, combinants. The relationships of these isolates were investigated by 729, 1551 and 864 nt, respectively. All of the motifs reported for ML method (Figure 2). The tree partitioned most of the sequences potyvirus genomes were found. into the same six consistent phylogenetic groups; Orchis, basal-B,

FIGURE 1 Graph showing SISCAN analysis of the polyprotein sequence of Turnip mosaic virus isolate UKR9 with that of isolates Rn98 (dotted line) and TUR9 (thin solid line). The sequences of Rn98 and TUR9 represent the likely parental sequences of UKR9. Window comparison involved subsequences of 100 nucleotides and a step between window positions of 50 nucleotides. Note the strong support (i.e., Z value > 3.0) for UKR9 being more closely related to Rn98 than TUR9 in the degapped nt 1–1307 (corresponds to nt 1–1374 in UK1 genome, Accession number AF169561) and the reverse in the degapped nt 1308–9703 (corresponds to nt 1375–9834 in UK1 genome). Arrow indicates the location of recombination site 434 | SHEVCHENKO et al.

FIGURE 2 Maximum likelihood tree showing phylogenetic relationships between the non-recombinant isolates of Turnip mosaic virus. The regions from nt 1375 to 3′ end in genomic sequences (correspond to the UK1 isolate, Accession number AF169561) were used for constructing tree. UKR9 isolate is shown in square SHEVCHENKO et al. | 435

TABLE 2 Comparison of nucleotide (below diagonal) and amino Asian-BR sequences not only in the P3–CI genomic region but acid (above diagonal) similarities of UKR9 and parental isolates also in the HC-Pro gene (Nguyen et al., 2013; Ohshima, Akaishi,

Isolate UKR9 TUR9 Rn98 Kajiyama, Koga, & Gibbs, 2010; Ohshima et al., 2007; Tomitaka & Ohshima, 2006; Yasaka et al., 2015, 2017). The latter gene is UKR9 – 0.03 0.06 known to be an RNA silencing suppressor of potyviruses (Valli, TUR9 0.103 – 0.069 Gallo, Rodamilans, López-Moya, & García, 2017). In this study, Rn98 0.168 0.182 – we found a novel interlineage recombination type pattern of The nucleotide and amino acid similarities were assessed by Kimura two- world-B and Asian-BR group parents (between nt 1374 and parameter and Dayhoff PAM250 matrices. 1375). World-B and Asian-BR recombination type pattern isolates were relatively easy to find in East Asia (Ohshima et al., Iranian, basal-BR, Asian-BR and world-B. All the phylogenetic group- 2007; Tomimura et al., 2003), whereas those were less common ings were supported by high bootstrap values. UKR9 isolate fell into in Europe. Furthermore, most part of the UKR9 genome was Asian-BR phylogenetic group with the major parental isolate TUR9 Asian-BR phylogenetic group (Figure 2). Therefore, our result in the ML tree. may show TuMV populations between Ukraine and East Asia had The nucleotide and amino acid sequence similarities between some relation in the past. UKR9 isolate and isolates Rn98 and TUR9 were calculated (Table 2). The simplest interpretation of the phylogenetic analyses of the The similarities showed that the UKR9 isolate was distinct from both worldwide isolates in earlier studies (Nguyen et al., 2013; Yasaka parental isolates. et al., 2017) is that the TuMV population probably originated from a virus of wild orchids in Europe. After TuMV populations were adapted to wild and domestic brassica crops in Southern European 4 | DISCUSSION and Asia Minor countries, they were then probably spread to Asia countries. The UKR9 isolate fell into Turkish isolates in Asian-BR In the present study, TuMV was shown to be spread in the Kyiv phylogenetic group (Figure 2) (Korkmaz et al., 2008; Yasaka et al., region. The host range test of a Ukrainian TuMV (UKR9) isolate 2017). This finding may show a Ukrainian isolate had relation to showed that it was able to infect systemically both Brassica and Turkish isolates, although we need to determine more genomic se- Raphanus plants; hence, this isolate belonged to BR host infecting quences of Ukraine isolates. type (Table 1). In summary, although world-B × Asian-BR interlineage recom- Brassica oleracea var. capitata (cabbage), R. sativus (radish), B. jun- binant of BR host infecting type was found in Kyiv in the present cea (mustard) and S. alba (white mustard) were the predominant study, it is still largely unknown whether this recombination type hosts for TuMV in Kyiv region (data not shown). Cabbage was re- pattern is dominant in Ukraine. Wide survey of TuMV in different garded the most important and widely cultivated brassica food plant regions of Ukraine is necessary to confirm this. However, to our in Ukraine, when other plants remained of seasonal use. TuMV was knowledge, the present study shows for the first time the occur- also found in a single sample of B. orientalis (hill mustard)—an inva- rence of TuMV in Ukraine and showed evolutionary relationships sive introduced species originating from Asia and occasionally used between a Ukraine isolate and the worldwide isolates previously as a fodder crop in some Soviet republics (Ukraine and Russia) in reported. 1970–1980 (Sozinov & Ryabchoun, 1995), which perhaps facilitated the spread of the species into new areas. Today, B. orientalis is an ACKNOWLEDGEMENTS abundant European weed and a known host for CMV and TuMV (Kobyłko, Maj, & Gajewski, 2009). Despite this, we were unable to This work was in part supported by Japan Society for the Promotion collect more samples of this species in present study and hence its of Science, Bilateral Collaborations (Joint Research Projects) be- role in TuMV epidemiology remains unclear. tween Japan and Ukraine. Recombination plays a major role in plant RNA and DNA virus variability and adaptive evolution. In the case of potyviruses, COMPLIANCE WITH ETHICS GUIDELINES 63% of the Potato virus Y genomes (Ogawa, Tomitaka, Nakagawa, & Ohshima, 2008) and 74% of the TuMV genomes (Ohshima et al., This article does not contain any studies with human or animal sub- 2007) were recombinants. The recombination sites in TuMV ge- jects performed by any of the authors. nomes were found throughout the genomes, and the P1 and CI-VPg genomic regions were identified as the statistically sup- AUTHOR CONTRIBUTIONS ported hotspots for recombination events. The recombination events of many interlineage recombinants with BR host infecting O.S. and K.O. designed the study, R.Y., O.S., O.T., T.S. and K.O. types were hypothesized to extend the host range of TuMV to conducted the experiments, O.S., K.O. and R.Y. analysed the data, generate the BR host infecting type, which is found mainly in and O.S. and K.O. wrote the manuscript. All authors reviewed the Asian countries. Interestingly, these recombinants generally had manuscript. 436 | SHEVCHENKO et al.

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