Journal of Pathology (2017), 99 (3), 703-706 Edizioni ETS Pisa, 2017 703

OCCURRENCE OF TURNIP MOSAIC IN PHALAENOPSIS sp. IN

G.H. Zheng1*, D.W. Peng2*, Q.X. Tong1, 3, Z.Z. Zheng1 and Y.L. Ming1, 3

1 Xiamen Overseas Chinese Subtropical Plant Introduction Garden, National Plant Introduction Quarantine Base, Xiamen Key Laboratory for Plant Introduction, Quarantine and Natural Products, Xiamen, 361002, Fujian, China 2 School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, Zhejiang, China 3 College of Chemical Engineering, Huaqiao University, Xiamen, 361021, Fujian, China * The authors have contributed equally to the this study and should therefore be regarded as equivalent authors

SUMMARY TuMV has great genetic versatility in adapting to dif- ferent environments and breaking host resistance. For A turnip mosaic virus (TuMV) isolate from asymptom- example, TuMV with just a single-nucleotide mutation in atic Phalaenopsis sp. was detected by indirect ELISA. Its the cylindrical inclusion gene (CI) can overcome the resis- presence was confirmed by RT-PCR with a pair of degen- tance gene TuRB01 (Walsh et al., 2002). A single mutation erate primers whose design was based on reported coat (+ 3394 T > C) in the viral P3 protein induces local necrotic protein gene sequences. Further analysis of the genomic lesions and overcomes extreme resistance in napus. sequence of this isolate designated as TuMV-ZH1 (Gen- Such a change results in systemic non-necrotic infection Bank accession No. KF246570) showed a high nucleotide when another mutation (+ 5447 T > C) in CI protein is in- sequence identity with isolate Lu2 (96.7%) and CHN 12 troduced (Jenner et al., 2002). (96.3%) from China. Phylogenetic analysis indicated that TuMV can infect 13 species of orchid , including TuMV-ZH1 belongs to the world-B lineage. To the best of Aceras anthropophorum, Anacamptis pyramidalis, Barlia lon- our knowledge, the above may be the first indication that gibracteata, Calanthe sp., Ophrys speculum, Ophrys tenthre- TuMV infects orchids in China. In addition, proteins P1 dinifera, Orchis italica, Orchis morio, Orchis militaris, Orchis and P3 of TuMV-ZH1 are highly mutated as previously papilionaceae, Orchis simia, Cymbidium sp. and Pescatorea reported for other TuMV isolates. sp., in which it induces mosaic symptoms (Lesemann and Vetten, 1985; Inouye, 1992). A TuMV isolate, denoted Keywords: TuMV, coat protein, genome, phylogenetic TuMV-ZH1, was identified in Phalaenopsis sp. in a sur- analysis. vey for orchid pathogens in China. This paper describes

INTRODUCTION

Turnip mosaic virus (TuMV) is transmitted by at least 89 species of aphids in a non-persistent manner and is an important member of the (Walsh and Jen- ner, 2002). This virus infects more than 318 plant species of 156 genera in 43 families and it damages Cruciferae. TuMV is widespread in temperate and subtropical regions of five continents and occurs to most regions of China. Studies have shown that TuMV isolates can be grouped into two pathotypes designated as Brassica (B) and Brassi- ca-Raphanus (BR), and further subdivided into four geno- groups called basal-B, basal-BR, -BR and world-B (Ohshima et al., 2002). Placing TuMV isolates into these groups has become the main classification method of this virus (Sanchez et al., 2007; Farzadfar et al., 2009; Wang et al., 2009).

Corresponding author: Y.L. Ming Fig. 1. Electron micrograph of virus particles purified from Fax: + 86.0592.2063215 infected Phalaenopsis sp. TuMV-ZH1 particles are filamen- E-mail: [email protected] tous and measure 15-18 × 700-800 nm. 704 Turnip mosaic virus in Phalaenopsis sp. Journal of Plant Pathology (2017), 99 (3), 703-706

Table 1. Primers for genomic sequence amplification of TuMV-ZH1.

Primers Sequence (5’→3’) Primers Sequence (5’→3’)

TuMV1-1 F AAAAATATAAAAACTCAACACA F CCACAGGTACACGCACT R TATAATTGGTTGCTGCGCTCT TuMV5-2 R TCCGTACACTTCTCTACCCAT F ATGGCAGCAGTTACATTCGCATC F CAAAGTTTTCCGAACCCGTA TuMV-1 R GCATCGTACAACTTGCCTTCGT TuMV6-1 R ACGAGGTCCTTATTAGCTCT F AAAAGAACGGACGAAATTCGCAA F TCCCAGAGCGAGAAAACGAA TuMV-2 R CCGATTGCAAGTTTCCGTGACC TuMV6-2 R ATCCAAGTATTTTCCCGTCT F AGAAAGGCTACACCCAATACGAG F ATTAGCACGAAAGACGGCCAA TuMV-3 R CACGCTGTATCTGCCGCCTA TuMV-7 R ACCATCAGCATCGCAATACACC F AGTCTACCAGAACTCGTCGAA F ATTATTTCGCTGAGTTCACACC TuMV3-3 R CGACATCCAATTTGAGCCTT TuMV8-1 R ATAGTTGACTGCCAGTATGACC F ATGTATTCAAGTTTATCAATGTGCT F AGTTTGATAGCTCGTTGTCACC TuMV4-1 R TGTCTTTGTCACTCTCGTGT TuMV8-2 R CTGCTTCTGCCTCTCTCGTTC F CTAGAGCAACTGCCACGGAA F GCCAGCTCAAGAGGATCTCACC TuMV4-2 R CCACTACCCTGTGCGTTC TuMV-9 R CACCACATGCGCTAACACCA F CACACCACCAGGTCGAGA F TCAAACCGCTCATTGACCAC TuMV5-1 R TCGTGCATACTCACTAGCTG TuMV-10 R GTCCCTTGCATCCTATCAAAT

Table 2. TuMV isolate sources analysed in this study.

GenBank Isolate Country Original host plant Group accession No. CHN 12 China unknown world-B AY090660 Lu2 China Brassica sp. world-B HQ446216 UK1 UK Brassica napus world-B AF169561 C42J Brassica rapa world-B AB093625 A1 Italy Alliaria officinalis basal-B AB093598 A102/11 Italy Anemone coronaria basal-B AB093597 Fig. 2. Genome organization and sequencing strategy of Cal1 Italy Campania officinalis basal-BR AB093601 TuMV-ZH1. The approximate position of each overlapping KYD81J Japan Raphanus sativus basal-BR AB093613 region amplified by primer sets is shown. SGD311J Japan Raphanus sativus Asian-BR AB093619 ND10J Japan Raphanus sativus Asian-BR AB252130 ringspot virus, odontoglossum ringspot virus, cymbid- ium mosaic virus, bean yellow mosaic virus, cucumber the identification process and the genomic sequence of mosaic virus and orchid fleck dichorhavirus) was also TuMV-ZH1 to clarify its origin and genetic features. performed.

Virion purification. TuMV particles were purified from MATERIALS AND METHODS asymptomatic or symptom-showing (Thompson et al., 1988), were negatively stained with phosphotungstic Collection of orchid plants. A total of 403 samples, acid (PTA) for 5 min and examined under a JEM2100 distributed among 19 orchid genera (Cymbidium, Dendro- transmission electron microscope (JEOL, Japan). bium, Cattleya, Paphiopedilum, Oncidium, Phalaenopsis, Vanda, Bulbophyllum, Coelogyne, Calanthe, Cephalan- RNA extraction and RT-PCR. Total plant RNAs thera, Phaius, Malaxis, Miltonia, Neofinetia, Gastrochilus, were extracted from TuMV-infected leaves with the Epidendrum, Rhynchostylis, Dendrochilum) were collected Total­RNAExtractor (Sangon Biotech, China) following from several Chinese main orchid producing areas, such the instructions of the manufacturer. cDNA was synthe- as Guangzhou, Zhuhai, Kunming and Xiamen. sized with the M-MuLV First Strand cDNA Synthesis Kit (Sangon Biotech, China). I-ELISA detection of TuMV. The polyclonal antibod- Degenerate primers were designed based on the re- ies to TuMV were purchased from AC Diagnostic Inc. ported coat protein (CP) gene (864 bp) of TuMV (CP-F (USA) and used at the working concentration of 1:2000. 5’-GCAGGYGARACRCTYGAYG-3’ and CP-R 5’-TA- Alkaline phosphatase (AP)-labelled IgG was purchased ACCCCTTAACGCCAAGTA-3’). PCR was performed from Beijing Biosynthesis Biotechnology Co. (China) with 2.5 μl 10 × PCR buffer, 1.0 μl dNTPs (10 mM each), and used at the working concentration of 1: 10000. The 2.0 μl cDNA, 1.0 μl (200 nM) of each primer, 0.5 μl of Fast- collected samples were tested for TuMV by I-ELISA ac- Pfu Fly DNA polymerase (TransGen Biotech, China) and cording to the method described by Hornbeck (1991). 17 μl of double distilled water. Thermocycling conditions Serological detection of other quarantine plant were 5 min at 94°C, 25 cylces of 30 s at 94°C, 20 s at 55°C, (cymbidium ringspot virus, tobacco mosaic virus, tomato 60 s at 72°C, and a final extension at 72°C for 5 min. PCR Journal of Plant Pathology (2017), 99 (3), 703-706 Zheng et al. 705

Fig. 3. Phylogenetic tree similarity of TuMV-ZH1 and other typical isolates based on genomic nucleotide sequence (A) and amino acid sequence (B). TuMV-ZH1 and several other isolates are clustered in the world-B lineage. products were analyzed by electrophoresis in 1% (w/v) virions from the infected Phalaenopsis were filamentous, agarose gels and stained by GoldView. 15-18 × 700-800 nm in size (Fig. 1). Other viruses detected The degenerate primers used for genome amplification by ELISA in the orchid samples tested were odontoglos- were designed by Oligo7 using conserved regions of the sum ringspot virus (25%) and cymbidium mosaic virus genomic sequence of TuMV (Table 1). The 5’ and 3’ termi- (14%). nal sequences were determined according to the sequence Sequence comparison revealed that the expected most related to the TuMV-ZH1 isolate. The schematic ge- 864 bp CP fragment had a 99% identity at the nucleo- nome amplification strategy is shown in Fig. 2. tide level with the homologous sequence of isolate BJ-B03 (KC119187). The complete genomic sequence of TuMV- Cloning and sequencing. The expected fragments were ZH1 obtained from assembled fragments (Fig. 2) was excised from the gel, cleaned by SanPrep DNA deposited in GenBank under accession No. KF246570. Gel Extraction Kit (Sangon Biotech, China) and ligated Its genome was 9,833 nt in length excluding the poly(A) to pUCm-T vector. After the transformation of Escherichia tail. Compared with other isolates, TuMV-ZH1 shared coli DH5α, plasmid insertion was confirmed by PCR and 96.7% and 98.2% sequence identity with isolate Lu2 at restriction enzyme digestion with PstI and BamHI. Recom- the nucleotide and amino acid levels, respectively; 96.3% binant clones were sequenced at Sangon Biotech (China). and 98.0% with isolate CHN 12, and 95.8% and 97.5% with isolate UK1. The nucleotide sequence identity of Sequence and phylogenetic analysis. The TuMV CP TuMV-ZH1 with other isolates from world-B group was sequence was matched against the international gene more than 95%, but less than 87% with other typical iso- sequence databases by BLAST. The sequences were as- lates from the remaining three groups. Phylogenetic trees sembled with DNAstar (DNASTAR, USA) and DNAman showed that TuMV isolates ZH1, CHN 12, LU2, UK1 (Lynnon Co., Canada) for further analyses including ge- and C42J grouped together (Fig. 3). A comparative amino netic variability. The genome sequence was aligned with acid sequence analysis of TuMV-ZH1 and isolates CHN those of other TuMV isolates retrieved from GenBank 12 and Lu2 showed two highly variable regions (13-266 (Table 2) with Clustal W in DNAStar. Phylogenetic analy- and 925-1171 aa) with 34 mutation sites accounting for ses were performed using neighbor-joining (NJ) method in 58.6% of total mutations (58 sites) between isolates ZH1 MEGA5 (Tamura et al., 2011). The statistical significance and CHN 12, and 40 mutation sites accounting for 63.5% of branches was obtained by applying bootstrap analy- of total mutations (63 sites) between isolates ZH1 and Lu2 sis with 1000 replicates. The genome sequence of Japa- (Fig. 4). The two highly variable regions are part of pro- nese yam mosaic virus (JYMV) (GenBank accession No. tein P1 (1-362 aa) and P3 (820-1175 aa). NC_000947) was used as outgroup (Ohshima et al., 2002). TuMV-ZH1 was closely genetically related to other iso- lates from China, suggesting it may have originated in this country. Proteins P1 and P3 are the most variable among RESULTS AND DISCUSSION TuMV-ZH1-encoded proteins. TuMV P3 is an important factor determining host range and symptomatology (Jen- A TuMV isolate (ZH1) from a symptomless Phalaenop- ner et al., 2003; Suehiro et al., 2004). Also, protein P1 of sis sp. plant was characterized in this study. The presence modulates replication and host defense (Pasin of TuMV was detected by ELISA in a symptomless. Pha- et al., 2014), and for Plum pox virus (PPV), a species in laenopsis sp. sample. Of the 403 orchid samples tested, the genus Potyvirus, variations in protein P1 are associ- Phalaenopsis was the only one that reacted for TuMV by ated with host-dependent pathogenicity (Maliogka et al., RT-PCR. As shown by electron micrographs, purified 2012). Based on these findings, it is predicted that proteins 706 Turnip mosaic virus in Phalaenopsis sp. Journal of Plant Pathology (2017), 99 (3), 703-706

Fig. 4. Amino acid sequence similarity analysis between isolates TuMV-ZH1, CHN 12 and Lu2. CHN 12 and Lu2 are the closest to TuMV-ZH1 in phylogenetic relationship. There are two highly variable regions in the genomes of 13-266 aa and 925-1171 aa, respectively.

P1 and P3 of TuMV-ZH1 could be involved in sustain- Maliogka V.I., Salvador B., Carbonell A., Sáenz P., León D.S., ing TuMV infection in a symptomless host. More work is Oliveros J.C., Delgadillo M.O., García J.A., Simón-Mateo C., needed to identify the amino acid residues involved in the 2012. Virus variants with differences in the P1 protein coex- interaction of TuMV-ZH1 and Phalaenopsis sp. ist in a Plum pox virus population and display particular host-dependent pathogenicity features. Molecular Plant Pa- thology 8: 877-886. ACKNOWLEDGEMENTS Ohshima K., Yamaguchi Y., Hirota R., Hamamoto T., Tomimu- ra K., Tan Z., Sano T., Azuhata F., Walsh J.A., Fletcher J., The authors thank Prof. Chang Chin-an (Graduate In- Chen J., Gera A., Gibbs A., 2002. Molecular evolution of stitute of Biochemical Sciences and Technology, Chaoy- Turnip mosaic virus: Evidence of host adaptation, genetic recombination and geographical spread. Journal of General ang University of Technology, Taichung, 41349, Taiwan) Virology : 1511-1521. for providing experimental materials and technical as- 83 sistance. We thank Peter Sol Reinach (Wenzhou Medi- Pasin F., Simón-Mateo C., García J.A., 2014. The hypervariable cal University) for providing language editing service of amino-terminus of P1 protease modulates potyviral replica- tion and host defense responses. PLoS Pathogens : e1003985. this manuscript. This work is supported by the grant from 3 Fujian Provincial Natural Science Foundation of China Sanchez F., Rodriguez-Mateos M., Tourino A., Fresno J., Go- (2011D002) and Xiamen Municipal Natural Science Foun- mez-Campo C., Jenner C.E., Walsh J.A., Ponz F., 2007. Iden- dation (3502Z20127015 & 3502Z20162014). tification of new isolates of Turnip mosaic virus that cluster with less common viral strains. Archives of Virology 152: 1061-1068. Suehiro N., Natsuaki T., Watanabe T., Okuda S., 2004. An im- REFERENCES portant determinant of the ability of Turnip mosaic virus to Farzadfar S., Tomitaka Y., Ikematsu M., Golnaraghi A.R., Pour- infect Brassica spp. And/or raphanus sativus is in its p3 pro- rahim R., Ohshima K., 2009. Molecular characterisation of tein. Journal of General Virology 85: 2087-2098. Turnip mosaic virus isolates from weeds. Euro- Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Ku- pean Journal of Plant Pathology 124: 45-55. mar S., 2011. Mega5: Molecular evolutionary genetics analy- Hornbeck P., 1991. Assays for antibody production. Current sis using maximum likelihood, evolutionary distance, and Protocols in Immunology 2.1.1-2.1.22. maximum parsimony methods. Molecular Biology and Evolu- Inouye N., 1992. On virus diseases of orchids in japan. Proceed- tion 28: 2731-2739. ings of Nagoya International Orchid Congress 1992, Nagoya, Thompson S., Fraser R.S., Barnden K.L., 1988. A beneficial Japan: 51-59. effect of trypsin on the purification of Turnip mosaic virus Jenner C.E., Tomimura K., Ohshima K., Hughes S.L., Walsh (TuMV) and other potyviruses. Journal of Virological Meth- J.A., 2002. Mutations in Turnip mosaic virus P3 and cylindri- ods 20: 57-64. cal inclusion proteins are separately required to overcome Walsh J.A., Jenner C.E., 2002. Turnip mosaic virus and the quest two Brassica napus resistance genes. Virology 300: 50-59. for durable resistance. Molecular Plant Pathology 3: 289-300. Jenner C.E., Wang X.W., Tomimura K., Ohshima K., Ponz F., Walsh J.A., Rusholme R.L., Hughes S.L., Jenner C.E., Bam- Walsh J.A., 2003. The dual role of the potyvirus P3 protein of bridge J.M., Lydiate D.J., Green S.K., 2002. Different class- Turnip mosaic virus as a symptom and avirulence determinant es of resistance to Turnip mosaic virus in Brassica rapa. Euro- in . Molecular Plant-Microbe Interactions 16: 777-784. pean Journal of Plant Pathology 108: 15-20. Lesemann D.E., Vetten H.J., 1985. The occurrence of Tobacco Wang H.Y., Liu J.L., Gao R., Chen J., Shao Y.H., Li X.D., 2009. rattle and Turnip mosaic viruses in Orchis spp. and of an unidentified potyvirus in Cypripedium calceolus. Acta Horti- Complete genomic sequence analyses of Turnip mosaic virus basal-BR isolates from China. Virus Genes : 421-428. culturae 164: 45-54. 38

Received April 17, 2017 Accepted September 4, 2017