Virus-like symptoms consisting of light mosaic, chlorotic spots and oak
chlorotic line patterns were observed on lilac plants (Syringa vulgaris L.) growing in a
public garden in Imola (Emilia Romagna region, Italy). The causal agent was identified
as cucumber mosaic virus (CMV) on the basis of biological, serological and nucleotide
sequence properties of partial coat protein and movement protein genes, and the
isolate was designated SYV. The CMV-SYV isolate caused mosaic symptoms on indicator
plants of Nicotiana tabacum cv. Xanthi-nc, N. rustica and Cucumis sativus, while
symptoms of local necrotic spots or pin points were observed on inoculated leaves of
Vigna unguiculata and Vicia faba. To assess genetic differences between CMV-SYV
and other known CMV isolates, phylogenetic analyses were carried out using 16 nucleotide
sequences of coat protein and movement protein genes, including for SYV. The
CMV-SYV isolate was most related to CMV subgroup IA isolates, and had 85.1-100%
nucleotide sequence similarity to subgroup I isolates. This is the first report of CMV
infecting S. vulgaris

Elisa TROIANO, Maria Grazia BELLARDI, Giuseppe PARRELLA

English

Virus-like symptoms consisting of light mosaic, chlorotic spots and oak
chlorotic line patterns were observed on lilac plants (Syringa vulgaris L.) growing in a
public garden in Imola (Emilia Romagna region, Italy). The causal agent was identified
as cucumber mosaic virus (CMV) on the basis of biological, serological and nucleotide
sequence properties of partial coat protein and movement protein genes, and the
isolate was designated SYV. The CMV-SYV isolate caused mosaic symptoms on indicator
plants of Nicotiana tabacum cv. Xanthi-nc, N. rustica and Cucumis sativus, while
symptoms of local necrotic spots or pin points were observed on inoculated leaves of
Vigna unguiculata and Vicia faba. To assess genetic differences between CMV-SYV
and other known CMV isolates, phylogenetic analyses were carried out using 16 nucleotide
sequences of coat protein and movement protein genes, including for SYV. The
CMV-SYV isolate was most related to CMV subgroup IA isolates, and had 85.1-100%
nucleotide sequence similarity to subgroup I isolates. This is the first report of CMV
infecting S. vulgaris

Elisa TROIANO

Maria Grazia BELLARDI

Giuseppe PARRELLA

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

Phytopathologia Mediterranea 58(2): 385-389, 2019Firenze University Press www.fupress.com/pmPhytopathologia MediterraneaThe international journal of the  Mediterranean Phytopathological UnionISSN 0031-9465 (print) | ISSN 1593-2095 (online) | DOI: 10.14601/Phytopathol_Mediter-10625Citation: Troiano E., Bellardi M.G., Parrella G. (2019) Syringa vulgaris is a new host for cucumber mosaic virus. Phytopathologia Mediterranea 58(2): 385-389. doi: 10.14601/Phytopathol_Mediter-10625Accepted: April 11, 2019Published: September 14, 2019Copyright: © 2019 Troiano E., Bel-lardi M.G., Parrella G. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/pm) and distributed under the terms of the Creative Com-mons Attribution License, which per-mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability Statement: All rel-evant data are within the paper and its Supporting Information files.Competing Interests: The Author(s) declare(s) no conflict of interest.Editor: Nihal Buzkan, Kahramanmaraş Sütçü Imam University, Turkey.New or Unusual Disease ReportsSyringa vulgaris is a new host for cucumber mosaic virusElisa TROIANO1, Maria Grazia BELLARDI1,2, Giuseppe PARRELLA1,*1 Istituto per la Protezione Sostenibile delle Piante del CNR, IPSP-CNR, Via Università 133, 80055 Portici (Napoli), Italy2 Dipartimento di Scienze e Tecnologie Agro-Alimentari (DISTAL), Patologia Vegetale, Alma Mater Studiorum, Università di Bologna, Viale G. Fanin 44, 40127 Bologna, Italy*Corresponding author: giuseppe.parrella@ipsp.cnr.it Summary. Virus-like symptoms consisting of light mosaic, chlorotic spots and oak chlorotic line patterns were observed on lilac plants (Syringa vulgaris L.) growing in a public garden in Imola (Emilia Romagna region, Italy). The causal agent was identified as cucumber mosaic virus (CMV) on the basis of biological, serological and nucleo-tide sequence properties of partial coat protein and movement protein genes, and the isolate was designated SYV. The CMV-SYV isolate caused mosaic symptoms on indica-tor plants of Nicotiana tabacum cv. Xanthi-nc, N. rustica and Cucumis sativus, while symptoms of local necrotic spots or pin points were observed on inoculated leaves of Vigna unguiculata and Vicia faba. To assess genetic differences between CMV-SYV and other known CMV isolates, phylogenetic analyses were carried out using 16 nucle-otide sequences of coat protein and movement protein genes, including for SYV. The CMV-SYV isolate was most related to CMV subgroup IA isolates, and had 85.1-100% nucleotide sequence similarity to subgroup I isolates. This is the first report of CMV infecting S. vulgaris.Keywords. Lilac, CMV, RT-PCR, subgroup identification.INTRODUCTIONSyringa vulgaris L. (lilac or common lilac; Oleaceae) is a popular spring-blooming ornamental shrub widely used in public and private gar-dens, for its large, showy and often fragrant inflorescences. Numerous lilac hybrids and cultivars have been developed for horticultural use. These selec-tions exhibit variation in flower colour, period of blooming, and growth habit, and they are generally vegetatively propagated by rooting softwood cuttings to maintain genetic constancy (Hartmann et al., 1990). Risks of transmitting infectious diseases through vegetative propagation, particularly those caused by viruses, can be important. To combat virus diseases carried through vegetative propagules, adoption of preventive phytosanitary mea-sures is essential, including correct pathogen diagnoses. Tomato mosaic virus (ToMV), arabis mosaic virus (ArMV), lilac leaf chlorosis virus (LLVC), toma-386 E. Troiano et aliito black ring virus (TBRV), tomato bushy stunt virus (TBSV), cherry leaf roll virus (CLRV), elm mottle virus (EMoV), strawberry latent ringspot virus (SLRSV), lilac ring mottle virus (LiRMoV) and tobacco rattle virus (TRV) have been previously described from lilac (Novák and Lanzová, 1975; Novák and Lanzová, 1977; van der Meer et al., 1976; van der Meer, 1976; Brunt, 1978; Canova and Betti, 1987; Castello et al., 1992; Cooper, 1993; Sharma-Poudyal et al., 2016). Most of these viruses elicit similar symptoms in lilac, consisting mainly of chlorotic spots and line pat-terns on the leaves (van der Meer, 1976). This symptom-atology is not indicative on the particular virus involved, and to get correct diagnoses, virus-specific detection methods are required. In this paper, we report the iden-tification and characterization of a cucumber mosa-ic virus (CMV) isolate belonging to subgroup IA, from symptomatic lilac plants showing chlorotic ringspots and line patterns. This is the first documented serologi-cal and molecular evidence for the occurrence of CMV in lilac, causing similar symptoms to other viruses asso-ciated with that plant host.MATERIALS AND METHODSSince 2016, lilac plants showing virus-like leaf symp-toms, including light mosaic, chlorotic ringspots and oak chlorotic line patterns (Figure 1), were observed in a public garden in Imola (Emilia Romagna region, Italy). Symptoms progressively and completely regressed after blooming, during the summer. In spring 2018, symp-tomatic leaves were collected from two plants, and were used in bioassay, serological and molecular tests.In bioassays, three plants each of Chenopodium qui-noa, Nicotiana rustica, N. tabacum cv. Xanthi and Vi- gna unguiculata cv. Black eye were mechanically inoc-ulated with sap obtained by macerating symptomatic lilac leaves in 0.03 M phosphate buffer (pH 7). The same leaves were tested for the presence of CMV, alfalfa mosa-ic virus (AMV) and ToMV, using double-antibody sand-wich (DAS)-ELISA commercial diagnostic kits (Bioreba AG; respectively, Art. Nr. 160675, 140575 and 152675 for the three viruses), including negative and positive controls. Samples from symptomatic and healthy leaves were also assessed for the presence of ilarviruses and nepovirus, using reverse transcription (RT)-PCR tests on total RNA extracted with the E.Z.N.A.® plant RNA kit (Omega Bio-tek, USA), and genus-specific primer pairs (Wei and Clover, 2008; Untiveros and Perez-Egusqui-za, 2010). For the (RT)-PCR tests, the procedure of Par-rella and Greco (2016) was used. Total RNA extracted from leaves of a healthy lilac plant was used as negative control, while total RNA extracted from tobacco plants infected with isolates of tomato ring spot virus (ToRSV) was used as positive control for nepovirus and parietar-ia mottle virus (PMoV) was used as positive control for ilarvirus (Parrella, 2002). Positive controls were derived from the collection of virus isolates at the Istituto per la Protezione Sostenibile delle Piante del CNR (IPSP-CNR), maintained as dehydrated leaves and stored over calci-um chloride at 4°C.In addition, and to confirm preliminary positive results obtained using CMV-specific ImmunoStrip® (Agdia Inc.) on symptomatic leaves from the two lilac plants, RT-PCR tests were also conducted using two pairs of CMV specific primers on total RNA isolated from infected and healthy lilac leaf tissues. Primer pair Figure 1. Natural symptoms on the leaves of Syringa vulgaris (lilac) plants infected by cucumber mosaic virus (CMV) isolate SYV, growing in a public garden (Northeast of Italy). Light mosaic (A), chlorotic spots (B) and oak chlorotic line patterns (C) were observed.387Lilac as a new host of CMVCMV1/CMV2 was used to amplify the partial coat pro-tein gene (CP), and part of the 3’-noncoding region of CMV RNA3 (Singh et al. 1995; Parrella and Sorrentino 2009), and primer pair CMVMP1 (5’-ATGGCTTTC-CAAGGTACCA-3’) and CMVMP2 (5’-CTAAAGAC-CGTTAACCACC-3’) was used to amplify the entire Open Reading Frame (ORF) of the movement protein gene (MP). PCR conditions were as follows: initial dena-turation cycle for 3 min at 92°C, followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C, extension for 1.5 min at 72°C. In the final cycle, the elongation time at 72°C was 7 min. PCR products of the expected length (approx. 500 bp for CMV1/CMV2 and 840 bp for CMVMP1/CMVMP2) were separated by electrophoresis in agarose gels and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). DNA sequencing was carried out at Eurofins Genom-ics. Nucleotide sequence data were assembled using BioEdit Version 5.0.9 (Hall, 1999), and then compared with corresponding regions of the other CMV isolates retrieved from the GenBank database. Multiple nucleo-tide sequence alignments were performed by using the Clustal W program, while phylogenetic and molecular evolutionary analyses were inferred using the Maximum Likelihood method (Felsenstein, 1981), both packaged in the MEGA X software (Kumar et al., 2018). Peanut stunt virus (PSV; NC002040) was used as the outgroup to root the phylogenetic tree.RESULTS AND DISCUSSIONMechanical inoculation from symptomatic lilac leaf samples induced local chlorotic lesions on C. quinoa and V. unguiculata observed 5 days post-inoculation, and vein clearing and light mosaic in Nicotiana spp. observed 14 days post-inoculation.In double-antibody sandwich (DAS)-ELISA tests, only CMV was identified from both lilac plants. These results were confirmed by RT-PCR with CMV-specific primer pairs, and amplification products were obtained from the naturally infected lilac plants and mechani-cally inoculated plants, as well as from the PAE1 isolate of CMV (Parrella and Sorrentino, 2009) used as posi-tive control. No amplification products were obtained from healthy plants (Figure 2). The sequences obtained from the two plants with the CMV1/CMV2 prim-ers were identical, as well as those obtained with the CMVMP1/CMVMP2 primers, and these were deposit-ed at GenBank (Acc. No. MH433673 for the partial CP gene and Acc. No. MH433674 for MP gene). The par-tial CP sequence of CMV-SYV was 96.5–98.6% similar to that of subgroup IA, 92.8–94.4% similar to subgroup IB and 77.0–76.5% similar to subgroup II, while the MP sequence was 96.4–99.0% similar to that of subgroup IA, 91.9–94.0% similar to subgroup IB and 79.2–79.6% similar to subgroup II. These results agree with those reported elsewhere concerning the nucleotide similari-ties within and among CMV isolate subgroups (Roossin-ck, 2002; Moury, 2004). Both of the CMV-SYV sequenc-es obtained showed greatest sequence similarity at the nucleotide level with the CMV-Fny isolate (98.6% for the partial CP gene and 99.0% for the MP gene).CMV1/CMV2 primers have been designed to pro-duce amplicons of different sizes to distinguish CMV isolates belonging to subgroups I or II (Singh et al., 1995; Parrella and Sorrentino, 2009). Based on the length of the amplicons obtained with these primers (487 bp), the CMV isolate from lilac (named SYV, from Syringa vul-garis) belonged to subgroup IA (Singh et al., 1995). This was further confirmed by the detection of one HpaII restriction site in the 487 bp sequence, as previously pro-posed for the CMV isolates belonging to subgroup IA (Parrella and Sorrentino, 2009).The bootstrap maximum-likelihood trees for nucleotide sequences of the CMV-SYV isolate and the two corresponding genomic regions of CMV isolates retrieved from the GenBank database and identified by the CMV1/CMV2 (partial CP gene) and CMVMP1/CMVMP2 (MP ORF) primer pairs, are presented Figure 2. Results obtained with CMV1/CMV2 (lanes C1, 1, 3 and 5) and CMVMP1/CMVMP2 (lanes C2, 2, 4, and 6) primer pairs in reverse transcription (RT)-PCR tests, using total RNA extracted from healthy and symptomatic leaves of Syringa vulgaris (lilac): lane Mk, DNA ladder; lanes C1 and C2, negative controls (healthy lilac plant); lanes 1 and 2, amplicons obtained with RNA extracted from the lilac plant n. 1; lanes 3 and 4, amplicons obtained with RNA extracted from the lilac plant n. 2; lanes 5 and 6, positive controls (RNA of the CMV-PAE isolate; Parrella and Sorrentino, 2009).388 E. Troiano et aliiin Figures 1A and 1B. Analyses for both fragments showed that CMV isolates could be divided into two subgroups, as subgroup I and II. As expected, sub-group I was further divided into subgroups IA and IB. For CMV-SYV, this analysis showed that it belongs to subgroup IA and is closely related to the CMV-Fny, in both the genetic regions studied. Therefore, these results further confirmed that CMV-SYV belongs to subgroup IA of CMV.Several viruses have been described in lilac (Brunt, 1978; Canova and Betti, 1987; Cooper 1993; Shar-ma-Poudyal et al., 2016). However, until now the pres-ence of CMV infections in this host have not been reported. Results of the present study indicate that the CMV-SYV isolate systemically infected lilac plants caus-ing symptoms including light mosaic, chlorotic ring-spots and oak chlorotic line patterns on the host leaves (Figure 1). CMV is efficiently transmitted by more than 75 species of aphids (Hemiptera: Aphididae), in a non-persistent, stylet-borne manner, and these insects are the main vectors disseminating CMV in nature (Jac-quemond, 2012). Nevertheless, since lilac is generally vegetatively propagated by basal shoots or root sprouts, it is important that mother-plants are carefully checked for virus infections to avoid spreading virus-infected material. To our knowledge, this is the first report of natural CMV infections in lilac.LITERATURE CITEDBrunt A.A., 1978. The occurrence, hosts and properties of lilac chlorotic leafspot virus, a newly recognized virus from Syringa vulgaris. Annals of Applied Biology 88: 383–391.Canova A., Betti L., 1987. Virus disease of ornamental shrubs. Ring mottling of dwarf lilac. Phytopathologia Mediterranea 26: 151–155.Castello J.D., Hibben C.R., Jacobi V., 1992. Isolation of tomato mosaic virus from lilac. Plant Disease 76: 696–699. Cooper J.I., 1993. Virus diseases of trees and shrubs, 2nd ed. Chapman and Hall, London, 205 pp. Felsenstein J., 1981. Evolutionary trees from DNA sequences: a maxi-mum likelihood approach. Journal of Molecular Evolution 17: 368–376.Hall T.A., 1999. BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.Hartmann H.T., Kester D.E., Davies F.T., 1990. Plant propagation principles and practices. Englewood Cliffs, New Jersey, USA: Prentice-Hall, 5th ed, 647 pp.Jacquemond M., 2012. Cucumber mosaic virus. Advances in Virus Research 84: 439–504.Figure 3. Phylogenetic trees derived from nucleotide sequences of the CMV RNA fragments obtained with the primer pairs CMV1/CMV2 and CMVMP1/CMVMP2 of SYV with the corresponding nucleotide regions of previously reported CMV isolates retrieved from Gen-Bank (accession numbers and, when available, natural host plants and geographic origins are reported in parentheses for each CMV iso-late): ZM (JN180311; Zea mais; South Korea); Rb (GU327365; Rudbeckia hirta var. pulcherrima; South Korea); Z1 (GU327368; Cucurbita pepo, South Korea); Fny (D10538; Solanum lycopersicum; United States of America); Y (D12499; Japan); Leg (D16405; Vigna unguiculata, Japan); Va (JX014248; Vigna angularis var. nipponensis, South Korea); Mf (AJ276481; South Korea); NT9 (D28780; Solanum lycopersicum; Taiwan); Tfn (Y19626; Solanum lycopersicum; Italy); Ix (U20219; Ixora sp.; Philippines); CTL (EF213025; Brassica chinensis; China); IA (AB042294; Indonesia); Q (M21464; Australia); Trk7 (L15336; Trifolium repens; Hungary); Ly (AF198103; Lupinus angustifolius; Australia); Ls (AF127976; USA); PAE1 (FN257306; Passiflora edulis; Italy); PSV (NC002040). The position of the SYV isolate is shown in the box. Pea-nut stunt virus (PSV) was used as an outgroup. Phylogenetic analyses were performed employing the maximum likelihood method pack-aged in the MEGA X software. Bootstrap values ≥ 70% are indicated at each node.389Lilac as a new host of CMVKumar S., Stecher G., Li M., Knyaz C., Tamura K., 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35: 1547–1549.Moury B., 2004. Differential selection of genes of cucum-ber mosaic virus subgroups. Molecular Biology and Evolution 21: 1602–1611.Novák J.B., Lanzová J., 1975. Etiology of two virus diseas-es of lilac (Syringa vulgaris L.) occurring in Czecho-slovakia. Biologia Plantarum 17: 226–227.Novák J.B., Lanzová J., 1977. Purification and properties of arabis mosaic and tomato bushy stunt viruses iso-lated from lilac (Syringa vulgaris L.). Biologia Planta-rum 19: 264–271.Parrella G., 2002. First report of parietaria mottle virus in Mirabilis jalapa. Plant Pathology 51: 401.Parrella G., Greco B., 2016. Sequence variation of block III segment identifies three distinct lineages with-in eggplant mottled dwarf virus isolates from Italy, Spain and Greece. Acta Virologica 60: 100–105.Parrella G., Sorrentino D., 2009. Identification of a cucumber mosaic virus isolate from Passiflora edulis in Southern Italy and validation of subgroup identi-fication by in silico restriction fragment length poly-morphism. Journal of Phytopathology 157: 762–767.Roossinck M.J., 2002. Evolutionary history of cucumber mosaic virus deduced by phylogenetic analysis. Jour-nal of Virology 76: 3382–3387.Sharma-Poudyal D., Osterbaue N.K., Putnam M.L., Scott S.W., 2016. First report of lilac ring mottle virus infecting lilac in the United States. Plant Health Prog-ress 17: 158–159.Singh Z., Jones R.A.C., Jones M.G.K., 1995. Identification of cucumber mosaic virus subgroup I isolates from banana plants affected by infectious chlorosis disease using RT- PCR. Plant Disease 79: 713–716.Untiveros M., Perez-Egusquiza M., 2010. PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae. Journal of Virological Methods 165: 97–104.van der Meer F.A., 1976. Observation on lilac ringspot. Acta Horticulturae 59: 105.van der Meer F.A., Huttinga H., Maat D.Z., 1976. Lilac ring mottle virus: isolation from lilac, some proper-ties, and relation to lilac ringspot disease. Netherland Journal of Plant Pathology 82: 67–80. Wei T., Clover G., 2008. Use of primers with 5’non-com-plementary sequences in RT-PCR for the detection of nepovirus subgroups A and B. Journal of Virological Methods 153: 16–21.

Syringa vulgaris is a new host for Cucumber mosaic virus

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Syringa vulgaris is a new host for Cucumber mosaic virus

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