Poinsettia Mosaic Virus Research Articles

Natural infection of purple passion fruit (Passiflora edulis f. edulis) by anovel member of the family Tymoviridae in Colombia.

Purple passion fruit is one of the most important fruit exports of Colombia, but its productivity is being compromised by the emergence of several viral diseases. High-throughput sequencing (HTS) surveys of viruses in purple passion fruit fields in the province of Antioquia suggested infection by anew member of the family Tymoviridae. In this work, we characterize the complete genome sequence of this virus, tentatively named purple passionfruit leaf deformation virus (PpLDV), and evaluate its distribution in Antioquia. PpLDV was assembled at high coverage in four datasets from different regions. The 6.1kb genome of PpLDV encodes asingle polyprotein with domains characteristic of the family Tymoviridae, contains amarafibox-like promoter and the 3'-UTR can fold into atRNA-like secondary structure with avaline anti-codon. Phylogenetic analysis of the polyprotein revealed that PpLDV is adistinct member of the family Tymoviridae, more closely related to the genus Tymovirus and the unclassified Poinsettia mosaic virus (PnMV). The presence of PpLDV was confirmed by RT-qPCR and RT-PCR in samples from commercial purple passion fruit fields, plantlets and seed sprouts collected in Antioquia using primers designed in this study. Keywords: high-throughput sequencing; Marafivirus; Passifloraceae; plant virology; RT-qPCR; Tymovirus.

Identification of Poinsettia mosaic virus (Tymovirus) in commercial varieties of Euphorbia pulcherrima

En México, el <em>Poinsettia mosaic virus</em> (PnMV) infecta dos variedades comerciales de nochebuena (<em>Euphorbia</em> <em>pulcherrima</em>); sin embargo, se desconoce la presencia del virus en otras variedades comercializadas en el país. Los objetivos de esta investigación fueron la identificación del PnMV en 20 variedades comerciales de nochebuena y evaluación de dos soluciones amortiguadoras en la transmisión mecánica del PnMV a especies diferenciales. La identificación del virus se realizó con pruebas serológicas y moleculares. Se inocularon cuatro especies diferenciales (<em>Nicotiana</em> <em>benthamiana</em>, <em>N. glutinosa</em>, <em>N. clevelandii</em> y <em>Chenopodium</em> <em>amaranticolor</em>) con PnMV. Se identificó a PnMV mediante DAS-ELISA en las 20 variedades comerciales, en 13 de ellas se corroboró la presencia por RT-PCR. En <em>N. benthamiana</em> se observaron verrugas y clorosis sistémica (solución de fosfatos + DIECA, pH 8.6). Se detectó el PnMV en 100% de las variedades comerciales de nochebuena evaluadas con la técnica serológica DAS-ELISA, aunque solo el 35% mostró síntomas característicos de la infección viral. <em>N. benthamiana</em> presentó síntomas putativos a la infección por el virus, pero no se corroboró molecularmente.

Identification of Poinsettia mosaic virus Associated with a Virus-like Mottle Symptom on Poinsettia in Ohio

These results represent the first confirmed report of Poinsettia mosaic virus (PnMV) in poinsettia in Ohio. They also serve to increase awareness of the virus among poinsettia growers so they may more effectively scout nursery stock for infected plants. Accepted for publication 22 May 2013. Published 13 August 2013.

First report of Tobacco mosaic virus (TMV) and Poinsettia mosaic virus (PnMV) in poinsettia (Euphorbia pulcherrima Willd. Ex Klotzch) in Mexico

Cite This Article T, O. O., Martínez, D. O., Rojas, S. R., Ponce, G. V., Díaz, C. N. (2013). First report of Tobacco mosaic virus (TMV) and Poinsettia mosaic virus (PnMV) in poinsettia (Euphorbia pulcherrima Willd. Ex Klotzch) in Mexico. Phyton-International Journal of Experimental Botany, 82(all), 235–241.

PRODUCTION OF TRANSGENIC POINSETTIA WITH RESISTANCE AGAINST POINSETTIA MOSAIC VIRUS (PNMV) USING AGROBACTERIUM-MEDIATED TRANSFORMATION

Genetic engineering plays a significant role for the improvement of ornamental crops. Using an Agrobacterium-mediated transformation approach, we have produced transgenic poinsettia conferring resistance against Poinsettia mosaic virus (PnMV). A. tumefaciens harbouring three hairpin (hp) RNA gene constructs to generate silencing-based resistance to PnMV was introduced into internode stem explants of poinsettia cultivar Millenium. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) revealed that resistance to mechanical inoculation of PnMV was found among the transgenic poinsettia lines, whereas the controls were susceptible. Grafting technique was used to re-introduce the free branching factor, phytoplasma, back to the selected desirable transgenic poinsettia lines. Subsequently, fresh cuttings derived from these lines with phytoplasma and branches were further investigated for the stability of PnMV resistance and their morphological performance compared with non-transformed controls. Currently, these lines are undergoing evaluation at the grower's facility for future commercialization.

Genetic heterogeneity found in the replicase gene of poinsettia mosaic virus isolates

The complete nucleotide sequences of five isolates of poinsettia mosaic virus (PnMV) from Japan (JN, JO1, JO2, JO4, and JO5) were determined. These isolates contained a single large open reading frame in their genomes and shared 96.6-97.8% identity at the nucleotide level and 91.3-98.1% identity at the amino acid level with two previously reported European isolates. Interestingly, the JO isolates were found to possess eight common translational frameshift sites in the interdomain region between the methyltransferase and protease domains, resulting in considerable variation in the interdomain region compared to the other isolates. This suggests that PnMV might have evolved by creating variations in its genome by such translational frameshifts.

Symptomless infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus

An infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus (PnMV) was generated. It consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kDa. Sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from Germany. RNA transcripts derived from this cDNA were infectious in Nicotiana benthamiana. However, plants did not present typical PnMV symptoms. Furthermore, RNA transcripts from this cDNA clone were not infectious in poinsettia. Serial propagation of this cDNA clone in N. benthamiana plants restored symptom induction in this host but did not re-establish infectivity in poinsettia.

Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

Detection of Poinsettia mosaic virus by RT-PCR in Euphorbia spp. in New Zealand.

Euphorbia pulcherrima (poinsettias) are commonly infected with Poinsettia mosaic virus (PnMV), which resembles the Tymovirus genus in its morphology and viral properties (2) but is closer to the Marafivirus genus at the sequence level (1). Symptoms induced by PnMV range from leaf mottling and bract distortion to symptomless (2). The presence of PnMV in plants imported into New Zealand had never been proven. Leaves of 10 E. pulcherrima samples and six samples from other Euphorbia spp. (E. atropurpurea, E. lambii, E. leuconeura, E. mellifera, E. milii, and E. piscatorial) were collected in the Auckland area, North Island in 2002. Isometric particles of 26 to 30 nm in diameter were observed with electron microscopy in 3 of 10 E. pulcherrima samples. These three samples produced systemic chlorosis and crinkling symptoms on mechanically inoculated Nicotiana benthamiana, which tested PnMV positive by double-antibody sandwich (DAS)-ELISA (Agdia, Elkart, IN). No particles or symptoms on N. benthamiana were observed with the other Euphorbia spp., which were also PnMV-negative by DAS-ELISA. A reverse transcription-polymerase chain reaction (RT-PCR) was developed to further characterize PnMV. Specific primers were designed from the PnMV complete genome sequence (Genbank Accession No. AJ271595) using the Primer3 web-based software (4). Primer PnMV-F1 (5'-CCTGTATTGTCTCTTGCCGTCC-3') and primer PnMV-R1 (5'-AGAGGAAAGGAAAAGGTGGAGG-3') amplified a 764-bp product from nt 5291 of the 5'-end RNA polymerase gene to nt 6082 of the 3'-untranslated region (UTR). Total RNA was extracted from leaf samples using the Qiagen Plant RNeasy Kit (Qiagen Inc., Chastworth, CA). RT was carried out by using PnMV-R1 primer and MMLV reverse transcriptase (Promega, Madison, WI). The PCR was performed in a 20-μl volume reaction containing 2 μl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM PnMV-F1 primer, and 1 U of Taq polymerase (Promega) with a denaturation step (94°C for 5 min), 30 amplification cycles (94°C for 30 s; 55°C for 30 s; 72°C for 1 min), and a final elongation (72°C for 5 min). The sequence of the RT-PCR product (Genbank Accession No. DQ462438) had 98.7% amino acid identity to PnMV. PCR products were obtained from two of three PnMV ELISA-positive E. pulcherrima and three of three PnMV ELISA-positive symptomatic N. benthamiana. The failure to amplify the fragment from all ELISA-positive PnMV is likely because of the presence of inhibitors and latex in E. pulcherrima (3) that make the RNA extraction difficult. Thus, while RT-PCR may be useful for further characterizing PnMV isolate sequences, ELISA may be more reliable for virus detection. In conclusion, to our knowledge, this is the first report of PnMV in E. pulcherrima but not in other Euphorbia spp. in New Zealand. E. pulcherrima plants have been imported into New Zealand for nearly 40 years, and the virus is probably widespread throughout the country via retail nursery trading.

First Report on Poinsettia mosaic virus in Korea

Most plants of commercial poinsettia cultivars grown from cuttings develop mosaic and chlorotic dot symptoms on leaves. Reverse transcription-polymerase chain reaction (RT-PCR) test showed that they were infected with Poinsettia mosaic virus (PnMV). In a survey of commercially grown poinsettias conducted in Korea, PnMV was detected in ten of ten poinsettia cultivars sampled and in 100％ of 178 samples tested. The virus has isometric particles and about 29 nm in diameter. Crystalline virus particles were observed in cytoplasm of cells of diseased plants by transmission electron microscopy. Nucleotide sequence of coat protein gene of PnMV- Kl showed 97.3％ homology with that of a German isolate. This is the first report on PnMV in Korea.

Detection of Poinsettia mosaic virus Infecting Poinsettias (Euphorbia pulcherrima) in Venezuela.

Poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, was detected in several cultivars of vegetatively propagated poinsettias grown in commercial nurseries in Estado Miranda, Venezuela. Symptoms associated with the affected plants consisted of severe mottling and distortion of leaves and bracteoles. The suspect virus was mechanically transmitted to Nicotiana benthamiana. Leaf extracts and thin sections of affected leaf tissue were analyzed by transmission electron microscopy. Spherical virus particles (30 nm diameter) were observed in samples from symptomatic poinsettia plants. Ultrastructural analyses of virus-infected cells revealed aggregates of virus particles in the cytoplasm and central vacuole. The virus was purified twice from infected N. benthamiana, resulting in yields as high as 12 mg/100 g. Dissociated coat protein contained a single 24-kDa protein species. The virus was not serologically related to Carnation mottle, Bean rugose mosaic, Cowpea mosaic, Cucumber mosaic, Pea enation mosaic, Prunus necrotic ringspot, Apple mosaic, Tobacco streak, Maize rayado fino, Tomato ringspot, Bean southern mosaic, Sowbane mosaic, Andean potato latent, Belladona mottle, Scrophularia or Turnip yellow mosaic viruses, but did react positively in enzyme-linked immunosorbent assay and western blot analysis with antiserum (ATCC PVAS-476) to PnMV. Based on these results, the virus is considered to be PnMV. To our knowledge, this is the first report of PnMV infecting poinsettias in Venezuela.

Sequence Analysis and Genome Organisation of Poinsettia Mosaic Virus (PnMV) Reveal Closer Relationship to Marafiviruses Than to Tymoviruses

Sequence comparison and genome organisation of poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, revealed a closer relationship to marafiviruses. The complete nucleotide sequence of PnMV was determined. The 6099-nt RNA genome encodes a putative 221-kDa polyprotein that lacks a stop codon between the replicase and the coat protein genes, as in most tymovirus RNAs. The genomic RNA has a poly(A) tail at its 3′-terminus in contrast to the tRNA-like structure found in the RNA of most tymoviruses, and no homology was observed to the conserved noncoding region of the tymoviral 3′-termini. The tymobox of PnMV, a 16-nt region of the subgenomic RNA (sgRNA) promoter shared by most tymoviruses, differs in 3 nt from the RNA sequence of tymoviruses but is identical to the sequence of marafiviruses. At least three sgRNAs were found in PnMV-infected Euphorbia pulcherrima and in isolated PnMV particles; one that is 650 nt long encodes the 21.4-kDa coat protein, and the others are about 3.5 and 1.7 kb and contain the 5′- and the 3′-terminal parts of genomic RNA, respectively. Like tymoviruses, PnMV particles sediment as top and bottom components. The particles of the top component contain the sgRNA (650 nt) encoding the coat protein, and those of bottom component contain both genomic and sgRNAs.

Two New Hosts for Poinsettia Mosaic Virus.

Poinsettia mosaic virus (PnMV), a possible member of the genus Tymovirus, commonly infects the potted flower crop Euphorbia pulcherrima Willd. ex Klotzsch (1). Two new host species for this virus were identified during grafting experiments with E. pulcherrima and other Euphorbia spp. E. cornastra (Dressler) A. Radcliffe-Smith was reciprocally grafted with PnMV-positive E. pulcherrima cv. Eckespoint Lilo. PnMV was detected by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) in E. cornastra leaves directly below the graft union 4 weeks after grafting. Infection was not fully systemic 6 weeks after grafting when screened by DAS-ELISA with antibodies specific to PnMV (DSMZ, Braunschweig, Germany). The symptomless infection in E. cornastra persisted in cuttings from grafted plants after a 1-year observation period. E. bubalina Boiss. anatomy differs from that of E. pulcherrima. The two species did not produce a viable graft union. However, in an experiment with two attempted graftings, the E. pulcherrima scions remained turgid for 14 to 18 days. As a result of one grafting, the E. bubalina rootstock tested positive for PnMV. The virus induced a mild mosaic in E. bubalina, but no reduction in growth. To confirm virus presence in E. cornastra and E. bubalina, both DAS-ELISA and immunosorbent electron microscopy were used. Non-grafted controls remained PnMV negative. PnMV was re-isolated from both species by sap inoculation to Nicotiana benthamiana. E. coulescens Haw., E. xylophyllides Brogn. ex Lem., E. marlothiana N. E. Br., and Ricinus communis L. were not infected by PnMV after similar grafting attempts. Reference: (1) A. A. Brunt et al., eds. 1996. Viruses of Plants. CAB Int., Wallingford, UK.

DISEASE NOTES OR NEW RECORDS: Poinsettia mosaic virus - first record in Queensland

Poinsettia mosaic virus has been identified for the first time in Queensland. It produces strong mosaic symptoms in outdoor plants of some dwarf poinsettia cultivars in the winter months, but symptoms became masked in the summer. Potted dwarf poinsettias obtained from interstate nurseries were also infected.

Investigations on the nature of a graft-transmissible agent in poinsettia

The free-branching poinsettia (Euphorbia pulcherrima) cultivar Annette Hegg Brilliant Diamond contains a free-branching agent that is graft-transmissible to the restricted-branching cultivar Eckespoint C-1 Red. Transmission electron microscopy failed to reveal evidence of bacteria, fungi, or mycoplasma-like organisms in either 'Brilliant Diamond' or 'C-1 Red' plants. Treatment of both cultivars with tetracycline-hydrochloride produced no differences in branching pattern or leaf morphology in either cultivar, indicating that the agent may not be a mycoplasma-like organism. Scions of a poinsettia mosaic virus indicator species (Euphorbia cyathophora) grafted onto 'Brilliant Diamond' and 'C-1 Red' stocks exhibited the mottling symptoms characteristic of poinsettia mosaic virus, while self-grafted E. cyathophora scions showed no mottling, indicating that poinsettia mosaic virus was not the agent. The agent was not transmitted by pin prick, carborundum, or dodder (Cuscuta sp.), and ribaviran did not eliminate expression of the branching agent from 'Brilliant Diamond' plants. No differences in double-stranded RNA banding patterns were found between extracts of free- and restricted-branching poinsettias by polyacrylamide gel electrophoresis. The double-stranded RNA was attributed to poinsettia mosaic virus and other unknown RNA viruses. Attempts to detect a specific DNA associated with free-branching were inconclusive. Key words: branching agent, Euphorbia pulcherrima, graft transformation, dsRNA, poinsettia mosaic virus.

Poinsettia Mosaic Virus — a Tymovirus?

Abstract Nicotiana benthamiana was found to be a useful indicator and propagation host for poinsettia mosaic virus (PoiMV). It is not susceptible to poinsettia cryptic virus. The isometric particles of PoiMV sedimented as two components with sedimentation coefficients (s°20,w) of 55 S (“top” component) and 117 S (“bottom” component) and, after fixation, with buoyant densities (g cm‐1) in caesium chloride of 1.29 (“top”) and 1.41 (“bottom”). The A260/A280 ratios were 1.8 and 1.0 for “top” and “bottom” components, respectively. “Top” and “bottom” component particles measured 26 and 29 nm in diameter when mounted in 2 % uranyl acetate; “top” component particles were severely disrupted by 2 % neutral potassium or sodium phosphotungstate or 2 % ammonium molybdate, a phenomenon which is not observed with recognised tymoviruses. The virus particles contained c. 37 % ss‐RNA (mol. wt 2.3° 106) with a base composition of 21 % guanine, 24% adenine, 31 % cytosine and 24% uracil. PoiMV particles were detected in the cytoplasm, vacuoles and nuclei of infected poinsettia and N. benthamiana cells. As in tymovirus infections, the particles within the nuclei were apparently devoid of nucleic acid. The virus induced conspicuous peripheral vesiculation of chloroplasts; the vesicles, however, unlike those induced by recognised tymoviruses, were bound by the inner membrane only of the double chloroplast envelope. PoiMV thus lacks a group‐specific character of the tymovirus group. Serologically it is not related to any of the 17 recognised tymoviruses. It is therefore probably best recognised as a possible member of the tymovirus group.

Poinsettia Mosaic Virus in British Columbia

Poinsettia Mosaic Virus in British Columbia