First Report of Cucumber mosaic virus Infecting Solanum jasminoides in Italy

Solanum jasminoides Paxton (potato vine or jasmine nightshade) is a vegetatively propagated ornamental species within the Solanaceae family. Recently, symptomless plants of this species were reported as natural hosts of the quarantine pest, Potato spindle tuber viroid (PSTVd) in Italy (1). In January 2008, approximately 1,000 potted, 2-year-old plants of S. jasminoides growing in an ornamental nursery in Sicily showed virus-like mosaic and malformation of leaves. Symptoms were observed on approximately 60% of the plants. Leaf tissue, collected from 30 symptomatic and 10 symptomless plants, was analyzed by double-antibody sandwich-ELISA with polyclonal antisera specific to Cucumber mosaic virus (CMV), Tomato spotted wilt virus, and Impatiens necrotic spot virus (Loewe Biochemica, Sauerlach, Germany). The same samples were also analyzed by tissue-printing hybridization with a PSTVd-specific digoxigenin-labelled riboprobe. All the symptomatic samples tested positive only with antisera against CMV, but negative in all other tests. The symptomless samples were negative in all the performed tests. To confirm the association of CMV with the diseased plants, total RNA was extracted from the same samples (RNeasy Plant Mini Kit; Qiagen, Hilden, Germany) and analyzed by reverse transcription (RT)-PCR using CMV-specific primers MP+5′-CATGGCTTTCCAAGGTACCAG-3′ and MP-5′-CTAAAGACCGTTAACCACCTGC-3′ that amplify a 844-bp fragment from the MP gene (2). The expected fragment was amplified only from samples of symptomatic tissue. CMV was also detected in mother plants grown in the same nursery and showing same mosaic symptoms. Definitive identification of the pathogen was obtained by cloning and sequencing the RT-PCR product. The obtained sequence (GenBank Accession No. EU828783) had 99 and 98% similarity with the subgroup I-A isolates CMV-LUN (GenBank Accession No. EU432183) and CMV-Fny (GenBank Accession No. DI0538), respectively. To our knowledge, this is the first report of CMV infecting S. jasminoides and it adds a new host to the more than 1,000 species (85 plant families) infected by this virus. The high incidence of the disease in the nursery could be due to propagation of cuttings from an infected source. References: (1) F. Di Serio. J. Plant Pathol. 89:297, 2007. (2) H. X. Lin et al. J. Virol. 78:6666, 2004.

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Lamium maculatum is a Natural Host for Cucumber mosaic virus

Plant Disease ◽

10.1094/pdis-08-12-0717-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 150-150 ◽

Cited By ~ 1

Author(s):

R. Bešta-Gajević ◽

A. Jerković-Mujkić ◽

S. Pilić ◽

I. Stanković ◽

A. Vučurović ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Impatiens Necrotic Spot Virus ◽

Natural Occurrence ◽

Rt Pcr ◽

Natural Ecosystems ◽

New Host ◽

Total Rna ◽

Negative Controls ◽

Das Elisa

Lamium maculatum L. (spotted dead-nettle) is a flowering perennial ornamental that is commonly grown as a landscape plant for an effective ground cover. In June 2010, severe mosaic accompanied by reddish brown necrosis and leaf deformation was noticed on 80% of L. maculatum growing in shade under trees and shrubs in Sarajevo (Bosnia and Herzegovina). Leaves from 10 symptomatic L. maculatum plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV), the most important viral pathogens of ornamental plants (1,2). Commercial positive and negative controls and extracts from healthy L. maculatum leaves were included in each assay. All samples tested negative for TSWV and INSV and positive for CMV. The virus was mechanically transmitted to test plants and young virus-free plants of L. maculatum using 0.01 M phosphate buffer (pH 7). The virus caused chlorotic local lesions on Chenopodium quinoa, while systemic mosaic was observed on Capsicum annuum ‘Rotund,’ Nicotiana rustica, N. glutinosa, N. tabacum ‘White Burley,’ and Phaseolus vulgaris ‘Top Crop.’ The virus was transmitted mechanically to L. maculatum and induced symptoms resembling those observed on the source plants. Inoculated plants were assayed by DAS-ELISA and all five inoculated plants of each species tested positive for CMV. The presence of CMV in L. maculatum as well as mechanically infected N. glutinosa plants was further confirmed by RT-PCR. Total RNA from symptomatic leaves was isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) following the manufacturer's instructions. The primer pair, CMVAu1u/CMVAu2d, that amplifies the entire coat protein (CP) gene and part of 3′- and 5′-UTRs was used for both amplification and sequencing (4). Total RNA obtained from the Serbian CMV isolate from pumpkin (GenBank Accession No. HM065510) and a healthy L. maculatum plant were used as positive and negative controls, respectively. All naturally and mechanically infected plants as well as the positive control yielded an amplicon of the expected size (850 bp). No amplicon was observed in the healthy control. The amplified product derived from isolate 3-Lam was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions and deposited in GenBank (JX436358). Sequence analysis of the CP open reading frame (657 nt), conducted with MEGA5 software, revealed that the isolate 3-Lam showed the highest nucleotide identity of 99.4% (99.1% amino acid identity) with CMV isolates from Serbia, Australia, and the USA (GQ340670, U22821, and U20668, respectively). To our knowledge, this is the first report of the natural occurrence of CMV on L. maculatum worldwide and it adds a new host to over 1,241 species (101 plant families) infected by this virus (3). This is also an important discovery for the ornamental industry since L. maculatum is commonly grown together with other ornamental hosts of CMV in nurseries and the urban environment as well as in natural ecosystems. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (4) I. Stankovic et al. Acta Virol. 55:337, 2011.

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First Report of Cucumber mosaic virus Infecting Peperomia tuisana in Serbia

Plant Disease ◽

10.1094/pdis-01-13-0089-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 1004-1004 ◽

Cited By ~ 2

Author(s):

K. Milojević ◽

I. Stanković ◽

A. Vučurović ◽

D. Ristić ◽

D. Milošević ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Disease Incidence ◽

Impatiens Necrotic Spot Virus ◽

Rt Pcr ◽

Test Species ◽

First Report ◽

Cp Gene ◽

Negative Controls ◽

Das Elisa

Peperomia tuisana C.DC. ex Pittier (family Piperaceae) is an attractive succulent grown as an ornamental. Despite its tropical origins, it can be successfully grown indoors in any climate. In March 2012, three samples of P. tuisana showing virus-like symptoms were collected from a commercial greenhouse in Zemun (District of Belgrade, Serbia) in which estimated disease incidence was 80%. Infected plants showed symptoms including necrotic ringspots and line patterns that enlarged and caused necrosis of leaves. A serious leaf drop led to growth reduction and even death of the plant. Leaves from three symptomatic P. tuisana plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against the most common viral pathogens of ornamentals: Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) (1,2). Commercial positive and negative controls were included in each ELISA. Serological analyses showed that all plants were positive for CMV and negative for TSWV and INSV. The ELISA-positive sample (isolate 1-12) was mechanically inoculated onto five plants each of three test species as well as of healthy young P. tuisana using 0.01 M phosphate buffer (pH 7). Chlorotic local lesions on Chenopodium quinoa and severe mosaic and leaf malformations were observed on all inoculated Nicotiana tabacum ‘Samsun’ and N. glutinosa. Also, the virus was successfully mechanically transmitted to P. tuisana that reacted with symptoms identical to those observed on the original host plants. All mechanically inoculated plants were positive for CMV in DAS-ELISA. For further confirmation of CMV infection, reverse transcription (RT)-PCR was performed on extracts made from symptomatic P. tuisana, N. tabacum ‘Samsun,’ and N. glutinosa leaf materials. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was carried out using One-Step RT-PCR Kit (Qiagen). A CMV-specific primer pair, CMVCPfwd and CMVCPrev (3), which amplifies an 871-bp fragment of the entire coat protein (CP) gene and part of 3′- and 5′-UTRs, were used for both amplification and sequencing. Total RNAs obtained from the Serbian CMV isolate (HM065510) and healthy P. tuisana were used as positive and negative controls, respectively. A product of the correct predicted size was obtained in all naturally and mechanically infected plants, as well as positive control. No amplicon was recorded in the healthy control. The amplified product derived from isolate 1-12 was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions, deposited in GenBank (KC505441), and analyzed by MEGA5 software (4). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 1-12 shared the highest nucleotide identity of 99.1% (99.5% amino acid identity) with the Japanese isolate (AB006813). To our knowledge, this is the first report on the occurrence of CMV in P. tuisana in Serbia. This is also an important discovery since P. tuisana is commonly grown together with other ornamental hosts of CMV, and thus could represent a serious threat for future expansion of CMV in the greenhouse floriculture industry in Serbia. References: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) S. Flasinski et al. Plant Dis. 79:843, 1995. (3) K. Milojevic et al. Plant Dis. 96:1706, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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Polygala myrtifolia as a New Natural Host of Cucumber mosaic virus

Plant Disease ◽

10.1094/pdis.2002.86.12.1403b ◽

2002 ◽

Vol 86 (12) ◽

pp. 1403-1403 ◽

Cited By ~ 3

Author(s):

M. Tessitori ◽

A. Reina ◽

V. Catara ◽

G. Polizzi

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Invasive Plant ◽

Leaf Tissue ◽

Impatiens Necrotic Spot Virus ◽

Enzyme Linked Immunosorbent Assay ◽

New Host ◽

Polyclonal Antisera ◽

Polygala Myrtifolia ◽

Das Elisa

Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) are among the most important viral pathogens of ornamental plants (1). Polygala myrtifolia L. (myrtle-leaf milkwort), originating from South Africa, and a member of the Polygalaceae, was recently introduced in Italy as a cultivated ornamental shrub for its fast and attractive free-flowering growth and drought-resistant characteristics. It can become an invasive plant and is now considered a serious problem in coastal areas of Australia where it was introduced as a garden plant. In Italy, P. myrtifolia is propagated by cuttings in commercial nurseries during the summer. In the winter of 2002, plants of P. myrtifolia growing in pots in an ornamental nursery in Sicily showed virus-like mosaic and malformation of leaves that appeared lanceolate with a lack of flowering. Leaf tissue was analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antisera to CMV, TSWV (Lettuce type), and INSV. Positive ELISA results were obtained only with the CMV polyclonal antisera. Complete remission of symptoms was observed on new flushes after pruning and incubation of infected plants at warm temperatures (30 and 20°C, day and night, respectively). This evidence led to the hypothesis that the disease or virus was disseminated by transportation and propagation of plants without visible symptoms during the hot season. A survey was also performed in two historical gardens of Catania (Sicily) where a group of apparently healthy P. myrtifolia plants, from the previously mentioned ornamental nursery in Sicily, were introduced as a single specimen or to form a hedge. These plants showed the same leaf malformations and mosaic symptoms observed in the nursery. DAS-ELISA confirmed the presence of CMV but not TSWV and INSV. To our knowledge, this is the first report of CMV on P. myrtifolia and it adds a new host to over 1,000 species (85 plant families) infected by this virus. Reference: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997.

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First Report of Cucumber mosaic virus on Jatropha curcas in India

Plant Disease ◽

10.1094/pdis-92-1-0171c ◽

2008 ◽

Vol 92 (1) ◽

pp. 171-171 ◽

Cited By ~ 16

Author(s):

S. K. Raj ◽

S. Kumar ◽

S. K. Snehi ◽

U. Pathre

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Jatropha Curcas ◽

Leaf Tissue ◽

Mosaic Disease ◽

Biodiesel Fuel ◽

Rt Pcr ◽

Specific Primers ◽

First Report ◽

Gel Diffusion

Jatropha curcas L. is a major commercial biodiesel fuel crop grown on 98 million acres (39.66 million ha) in India. Severe mosaic disease accompanied by yellow spots was noticed on 15% of J. curcas growing in the experimental plots of NBRI, Lucknow, India, during October of 2006. Inoculations with sap from symptomatic plants resulted in systemic mosaic on three of seven J. curcas seedlings. Gel diffusion tests were performed with antiserum to Cucumber mosaic virus (CMV), Tobacco ringspot virus, and Chrysanthemum virus B (PVAS-242a, PVAS-157, and PVAS-349, respectively; ATCC, Manassas, VA). Leaf sap of infected plants reacted only with PVAS-242a, indicating the presence of CMV. Reverse transcription (RT)-PCR assays with CMV coat protein gene specific primers (Genbank Accession Nos. AM180922 and AM180923) and total nucleic acid extracted from symptomatic J. curcas leaf tissue yielded the expected ~650-bp amplicon, which was cloned and sequenced (GenBank Accession No. EF153739). BLAST analysis indicated 98 to 99% nucleotide identity with CMV isolates (GenBank Accession Nos. DQ914877, DQ640743, AF350450, AF281864, X89652, AF198622, DQ152254, DQ141675, and DQ028777). Phylogenetic analysis showed that the J. curcas isolate was more closely related to Indian isolates of CMV belonging to subgroup Ib. Literature surveys revealed records of Jatropha mosaic virus on J. gossypiifolia in Puerto Rico (1) and on J. curcas in India (2). To our knowledge, this is the first report of CMV on J. curcas. References: (1) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (2) D. S. A. Narayana et al. Curr. Sci. 91:584, 2006.

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First Report of Natural Infection of Penstemon acuminatus with Cucumber mosaic virus in the Treasure Valley Region of Idaho and Oregon

Plant Disease ◽

10.1094/pdis-93-7-0762a ◽

2009 ◽

Vol 93 (7) ◽

pp. 762-762 ◽

Cited By ~ 1

Author(s):

R. K. Sampangi ◽

C. Almeyda ◽

K. L. Druffel ◽

S. Krishna Mohan ◽

C. C. Shock ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Great Basin ◽

Natural Infection ◽

The United States ◽

Native Plant ◽

Rt Pcr ◽

Cmv Infection ◽

Spot Virus ◽

First Report

Penstemons are perennials that are grown for their attractive flowers in the United States. Penstemon species (P. acuminatus, P. deustus, and P. speciosus) are among the native forbs considered as a high priority for restoration of great basin rangelands. During the summer of 2008, symptoms of red spots and rings were observed on leaves of P. acuminatus (family Scrophulariaceae) in an experimental trial in Malheur County, Oregon where the seeds from several native forbs were multiplied for restoration of range plants in intermountain areas. These plants were cultivated as part of the Great Basin Native Plant Selection and Increase Project. Several native wildflower species are grown for seed production in these experimental plots. Plants showed red foliar ringspots and streaks late in the season. Fungal or bacterial infection was ruled out. Two tospoviruses, Impatiens necrotic spot virus and Tomato spotted wilt virus, and one nepovirus, Tomato ring spot virus, are known to infect penstemon (2,3). Recently, a strain of Turnip vein-clearing virus, referred to as Penstemon ringspot virus, was reported in penstemon from Minnesota (1). Symptomatic leaves from the penstemon plants were negative for these viruses when tested by ELISA or reverse transcription (RT)-PCR. However, samples were found to be positive for Cucumber mosaic virus (CMV) when tested by a commercially available kit (Agdia Inc., Elkhart, IN). To verify CMV infection, total nucleic acid extracts from the symptomatic areas of the leaves were prepared and used in RT-PCR. Primers specific to the RNA-3 of CMV were designed on the basis of CMV sequences available in GenBank. The primer pair consisted of CMV V166: 5′ CCA ACC TTT GTA GGG AGT GA 3′ and CMV C563: 5′ TAC ACG AGG ACG GCG TAC TT 3′. An amplicon of the expected size (400 bp) was obtained and cloned and sequenced. BLAST search of the GenBank for related sequences showed that the sequence obtained from penstemon was highly identical to several CMV sequences, with the highest identity (98%) with that of a sequence from Taiwan (GenBank No. D49496). CMV from infected penstemon was successfully transmitted by mechanical inoculation to cucumber seedlings. Infection of cucumber plants was confirmed by ELISA and RT-PCR. To our knowledge, this is the first report of CMV infection of P. acuminatus. With the ongoing efforts to revegetate the intermountain west with native forbs, there is a need for a comprehensive survey of pests and diseases affecting these plants. References: (1) B. E. Lockhart et al. Plant Dis. 92:725, 2008. (2) D. Louro. Acta Hortic. 431:99, 1996. (3) M. Navalinskiene et al. Trans. Estonian Agric. Univ. 209:140, 2000.

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First Report of Tomato torrado virus Infecting Tomato in Colombia

Plant Disease ◽

10.1094/pdis-11-11-1000 ◽

2012 ◽

Vol 96 (4) ◽

pp. 592-592 ◽

Cited By ~ 9

Author(s):

M. Verbeek ◽

A. M. Dullemans

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Polyclonal Antibodies ◽

Sandwich Elisa ◽

Potato Virus X ◽

Rt Pcr ◽

Leaf Extracts ◽

Serological Tests ◽

First Report ◽

Initial Symptoms

Tomato (Solanum lycopersicum L.) plants grown in plastic greenhouses near Villa de Leyva, northeast of Bogota, Colombia showed necrotic spots on the leaves in September 2008. Initial symptoms were necrosis beginning at the base of leaflets that were surrounded by yellow areas. These symptoms resembled those described for Tomato torrado virus (ToTV; family Secoviridae, genus Torradovirus), which was first found in Spain (2). Other (tentative) members of the genus Torradovirus, Tomato marchitez virus (ToMarV), Tomato chocolate spot virus (ToChSV), and Tomato chocolàte virus (ToChV) (3) induce similar symptoms on tomato plants. One sample, coded T418, was stored in the freezer and brought to our lab in 2011. Serological tests (double-antibody sandwich-ELISA) using polyclonal antibodies (Prime Diagnostics, Wageningen, The Netherlands) on leaf extracts showed the absence of Pepino mosaic virus (PepMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY). Leaf extracts were mechanically inoculated onto the indicator plants Physalis floridana, Nicotiana hesperis ‘67A’, and N. occidentalis ‘P1’ (six plants in total) and were kept in a greenhouse at 20°C with 16 h of light. Necrotic symptoms appeared 4 to 5 days postinoculation and resembled those described for ToTV (2). Two dip preparations of systemically infected P. floridana and N. occidentalis leaves were examined by electron microscopy, which revealed the presence of spherical virus particles of approximately 30 nm. To confirm the presence of ToTV, total RNA was extracted from the original leaf material and an inoculated P. floridana and N. occidentalis plant using the Qiagen Plant Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. ToTV-specific primer sets ToTV-Dp33F/ToTV-Dp20R (5′-TGCTCAATGTTGGAAACCCC-3′/5′-AGCCCTTCATAGGCTAGCC-3′, amplifying a fragment of the RNA1 polyprotein with an expected size of 751 bp) and ToTV-Dp1F/ToTV-Dp2R (5′-ACAAGAGGAGCTTGACGAGG-3′/5′-AAAGGTAGTGTAATGGTCGG-3′, amplifying a fragment on the RNA2 movement protein region with an expected size of 568 bp) were used to amplify the indicated regions in a reverse transcription (RT)-PCR using the One-Step Access RT-PCR system (Promega, Madison, WI). Amplicons of the predicted size were obtained in all tested materials. The PCR products were purified with the Qiaquick PCR Purification Kit (Qiagen) and sequenced directly. BLAST analyses of the obtained sequences (GenBank Accession Nos. JQ314230 and JQ314229) confirmed the identity of isolate T418 as ToTV, with 99% identity to isolate PRI-ToTV0301 in both fragments (GenBank Accession Nos. DQ388879 and DQ388880 for RNA1 and RNA 2, respectively). To our knowledge, this is the first report of ToTV in Colombia, and interestingly, since ToTV has been found only in Europe and Australia (1) so far, this is the first report of ToTV on the American continent. References: (1) C. F. Gambley et al. Plant Dis. 94:486, 2010. (2) M. Verbeek et al. Arch. Virol. 152:881, 2007. (3) M. Verbeek et al. Arch. Virol. 155:751, 2010.

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First Report of Cucumber mosaic virus Subgroup IA Isolate Infecting Yucca aloifolia in Italy

Plant Disease ◽

10.1094/pdis-03-14-0324-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1284-1284 ◽

Cited By ~ 1

Author(s):

G. Parrella ◽

B. Greco

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Reverse Transcription ◽

Ornamental Plants ◽

Aphid Transmission ◽

Rt Pcr ◽

Cmv Infection ◽

Campania Region ◽

First Report ◽

Pcr Products

Yucca aloifolia L. (Spanish bayonet), family Asparagaceae, is the type species of the genus Yucca. It is native to Mexico and the West Indies and is appreciated worldwide as an ornamental plant. In 2013, during a survey for viruses in ornamental plants in the Campania region of southern Italy, symptoms consisting of bright chlorotic spots and ring spots 1 to 3 mm in diameter with some necrotic streaks were observed on leaves of two plants of Y. aloifolia growing in a nursery located in the Pignataro Maggiore municipality, Caserta Province. Cucumber mosaic virus (CMV) infection was suspected because the symptoms resembled those caused by CMV in Yucca flaccida (1). A range of herbal plant indicators was inoculated with sap extracts of symptomatic Y. aloifolia plants and developed symptoms indicative of CMV. Furthermore, 30 nm isometric virus particles were observed in the same Y. aloifolia sap extracts by transmission electron microscopy. The identity of the virus was confirmed by positive reaction in ELISA tests with CMV polyclonal antisera (Bioreba) conducted on sap extracts of symptomatic Y. aloifolia plants and systemically infected symptomatic hosts (i.e., Nicotiana tabacum, N. glutinosa, Cucumber sativus cv. Marketer, Solanum lycopersicum cv. San Marzano). The presence of CMV in the two naturally infected Y. aloifolia and other mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. RT-PCR was carried out with the ImProm-II Reverse Transcription System first-strand synthesis reaction (Promega) using the primer pair CMV1 and CMV2 (2). These primers amplify part of the CP gene and part of the 3′-noncoding region of CMV RNA3 and were designed to produce amplicons of different sizes to distinguish CMV isolates belonging to subgroups I or II (3). RT-PCR products were obtained from both naturally infected Y. aloifolia and mechanically inoculated plants as well as from PAE1 isolate of CMV (2), used as positive control, but not from healthy plants. Based on the length of the amplicons obtained (487 bp), the CMV isolate from Y. aloifolia (named YAL) belonged to subgroup I (3). The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen), cloned in the pGEMT vector (Promega), and three independent clones were sequenced at MWG (Ebersberg, Germany). Sequences obtained from the two CMV-infected Y. aloifolia plants were identical. This sequence was deposited at GenBank (Accession No. HG965199). Multiple alignments of the YAL sequence with sequences of other CMV isolates using MEGA5 software revealed highest percentage of identity (98.9%) with the isolates Z (AB369269) and SO (AF103992) from Korea and Japan, respectively. Moreover, the YAL isolate was identified as belonging to subgroup IA, based on the presence of only one HpaII restriction site in the 487-bp sequence, as previously proposed (2). Although CMV seems to not be a major threat currently for the production of Y. aloifolia, because the farming of this plant is performed using vegetative propagation, particular attention should be given to the presence of the virus in donor mother plants in order to avoid the dispersion of infected plants that could serve as sources for aphid transmission to other susceptible plant species. To our knowledge, this is the first report of CMV infection of Y. aloifolia in the world. References: (1) I. Bouwen et al. Neth. J. Plant Pathol. 84:175, 1978. (2) G. Parrella and D. Sorrentino. J. Phytopathol. 157:762, 2009. (3) Z. Singh et al. Plant Dis. 79:713, 1995.

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First Report of Cucumber mosaic virus Associated with Capsicum chinense var. Scotch Bonnet in Florida

Plant Disease ◽

10.1094/pdis-12-13-1276-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1016-1016 ◽

Cited By ~ 1

Author(s):

B. Babu ◽

H. Dankers ◽

M. L. Paret

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Yellow Mosaic Virus ◽

Hot Pepper ◽

Crystalline Inclusion ◽

Post Inoculation ◽

Capsicum Chinense ◽

Rt Pcr ◽

First Report ◽

Pcr Assays

Scotch bonnet (Capsicum chinense) is a tropical hot pepper variety that is grown in South America, the Caribbean Islands, and in Florida, and is an important cash crop. In Florida, scotch bonnet is grown on ~100 acres annually. Virus-like leaf symptoms including mosaic and yellow mottling were observed on scotch bonnet plants in a field at Quincy, FL, with a disease incidence of ~5%. Two symptomatic and one non-symptomatic plant sample were collected from this field for identification of the causal agent associated with the symptoms. Viral inclusion assays (2) of the epidermal tissues of the symptomatic scotch bonnet samples using Azure A stain indicated the presence of spherical aggregates of crystalline inclusion bodies. Testing of the symptomatic samples using lateral flow immunoassays (Immunostrips, Agdia, Elkhart, IN) specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV), showed a positive reaction only to CMV. The sap from an infected leaf sample ground in 0.01 M Sorensons phosphate buffer (pH 7.0) was used to mechanically inoculate one healthy scotch bonnet plant (tested negative for CMV with Immunostrip) at the 2- to 3-leaf stage. The inoculated plant developed mild mosaic and mottling symptoms 12 to 14 days post inoculation. The presence of CMV in the mechanically inoculated plant was further verified using CMV Immunostrips. Total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from the previously collected two symptomatic and one non-symptomatic scotch bonnet samples. The samples were subjected to reverse-transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, Grand Island, NY), and using multiplex RT-PCR primer sets (1). The primers were designed to differentiate the CMV subgroup I and II, targeting the partial coat protein gene and the 3′UTR. The RT-PCR assays using the multiplex primers produced an amplicon of 590 bp, with the CMV subgroup I primers. The RT-PCR product was only amplified from the symptomatic leaf samples. The obtained amplicons were gel eluted, and directly sequenced bi-directionally (GenBank Accession Nos. KF805389 and KF805390). BLAST analysis of these sequences showed 97 to 98% nucleotide identities with the CMV isolates in the NCBI database. The isolates collected in Florida exhibited highest identity (98%) with the CMV isolate from tomato (DQ302718). These results revealed the association of CMV subgroup I with symptomatic scotch bonnet leaf samples. Although CMV has been reported from scotch bonnet, this is the first report of its occurrence in Florida. References: (1) S. Chen et al. Acta Biochim Biophys Sin. 43:465, 2011. (2) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986.

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First Report of Lettuce big-vein associated virus (Varicosavirus) Infecting Lettuce in Mexico

Plant Disease ◽

10.1094/pdis-07-13-0761-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 573-573 ◽

Cited By ~ 3

Author(s):

D. L. Ochoa-Martínez ◽

J. Alfonsina-Hernández ◽

J. Sánchez-Escudero ◽

D. Rodríguez-Martínez ◽

J. Vera-Graziano

Keyword(s):

Mosaic Virus ◽

Consensus Sequence ◽

Leaf Tissue ◽

Plant Viruses ◽

Australian Isolate ◽

Healthy Plant ◽

Rt Pcr ◽

First Report ◽

Chlorotic Spot ◽

Spanish Isolate

Lettuce (Lactuca sativa) is a common consumed vegetable and a major source of income and nutrition for small farmers in Mexico. This crop is infected with at least nine viruses: Mirafiori lettuce big-vein virus (MiLBVV), Lettuce big-vein associated virus (LBVaV), both transmitted by the soil-borne fungus Olpidium brassicae; Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV), Lettuce mottle virus (LMoV), Cucumber mosaic virus (CMV), Bidens mosaic virus (BiMV), and Lettuce mosaic virus (LMV) (1). From March to May 2012, a disease on lettuce was observed in the south region of Mexico City displaying mild to severe mosaic, leaf deformation, reduced growth, slight thickening of the main vein, and plant death. At the beginning of the epidemic there were just a few plants with visible symptoms and 7 days later the entire crop was affected, causing a loss of 93% of the plants. It was estimated by counting the number of severely affected or dead plants in three plots. No thrips, aphids, or whiteflies were observed in the crop during this time. Twenty plants with similar symptoms were collected and tested by RT-PCR using the primers LBVaVF 5′-AACACTATGGGCATCCACAT-3′ and LBVaVR 5′-GCATGTCAGCAATCAGAGGA-3′ specific for the coat protein gene of LBVaV, amplifying a 322-bp fragment. Primers CP829F 5′-CCWACTTCATCAGTTGAGCGCTG-3′ and CP1418R 5′-TATCAGCTCCCTACACTATCCTCGC-3′ were used to detect MiLBVV (2). No amplification was obtained for MiLBVaV in any plants tested. PCR products of approximately 300 bp were obtained from four out of 20 symptomatic lettuce samples tested for LBVaV, but not from healthy plant and water controls. These results suggest the presence of another virus in symptomatic lettuce plants. Amplicons were gel-purified and sequenced using LBVaVF and LBVaVR primers. A consensus sequence was generated using the Bioedit v. 5 program. Both sequences of these Mexican lettuce isolates were 100% identical (Accession Nos. KC776266.1 and KC776267.1) and had identities between 94 and 99% to all sequences of LBVaV available in GenBank. Additionally, when alignments were made using ClustalW, these sequences showed identities of 99.7% to Almeria-Spanish isolate (Accession No. AY581686.1); 99.4% to Granada-Spanish isolate (AY581689.1); 99.1% to Dutch isolate (JN710441.1), Iranian isolate (JN400921.1), Australian isolate (GU220725.1), Brazilian isolate (DQ530354.1), England isolate (AY581690.1), and American isolate (AY496053.1); 96.2% to Australian isolate (GU220722.1); 96.3% to Japanese isolate (AB190527.1); and 92.8% to Murcia-Spanish isolate (AY581691.1). Twenty lettuce plants were mechanically inoculated with leaf tissue taken from the four plants collected in the field and tested positive for LBVaV by RT-PCR; 12 days after inoculation, mosaic symptoms were observed in all inoculated plants and six of them were analyzed individually by RT-PCR obtaining a fragment of the expected size. To our knowledge, this is the first report of LBVaV infecting lettuce in Mexico. Further surveys and monitoring of LBVaV incidence and distribution in the region, vector competence of olpidium species, and impact on the crop quality are in progress. References: (1) P. M. Agenor et al. Plant Viruses 2:35, 2008. (2) R. J. Hayes et al. Plant Dis. 90:233, 2006.

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First Report of Cucumber mosaic virus on Vigna marina in Taiwan

Plant Disease ◽

10.1094/pdis-06-10-0459 ◽

2010 ◽

Vol 94 (10) ◽

pp. 1267-1267 ◽

Cited By ~ 1

Author(s):

T.-C. Deng ◽

C.-H. Tsai ◽

H.-L. Tsai ◽

J.-Y. Liao ◽

W.-C. Huang

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Broad Bean ◽

Solomon Islands ◽

Natural Host ◽

Vector System ◽

Rt Pcr ◽

First Report ◽

Wilt Virus ◽

Broad Bean Wilt Virus

Vigna marina (Burm.) Merr., the dune bean or notched cowpea, is a tropical creeping vine that grows on sand dunes along the coastal regions of Taiwan. Although V. marina is a weed, some varieties are also grown for fodder and food. This legume is a natural host of Bean common mosaic virus in the Solomon Islands (1) and Alfalfa mosaic virus or Beet western yellows virus in Australia (2). In April 2009, plants of V. marina showing severe mosaic and chlorotic ringspots on the foliage were found in the coastal region of Hualien County in eastern Taiwan. Indirect ELISA on a single diseased plant showed positive results with antibodies against the cucumber isolate of Cucumber mosaic virus (CMV) but negative to Broad bean wilt virus-1, Broad bean wilt virus-2, and some potyviruses (Agdia Inc., Elkhart, IN). A pure isolate of CMV was obtained from V. marina through three successive passages of single lesion isolation in sap-inoculated Chenopodium quinoa. Results of mechanical inoculations showed that the CMV-V. marina isolate was successfully transmitted to C. amaranticolor, C. murale, C. quinoa, Chrysanthemum coronarium, Gomphrena globosa, Nicotiana benthamiana, N. tabacum cv. Vam-Hicks, Phaseolus limensis, P. lunatus, P. vulgaris, Tetragonia tetragonioides, V. marina, V. radiata, and V. unguiculata subsp. sesquipedalis. These results of artificial inoculations were confirmed by ELISA. Homologous reactions of the CMV-V. marina isolate with a stock polyclonal antiserum against the CMV-cucumber isolate (4) were observed in sodium dodecyl sulfate-immunodiffusion. To determine the specific CMV subgroup, total RNA was extracted from inoculated leaves of C. quinoa using the Total Plant RNA Extraction Miniprep System (Viogene, Sunnyvale, CA). A DNA fragment of 940 bp covering the 3′ end of the coat protein gene and C-terminal noncoding region of RNA-3 was amplified using the Cucumovirus-specific primers (3) after reverse transcription (RT)-PCR with AccuPower RT/PCR PreMix Kit (Bioneer, Daejeon, Korea). The product was gel purified by Micro-Elute DNA/Clean Extraction Kit (GeneMark Technology Co., Tainan, Taiwan) and cloned in yT&A Cloning Vector System (Yeastern Biotech Co., Taipei, Taiwan) for sequencing (Mission Biotech Co., Taipei, Taiwan) and the sequence was submitted to GenBank (No. HM015286). Pairwise comparisons of the sequence of CMV-V. marina isolate with corresponding sequences of other CMV isolates revealed the maximum (95 to 96%) nucleotide identities with CMV subgroup IB isolates (strains Nt9 and Tfn) compared with 94 to 95% identities with subgroup IA isolates (strains Y and Fny) or 77 to 78% identities with subgroup II (strains LS and Q). These results suggest that CMV is the causal agent for the mosaic disease of V. marina in Taiwan and the isolate belongs to subgroup I. To our knowledge, this is the first report of V. marina as a natural host of CMV. This strain of CMV with specific pathogenicity could threaten crop production in the coastal zones. In addition, V. marina associated with native coastal vegetation was injured by CMV infection, which might lead to ecological impacts on shoreline fading. References: (1) A. A. Brunt. Surveys for Plant Viruses and Virus Diseases in Solomon Islands. FAO, Rome, 1987. (2) C. Büchen-Osmond, ed. Viruses of Plants in Australia. Retrieved from http://www.ictvdb.rothamsted.ac.uk/Aussi/aussi.htm . September, 2002. (3) S. K. Choi et al. J. Virol. Methods 83:67, 1999. (4) S. H. Hseu et al. Plant Prot. Bull. (Taiwan) 29:233, 1987.

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