First Report of Chrysanthemum stunt viroid in Various Cultivars of Argyranthemum frutescens in France

Described for the first time in Chrysanthemum indicum in the United States, Chrysanthemum stunt viroid (CSVd) was reported to naturally infect species in the Asteraceae family (1,3), as well a few hosts in other families. In May 2010 in a nursery in southwest France, the occurrence of stunted A. frutescens plantlets of cv. Butterfly showing yellow deformed leaves with terminal necrosis, which resembled the growth reduction, flower distortion or leaf necrosis symptoms reported for CSVd in Argyranthemum spp. (3), was reported. Mother plants from which the plantlets originated were asymptomatic. Reverse transcription (RT)-PCR with universal pospiviroid primers Pospi1-FW/RE (4) was performed on five symptomatic plants. A fragment of expected size (197 bp) was obtained in all cases. Viroid infection was confirmed by RT-PCR with two sets of primers: Vid-FW/RE using a 59°C annealing temperature instead of the recommended 62°C (4) and Vir-plus/minus that allows the amplification of the full-length viroid genome (2). Sequences of the three different uncloned amplicons were determined and a 355-nt contig was assembled (GenBank No. JF938538). A BLAST analysis of this full-length sequence revealed 99% identity with CSVd isolates from Chrysanthemum from Korea and Germany (GenBank Accession Nos. AF394452 and X16408). The Argyranthemum CSVd sequence differed from the Chrysanthemum ones by an A insertion at position 289 and substitutions (A to T) at positions 65 and 299. The insertion at position 289 is currently unique among CSVd sequences in GenBank. Thirty-five symptomless mother plants of A. frutescens cv. Butterfly were tested by PCR and all were shown to be infected. The difference in symptomatology observed between the mother plants and the commercial potting plants cannot be explained at this stage, but may reflect the different physiologies or growing conditions of the two kinds of plants, since these are known to affect CSVd symptoms in other hosts (1). To estimate the extent of CSVd contamination in A. frutescens, samples of 11 other cultivars originating from different nurseries were similarly analyzed. In addition to Butterfly, cvs. Sonnenstral, Maya Bofinger, Lili, Blanc Double, and Daisy Solenio were found to be infected by CSVd in the absence of clear symptomatology. The CSVd-free cultivars were Angelic Bordeaux, Dark Pink, Pink Delight, Angelic White, Dana, and Summer. The Pospi1-FW/RE amplicons from Blanc Double, Lili, and Daisy Solenio were identical to the Butterfly isolate sequence while the Maya Bofinger sequence showed one substitution (C to T) at position 256 and Sonnenstral had one substitution (T to A) at position 254. Although CSVd infection of Butterfly had been reported from Germany (3), to our knowledge, the results reported here represent the first report of CSVd in Argyranthemum for France and implicate a range of cultivars. CSVd being classified as a quarantine pest in Chrysanthemum spp. in the European Union, the finding of its significant prevalence in A. frutescens cultivars, frequently in the absence of clear symptomatology, raises the possibility that contaminated Argyranthemum may constitute a reservoir for future Chrysanthemum contamination. References: (1) I. Bouwnen and A. van Zaayen. Page 281 in: Viroids. Science Publishers, Enfield, NH, 2003. (2) T. Candresse et al. Plant Dis. 91:330, 2007. (3) W. Mentzel and E. Maiss. Z. Pflanzenk. Pfanzenschutz 107:548, 2000. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

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First Report of Barley virus G in the United States and California

Plant Disease ◽

10.1094/pdis-03-21-0478-pdn ◽

2021 ◽

Author(s):

Anna Christine Erickson ◽

Bryce Falk

Keyword(s):

United States ◽

Viral Genome ◽

The United States ◽

Full Length ◽

Yellow Dwarf ◽

Rt Pcr ◽

First Report ◽

Query Coverage ◽

Full Length Sequence ◽

The U.S

Barley (Hordeum vulgare) is a valuable annual cereal crop grown widely throughout the United States and the world. The majority of barley grown commercially in California and throughout the U.S. is used for livestock feed, with the remainder being used by the malting industry and, to a lesser extent, direct food consumption; it is also often employed as a cover crop (Lazicki et al. 2016). Yellow dwarf viruses (YDVs), in the family Luteoviridae, that infect barley and other cereal crops are common and widely distributed throughout California and the U.S. (Griesbach et al. 1989; Seabloom et al. 2009). In April 2018, five barley samples exhibiting typical symptoms of YDV infection (primarily yellowing of leaf margins and tips) collected from fields in Yolo county planted with cultivar Butta 12 , were tested for viruses. Total RNA was extracted from leaf tissue using Trizol reagent, according to the manufacturer’s protocol. RNA was used as template in a multiplexed RT-PCR assay designed for the generic detection of barley and cereal infecting YDVs, using the protocol established by Malmstrom and Shu (2004). A 372 basepair amplicon indicative of Polerovirus infection was amplified from two of the samples and sequenced (Quintara Biosciences), and the resulting data analyzed via a BLASTn search. No further testing or work was done with the three samples that tested negative. Not unexpectedly, the top result returned for one of the positive samples was Cereal yellow dwarf virus-RPV (CYDV-RPV; 98% identity), a virus common to cereals in California and the U.S.. Unexpectedly, however, the top result returned for the other sample was Barley virus G (BVG), sharing 98.43% identity with the Uiseong BVG isolate (GenBank accession LC259081). To further confirm the presence of BVG in the sample, the full-length viral genome was amplified using two-step RT-PCR with primers targeting the extreme 5’ and 3’ ends of the viral genome, using the PrimeScript RT and PrimeSTAR GXL DNA Polymerase kits (Takara Bio), cloned into the binary vector pJL89 and a BLAST search of the resulting 5621 nucleotide full-length sequence (100% query coverage) once again returned results showing the YDV to be BVG. The full-length sequence was deposited into GenBank (MW853785). Nucleotide sequence comparisons showed that the CA BVG isolate shares 96.62%, 96.57%, and 96.02% identity with the sequences of the BVG-Gimje (KT962089), BVG-Uiseong (LC259081), and BVG-Aus8 (LC500836) isolates, respectively. To our knowledge, this is the first report of barley virus G in California and in the United states. Currently the prevalence, host range and mode and timing of introduction of BVG in California and the U.S. are unknown; its impact on cereal production and yield in any location in which it has been identified thus far is also unknown and may warrant further investigation.

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First Report of Hop stunt viroid in Apricot in China

Plant Disease ◽

10.1094/pd-90-0828c ◽

2006 ◽

Vol 90 (6) ◽

pp. 828-828 ◽

Cited By ~ 5

Author(s):

Y. A. Yang ◽

H. Q. Wang ◽

R. Guo ◽

Z. M. Cheng ◽

S. F. Li ◽

...

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Rt Pcr ◽

First Report ◽

Republic Of China ◽

Stunt Viroid ◽

Hop Stunt Viroid ◽

Viroid Infection ◽

Wide Range ◽

Irregular Border ◽

Apricot Tree

Hop stunt viroid (HSVd), a member of the family Pospiviroidae, was first described as the causal agent of hop stunt disease in Japan. It has since been found in a wide range of hosts including herbaceous and woody hosts (e.g., hop, cucumber, grapevine, citrus, plum, peach, pear, apricot, almond, and pomegranate). It was also detected and characterized in apricot where infection appears to be latent (1). The viroid occurs frequently in apricot. In southeastern Spain, the presence of HSVd was found to infect 81% of apricot trees (2). Apricots originated in China and are extensively cultivated, but HSVd infection in this host has not been reported. In September 2005, a single symptomatic apricot tree, ‘Yin Bai’, one of the most popular and widely grown cultivars in China, was discovered at the Institute of Fruit Science in Changping District in Beijing, Peoples Republic of China. Observed symptoms included a number of yellow spots with an irregular border that scattered in an irregular manner over the leaf surface. Total RNA was extracted and used for return-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) (4). Results of both assays were positive for HSVd. A 297-bp full-length DNA fragment was amplified by RT-PCR using primers R1 (5′-GCTGGATTCTGAGAAGAGTT-3′) complementary to HSVd residues 87–106 for the RT reaction, followed by R2 (5′-AACCCGGGGCTCCTTTCTCA-3′) complementary to HSVd residues 67–84 and forward primer F3 (5′-AACCCGGGGCAACTCTTCTC-3′) residues 79–96 for PCR. The primers are located in the strictly conserved central region of the conserved HSVd group and contain the unique endonuclease restriction site SmaI. The amplified products were cloned into pGEM-T (Promega, Madison, WI) and selected for further analysis on the basis of the results of restriction digests. Six individual clones were sequenced and three different sequences were obtained. Nucleic acid sequence (GenBank Accession No. DQ362901) obtained from one clone was 99.3% (nucleotide changes T206→C, C233→T) identical to HSVd.apr8 (GenBank Accession No. Y09349) (3). Sequence (GenBank Accession No. DQ362904) obtained from three clones was 99.7% (nucleotide change C233→T) and a third sequence (GenBank Accession No. DQ362905) obtained from two clones was 99.3% (nucleotide changes G107→A, C233→T) identical to HSVd.apr8. Further investigation is necessary to determine whether the symptoms observed are associated with the viroid infection. To our knowledge, this is the first report of HSVd isolated from apricot in China. References: (1) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (2) M. C. Cañzres et al. Acta Hortic. 472:581, 1998. (3) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997. (4) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

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First Report of Natural Infection of Greenhouse Tomatoes by Potato spindle tuber viroid in the United States

Plant Disease ◽

10.1094/pdis-07-10-0516 ◽

2010 ◽

Vol 94 (11) ◽

pp. 1376-1376 ◽

Cited By ~ 9

Author(s):

K.-S. Ling ◽

D. Sfetcu

Keyword(s):

United States ◽

Potato Spindle Tuber Viroid ◽

The United States ◽

Mechanical Transmission ◽

Rt Pcr ◽

Tomato Plants ◽

First Report ◽

Viroid Infection ◽

Plant Pathol ◽

Greenhouse Tomatoes

In April 2009, a large number of tomato plants (Solanum lycopersicum L.) grown in a commercial greenhouse facility near Los Angles, CA exhibited general plant stunting (short internodes) and foliar symptoms that included distortion, chlorosis, and scattered necrotic spotting. Over time, the leaves began to exhibit a purple color and curling. Diseased plants were often elongated and frail with spindly shoots. The disease resulted in a significant yield loss due to reduced fruit size. Disease symptoms described above are generally different from those of Pepino mosaic virus (PepMV) infection, which causes yellow mosaic or patches on leaves and marbling of fruits. The disease was initially localized in certain areas in a greenhouse despite using a number of cultural management efforts including vigorous scouting, roguing of diseased plants, and strict hygiene and cleaning practices. The disease was also observed in neighboring greenhouses by the spring of 2010. A standard panel of tests for common tomato viruses and viroids were conducted using the appropriate serological or PCR assays. Reverse transcription (RT) PCR analysis of nine symptomatic plants with pospiviroid-specific primers, Pospil-RE and Pospil-FW (3), produced an amplicon of the expected size (~196 bp) while three healthy looking tomato plants did not. Subsequently, full viroid genomic sequences were obtained through RT-PCR with primer sets specific for Potato spindle tuber viroid (PSTVd), 3H1/2H1 (2), as well as for the pospiviroid genus, MTTVd-F and MTTVd-R (1). Sequences obtained from direct sequencing of amplicons or cloned PCR products from one isolate were identical and consisted of a full viroid genome of 358 nt, which was named PSTVd-CA1 (GenBank Accession No. HM753555). BLASTn queries of the NCBI database showed that this isolate had a high sequence identity (98%) to other PSTVd isolates (i.e., EF044304, X52037, and Y09577). The disease was reproducible upon mechanical transmission (1) on three tomato ‘Moneymaker’ plants, which expressed symptoms that were similar to those on the source plants. Recovery of PSTVd on the inoculated tomato plants was confirmed by RT-PCR and sequencing. Because of its susceptibility to viroid infection, tomato ‘Moneymaker’ plants are commonly used as indicators for the study of pospiviroids, including PSTVd. Natural PSTVd infection on greenhouse tomatoes has been reported in Europe (3) and New Zealand. Although a number of reports in the United States have been published on naturally occurring PSTVd infections of potatoes, to our knowledge, this is the first report of a natural PSTVd infection on tomatoes in the United States. The exact source of the PSTVd inoculum in the current disease outbreak is unknown, but it could have been introduced from infected potato or ornamental plants (4) or through infected tomato seeds. The disease epidemic might have been enhanced by frequent hands-on activities in greenhouse tomato production and the environmental conditions (high temperature and intense lighting) in the greenhouse that favor symptom expression. References: (1) K.-S. Ling and W. Zhang, Plant Dis. 93:1216, 2009. (2). A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 59:3, 2010.

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First Report of Potato virus M and Chrysanthemum stunt viroid in Solanum jasminoides

Plant Disease ◽

10.1094/pd-90-1359a ◽

2006 ◽

Vol 90 (10) ◽

pp. 1359-1359 ◽

Cited By ~ 16

Author(s):

J. Th. J. Verhoeven ◽

C. C. C. Jansen ◽

J. W. Roenhorst

Keyword(s):

Plant Species ◽

Potato Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Test Plant ◽

Rt Pcr ◽

First Report ◽

Chrysanthemum Stunt Viroid ◽

Stunt Viroid ◽

Local Lesions ◽

Potato Virus M

In 2005, a plant of the ornamental crop Solanum jasminoides from the Netherlands was submitted for testing on viruses and viroids because of its intended use for propagation. Sap from this plant was mechanically inoculated to the test plant species Chenopodium quinoa, Nicotiana benthamiana, N. hesperis-67A, and N. occidentalis-P1 (3). N. hesperis-67A showed chlorotic local lesions and rugosity followed by vein necrosis, N. occidentalis-P1 showed necrotic local lesions and systemic leaf distortion, and the two other test plant species remained symptomless. Potato virus M (PVM) was identified by double antibody sandwich enzyme-linked immunosorbent assay using leaves from S. jasminoides and N. hesperis-67A. The plant of S. jasminoides was also tested for the presence of viroids by reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2). This reaction yielded an amplicon of the expected size of 198 bp. The sequence showed 100% identity to an isolate of Chrysanthemum stunt viroid (CSVd; NCBI GenBank Accession No. AF394453). Subsequently, the complete sequence of our viroid isolate (GenBank Accession No. DQ406591) was determined from the amplicon obtained after RT-PCR using specific primers for the detection of CSVd (1). The viroid isolate from S. jasminoides consisted of 354 nucleotides and showed the highest identity (98.6%) to a chrysanthemum isolate of CSVd (GenBank Accession No. AB055974). Therefore, the viroid was identified as CSVd. To our knowledge, this is the first report of PVM and CSVd in S. jasminoides. Reference: (1) R. Hooftman et al. Acta Hortic. 432:120, 1996. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst, EPPO Bull. 33:305, 2003.

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First Report of Apricot latent virus and Plum bark necrosis stem pitting-associated virus in Apricot from Spain

Plant Disease ◽

10.1094/pdis-94-2-0275a ◽

2010 ◽

Vol 94 (2) ◽

pp. 275-275 ◽

Cited By ~ 8

Author(s):

A. García-Ibarra ◽

P. Martínez-Gómez ◽

M. Rubio ◽

F. Dicenta ◽

A. Soler ◽

...

Keyword(s):

The United States ◽

Full Length ◽

Latent Virus ◽

Stone Fruit ◽

Pcr Analysis ◽

Rt Pcr ◽

First Report ◽

Cp Gene ◽

Fruit Samples ◽

Stem Pitting

Representing 2% of world production, 20,000 ha of apricot (Prunus armeniaca L.), are cultivated in Spain, primarily in the southeast. A survey was conducted during the spring of 2008 in orchards in the region of Murcia to assess the incidence of several stone fruit viruses. Leaf and fruit samples from 160 trees from 40 orchards were collected randomly for reverse transcription (RT)-PCR analysis. Total RNA extracted (3) from leaves and fruits was tested by a multiplex one-step RT-PCR protocol with a mix of primers that detect eight distinct viruses (4). Amplicons of 250 bp expected for Plum bark necrosis stem pitting-associated virus (PBNSPaV), corresponding to part of the heat shock 70 protein gene, were obtained from four trees and amplicons of 700 bp expected for Apricot latent virus (ApLV), corresponding to part of the coat protein (CP) gene, were obtained from two trees. In all cases, amplicons were obtained using RNA extracted from leaf and fruit tissues. RT-PCR results were confirmed by uniplex RT-PCR with primers specific for each virus and dot-blot hybridization with virus-specific digoxygenin-labeled RNA probes (2). To further characterize the new viruses, we designed primers to amplify specifically the CP gene of ApLV (5′-CCCGACCATGGCTACAAGC-3′ and 5′-TTGCCGTCCCGATTAGGTTG-3′) and the minor CP gene of PBNSPaV (5′-GAACAAACTACAGCAGCACC-3′ and 5′-CAAGGGTAGGACGGGTAACGC-3′). Amplicons of 1,500 and 950 bp corresponding to the ApLV and PBNSPaV CP genes, respectively, were purified from agarose gels and cloned in the pTZ57R plasmid (Fermentas, Burlington, Ontario, Canada). Blastp analysis of the full-length ApLV CP sequence from one infected tree (GenBank Accession No. GQ919051) revealed 86% amino acid (aa) similarity to the single full-length ApLV CP sequence available (No. AAC16234) and 79 and 66.9% similarity to Peach sooty ringspot virus (No. AAG48314) and Apple stem pitting virus (No. NP604468), respectively. Identity/similarity analysis of the full-length PBNSPaV minor CP genes using the Matrix Global Alignment Tool software, version 2.02 (1), revealed 98.8 to 99.6% aa similarity between the Spanish PBNSPaV isolates (Nos. GQ919047, GQ919048, GQ919049, and GQ919050) and 97.1 to 97.4% with the PBNSPaV isolate from the United States (No. EF546442). None of the six infected trees were associated with any particular field symptoms. Five infected trees were cv. Búlida and one was native cv. Murciana, which was infected with ApLV. All infected trees were located in geographically separated orchards. The incidence of ApLV and PBNSPaV was 1.25 and 2.5%, respectively. The low incidence of both viruses together with the scattered geographic distribution could be due to the recent introduction of virus-contaminated plants, although we cannot exclude that it is a consequence of a low dissemination rate. Even though no symptoms were observed, we cannot discard that the infection could affect fruit production or flowering or even cause a synergistic effect in mixed infection with other stone fruit viruses, a risk especially relevant considering the total area of cultivated apricot. To our knowledge, this is the first report of ApLV and PBNSPaV in Spain. References: (1) J. J. Campanella et al. BMC Bioinformatics 4:29, 2003. (2) M. C. Herranz et al. J. Virol. Methods 124:49, 2005. (3) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (4) J. A. Sánchez-Navarro et al. Eur. J. Plant Pathol. 111:77, 2005.

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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 Okra yellow mosaic Mexico virus in Okra in the United States

Plant Disease ◽

10.1094/pdis-94-7-0924b ◽

2010 ◽

Vol 94 (7) ◽

pp. 924-924 ◽

Cited By ~ 7

Author(s):

C. Hernandez-Zepeda ◽

T. Isakeit ◽

A. Scott ◽

J. K. Brown

Keyword(s):

United States ◽

The United States ◽

Full Length ◽

Bipartite Begomoviruses ◽

Economic Losses ◽

Growth Stages ◽

First Report ◽

Total Dna ◽

Hidalgo County ◽

Whitefly Vector

During the okra growing season from August to November of 2009, symptoms reminiscent of geminivirus infection were observed on 75% of ‘Green Emerald’ Abelmoschus esculentus (L.) Moench, plants in a 0.2-km2 field in Hidalgo County, TX. Visible symptoms consisted of irregular yellow patches on leaves, distinctive yellow borders on leaf edges, and chlorosis of subsequently developing leaves. The whitefly vector of begomoviruses, Bemisia tabaci (Genn.), infested okra plants in the early growth stages during late July 2009. Total DNA was isolated from the leaves of three symptomatic okra plant samples (1) and used as the PCR template to amplify a 575-bp fragment of the coat protein gene (CP) using the universal begomovirus primers AV494 and AC1048 (2). PCR products of the expected size were cloned into the pGEM-T Easy (Promega, Madison, WI) and sequenced using the universal M13F and M13 R primers. ClustalV alignment indicated 99 to 100% shared nucleotide (nt) identity, and BLAST analysis revealed that the closest relative was Okra yellow mosaic Mexico virus - Tetekalitla (OkYMMV) (GenBank Accession No. EF591631) at 98%. To amplify the full-length DNA-A and a possible cognate DNA-B component, one plant that was positive by CP-PCR and DNA sequencing was selected for further analysis. Total DNA from this plant was used as template for a second detection method that consisted of rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA is a non-sequence-specific approach that permits amplification of circular DNA. The RCA products were linearized to release unit length ~2.6 kb DNA-A and DNA-B components using BamHI, and EcoRI, respectively. These products were cloned into pGEM3zf+ (Promega) and sequenced using M13F and M13 R primers and then by primer walking (>300 base overlap). Full-length DNA-A and DNA-B components were obtained, respectively, at 2,613 bp (GenBank Accession No. HM035059) and 2,594 bp (GenBank Accession No HM035060). Alignment of the DNA-A component using ClustalV (MegAlign, DNASTAR, Madison, WI) with begomoviral sequences available in GenBank indicated that it was 99% identical to OkYMMV DNA-A (GenBank Accession No. DQ022611). The closest relative to the DNA-B component (ClustalV) was Sida golden mosaic virus (SiGMV) (GenBank Accession No. AJ250731) at 73%. The nt identity of the 172-nt ‘common region’ present in the DNA-A and DNA-B components was 99%, and the iterons (predicted Rep binding motif) were identical for the two components, indicating that they are a cognate pair. The genome organization was typical of other New World bipartite begomoviruses. The economic losses due to infection by this virus could not be determined because an early freeze killed the plants. Hidalgo County is adjacent to Tamaulipas, Mexico, where ~50 km2 of okra are grown and the whitefly vector is also present. The identification of OkYMMV based on two independent detection methods, and the presence of begomovirus-like symptoms together with the whitefly vector, provide robust evidence for the association of OkYMMV-TX with diseased okra plants. To our knowledge, this is the first report of OkYMMV-TX infecting okra crops in Texas and in the continental United States. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) S. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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First Report of Grapevine leafroll associated virus-3 in American Vitis spp. Grapevines in Washington State

Plant Disease ◽

10.1094/pd-90-1461a ◽

2006 ◽

Vol 90 (11) ◽

pp. 1461-1461 ◽

Cited By ~ 8

Author(s):

M. J. Soule ◽

K. C. Eastwell ◽

R. A. Naidu

Keyword(s):

New York ◽

Economic Impact ◽

Virus Disease ◽

The United States ◽

Washington State ◽

The State ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Table Grapes

Washington State is the largest producer of juice grapes (Vitis labruscana ‘Concord’ and Vitis labrusca ‘Niagara’) and ranks second in wine grape production in the United States. Grapevine leafroll disease (GLD) is the most wide spread and economically significant virus disease in wine grapes in the state. Previous studies (2) have shown that Grapevine leafroll associated virus-3 (GLRaV-3) is the predominant virus associated with GLD. However, little is known about the incidence and economic impact of GLD on juice and table grapes. Because typical GLD symptoms may not be obvious among these cultivars, the prevalence and economic impact of GLD in Concord and Niagara, the most widely planted cultivars in Washington State, has received little attention from the grape and nursery industries. During the 2005 growing season, 32 samples from three vineyards and one nursery of ‘Concord’ and three samples from one nursery of ‘Niagara’ were collected randomly. Petiole extracts were tested by single-tube reverse transcription-polymerase chain reaction (RT-PCR; 3) with primers LC 1 (5′-CGC TAG GGC TGT GGA AGT ATT-3′) and LC 2 (5′-GTT GTC CCG GGT ACC AGA TAT-3′), specific for the heat shock protein 70 homologue (Hsp70h gene) of GLRaV-3 (GenBank Accession No. AF037268). One ‘Niagara’ nursery sample and eleven ‘Concord’ samples from the three vineyards tested positive for GLRaV-3, producing a single band of the expected size of 546 bp. The ‘Niagara’ and six of the ‘Concord’ RT-PCR products were cloned in pCR2.1 (Invitrogen Corp, Carlsbad, CA) and the sequences (GenBank Accession Nos. DQ780885, DQ780886, DQ780887, DQ780888, DQ780889, DQ780890, and DQ780891) compared with the respective sequence of a New York isolate of GLRaV-3 (GenBank Accession No. AF037268). The analysis revealed that GLRaV-3 isolates from ‘Concord’ and ‘Niagara’ share nucleotide identities of 94 to 98% and amino acid identities and similarities of 97 to 98% with the Hsp70h gene homologue of the New York isolate of GLRaV-3. Additional testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using antibodies specific to GLRaV-3 (BIOREBA AG, Reinach, Switzerland) further confirmed these results in the ‘Niagara’ and two of the ‘Concord’ isolates. GLRaV-3 has previously been reported in labrusca cvs. Concord and Niagara in western New York (4) and Canada (1), but to our knowledge, this is the first report of GLRaV-3 in American grapevine species in the Pacific Northwest. Because wine and juice grapes are widely grown in proximity to each other in Washington State and grape mealybug (Pseudococcus maritimus), the putative vector of GLRaV-3, is present in the state vineyards, further studies will focus on the role of American grapevine species in the epidemiology of GLD. References: (1) D. J. MacKenzie et al. Plant Dis. 80:955, 1996. (2) R. R. Martin et al. Plant Dis. 89:763, 2005. (3) A. Rowhani et al. ICGV, Extended Abstracts, 13:148, 2000. (4) W. F. Wilcox et al. Plant Dis. 82:1062, 1998.

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First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States

Plant Disease ◽

10.1094/pdis-05-21-0917-pdn ◽

2021 ◽

Author(s):

Gardenia Orellana ◽

Alexander V Karasev

Keyword(s):

United States ◽

Leaf Tissue ◽

The United States ◽

Universal Primers ◽

Rt Pcr ◽

Universal Primer ◽

Tissue Samples ◽

First Report ◽

Specific Pcr ◽

Coleus Blumei

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

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First Report of Plum pox virus (Sharka Disease) in Prunus persica in the United States

Plant Disease ◽

10.1094/pdis.2000.84.2.202b ◽

2000 ◽

Vol 84 (2) ◽

pp. 202-202 ◽

Cited By ~ 65

Author(s):

L. Levy ◽

V. Damsteegt ◽

R. Welliver

Keyword(s):

Prunus Persica ◽

Virus Disease ◽

Sandwich Elisa ◽

Pcr Amplification ◽

The United States ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

Peach Fruit ◽

First Report

Plum pox (Sharka) is the most important virus disease of Prunus in Europe and the Mediterranean region and is caused by Plum pox potyvirus (PPV). In September 1999, PPV-like symptoms were observed in peach fruit culls in a packinghouse in Pennsylvania. All symptomatic fruit originated from a single block of peach (P. persica cv. Encore) in Adams County. Trees in the block exhibited ring pattern symptoms on their leaves. A potyvirus was detected in symptomatic fruit using the Poty-Group enzyme-linked immunosorbent assay (ELISA) test from Agdia (Elkhart, IN). Reactions for symptomatic peach fruit and leaves also were positive using triple-antibody sandwich ELISA with the PPV polyclonal antibody from Bioreba (Carrboro, NC) for coating, the Poty-Group monoclonal antibody (MAb; Agdia) as the intermediate antibody, and double-antibody sandwich ELISA with PPV detection kits from Sanofi (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and Agdia and the REAL PPV kit (Durviz, Valencia, Spain) containing universal (5B) and strain typing (4DG5 and AL) PPV MAbs (1). PPV also was identified by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) amplification and subsequent sequencing of the 220-bp 3′ noncoding region (2) (>99% sequence homology to PPV) and by IC-RT-PCR amplification of a 243-bp product in the coat protein (CP) gene (1). The virus was identified as PPV strain D based on serological typing with strainspecific MAbs and on PCR-restriction fragment length polymorphism of the CP IC-RT-PCR product with Rsa1 and Alu1 (1). This is the first report of PPV in North America. References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) L. Levy and A. Hadidi. EPPO Bull. 24:595, 1994.

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