First Report of Cucurbit Yellow Stunting Disorder Virus (Genus Crinivirus) in North America

In late summer 1999, field- and greenhouse-grown melon plants (Cucumis melo) showing severe stunting and yellowing symptoms were observed near Donna in southern Texas and near the town of Reynosa in northern Mexico. Symptoms were typical of those caused by viruses in the genus Crinivirus, family Closteroviridae. High populations of Bemisia spp. whiteflies were associated with these plantings, with many plants showing heavy infestation. Laboratory analyses showed that melon plants from both locations were infected by the whitefly-transmitted Cucurbit yellow stunting disorder virus (CYSDV). Positive hybridization reactions with digoxigenin-UTP-labeled transcript probes corresponding to the CYSDV heat shock protein 70 (HSP70) homolog coding region (1) were obtained for RNAs extracted from symptomatic plants. Similar probes for the related Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV), two whitefly-transmitted viruses previously reported from North America (2), did not hybridize with the RNAs. Definitive confirmation of CYSDV was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for two distinct CYSDV coding regions. RT-PCR with primers corresponding to CYSDV, but not LIYV or BPYV HSP70 homolog coding regions, gave specific (≈500 bp) products from corresponding test plants. RNAs from healthy control plants gave no RT-PCR product. Because the HSP70 coding region is highly conserved (2), we also performed RT-PCR with primers designed for the Spanish CYSDV capsid protein gene (GenBank accession AJ243000). Positive RT-PCR products of ≈700 bp were obtained only from the Texas and Mexico melon plants. CYSDV is a widespread and damaging virus of cucurbits in southern Europe and the Middle East (2). This is the first report of this important virus in North America. References: (1) Tian et al. Phytopathology 86:1167, 1996. (2) Rubio et al. Phytopathology 89:707, 1999.

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First Report of Grapevine leafroll-associated virus 2 in a Hybrid Grape ‘Vidal Blanc’ in Missouri

Plant Disease ◽

10.1094/pdis-10-11-0834 ◽

2012 ◽

Vol 96 (3) ◽

pp. 462-462 ◽

Cited By ~ 3

Author(s):

S. Lunden ◽

W. Qiu

Keyword(s):

New York ◽

Leaf Roll ◽

Late Summer ◽

The United States ◽

Coding Region ◽

Rt Pcr ◽

Protein Coding ◽

First Report ◽

Stem Lesions ◽

Grape Cultivars

Grapevine leaf roll disease (GLRD) is one of the most prevalent viral diseases in vineyards worldwide. At least 10 Grapevine leafroll-associated viruses (GLRaV-1 to -7, and -9, -10, and -11) are associated with GLRD. GLRaV-2 has a number of distinct isolates that are associated not only with GLRD, but also with graft incompatibility, young vine decline, and rootstock stem lesions. In the United States, GLRaV-2 isolates have been reported in the states of California (4), New York (1), Oregon, and Washington (3). A survey of grapevine viruses was conducted on seven grape cultivars, Norton, Chambourcin, Chardonel, Vignoles, Vidal Blanc, Traminette, and Cayuga White, in a vineyard located at Mountain Grove, MO during the falls of 2009 and 2010 and late summer of 2011. Vines from each cultivar were assigned into three blocks. From each block, six individual vines were randomly selected. Phloem scrapings were collected for detecting viruses by ELISA and reverse-transcription (RT)-PCR. To test for GLRaV-2, a set of primers, GLRaV-2-Forward (5′-GGTGATAACCGACGCCTCTA, nt 6745 to 6764) and GLRaV-2-Reverse (5′-CCTAGCTGACGCAGATTGCT, nt 7268 to 7287), were designed from the coat protein coding region as reported previously (2). GRLaV-2 was detected only in the samples of ‘Vidal Blanc’. The detection of GLRaV-2 was further verified by ELISA using GRLaV-2-specific antisera (Bioreba AG, Reinach, Switzerland). The GLRaV-2-infected ‘Vidal Blanc’ vines did not exhibit visible symptoms. The RT-PCR amplified 543-bp cDNA fragments were isolated from agarose gel and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from three individual clones and sequenced from both directions. Comparison of the 543-bp sequences showed that the sequences shared 99% nt identity with the corresponding regions of 21 GLRaV-2 isolates, including PMC-083 isolate from Croatia (GenBank Accession No. HM185277), LN isolate from China (GenBank No. FJ786017), SE isolate from Brazil (GenBank No. EU204909), and four isolates from Washington (GenBank Nos. EU760836, 760838, 760843, and 760848). ‘Vidal Blanc’ is a white interspecific hybrid grape. Absence of visible symptoms on this cultivar emphasizes the importance of planting certified grapevines that have been indexed for viruses. Discovery of GLRaV-2 in asymptomatic white grape cultivars will warn growers on the potential damages and epidemics of GLRD in Midwest vineyards and promote planting clean grapevines. To our knowledge, this is the first report of GLRaV-2 in grapevines in Missouri. References: (1) M. Fuchs et al. Plant Dis. 93:395, 2009. (2) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (3) R. R. Martin et al. Plant Dis. 89:763, 2005. (4) A. Rowhani et al. Phytopathology (Abstr.) 92:(suppl.):S71, 2002.

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First Report of Beet pseudo-yellows virus and Strawberry pallidosis associated virus in Strawberry in Peru

Plant Disease ◽

10.1094/pd-90-1457c ◽

2006 ◽

Vol 90 (11) ◽

pp. 1457-1457 ◽

Cited By ~ 5

Author(s):

W. M. Wintermantel ◽

S. Fuentes ◽

C. Chuquillanqui ◽

L. F. Salazar

Keyword(s):

Coat Protein ◽

Pcr Amplification ◽

Trialeurodes Vaporariorum ◽

Rt Pcr ◽

Greenhouse Whitefly ◽

First Report ◽

Wide Range ◽

Pcr Products ◽

Sequencing And Expression ◽

Beet Pseudo Yellows Virus

During a 2006 survey for the presence of criniviruses in Peru, large numbers of greenhouse whitefly (Trialeurodes vaporariorum) were observed infesting strawberry (Fragaria × ananassa) fields near Huaral on the central coast of Peru. Plants exhibited a wide range of symptoms including stunting and reddening of leaves. These symptoms are characteristic of those induced by the presence of the criniviruses Beet pseudo-yellows virus (BPYV) and/or Strawberry pallidosis associated virus (SPaV) together with any of a number of different strawberry-infecting viruses (1,3). The virus complex causes older leaves to develop a red color, vein and petiole reddening, roots become stunted, and plants fail to develop. Leaf samples with varying symptoms were collected from 22 plants from 2 fields, each planted with a different cultivar. Total nucleic acid was extracted, spotted onto positively charged nylon membranes, and tested by hybridization with probes specific to the minor coat protein (CPm) gene of BPYV (2) and coat protein (CP) gene of SPaV (4). Results identified the presence of BPYV, SPaV, or both viruses in mixed infections in symptomatic strawberry, while control plants were infected with each virus individually. No signal was detected in virus-free strawberry. Secondary confirmation was obtained using probes specific to the RNA-dependent RNA polymerase (RdRp) genes of SPaV and BPYV. The SPaV probe corresponded to nucleotides 6116–6599 of SPaV RNA1 (GenBank Accession No. NC_005895), whereas the BPYV probe corresponded to nucleotides 6076–6447 of BPYV RNA1 (GenBank Accession No. NC_005209). All probes were generated by reverse-transcription polymerase chain reaction (RT-PCR) amplification using sequence-specific primers, cloning of RT-PCR products into pGEM-T Easy (Promega, Madison, WI), confirmation by sequencing, and expression as digoxygenin-labeled transcript probes (Roche, Indianapolis, IN). Field 1, containing cv. Fern Sancho, had the largest number of symptomatic and infected plants (5 of 12 BPYV, 6 of 12 SPaV, and 4 of 12 with both). Only 1 of 10 plants from field 2 containing cv. Tajo Holandesa was infected, but with both SPaV and BPYV. BPYV and SPaV are transmitted by the greenhouse whitefly (T. vaporariorum), although BPYV is transmitted much more efficiently and has a broader host range than SPaV (4). Movement of these viruses in Peru is likely a result of both propagation by runners and vector transmission. To our knowledge, this is the first report of either virus in Peru. References: R. R Martin and I. E. Tzanetakis. Plant Dis. 90:384, 2006. (2) I. E. Tzanetakis and R. R. Martin. Plant Dis. 88:223, 2004. (3) I. E. Tzanetakis et al. Plant Dis. 87:1398, 2003. (4) I. E. Tzanetakis et al. Plant Dis. 90:1343, 2006.

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First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽

10.1094/pdis.2003.87.1.102c ◽

2003 ◽

Vol 87 (1) ◽

pp. 102-102 ◽

Cited By ~ 3

Author(s):

S. Adkins ◽

L. Breman ◽

C. A. Baker ◽

S. Wilson

Keyword(s):

North America ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Sequences ◽

South Florida ◽

N Gene ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

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First Report of Yam mild mosaic virus in Yam in Guangxi Province, China

Plant Disease ◽

10.1094/pdis-04-11-0350 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1320-1320 ◽

Cited By ~ 4

Author(s):

C. Zou ◽

J. Meng ◽

Z. Li ◽

M. Wei ◽

J. Song ◽

...

Keyword(s):

Mosaic Virus ◽

Viral Genome ◽

Terminal Region ◽

Mild Mosaic ◽

Rt Pcr ◽

Degenerate Primers ◽

Germplasm Exchange ◽

First Report ◽

Pcr Products ◽

Mild Mosaic Virus

Yams (Dioscorea spp.) are widely grown in China as vegetables and herbal medicine. However, studies on viral diseases on yams are still limited. As a pilot project of a government initiative for improving yam productivity, a small study was conducted in Guangxi, a southern province of China, on viral disease in yams. Incidence of virus-like disease for the three extensively grown D. alata cultivars, GH2, GH5, and GH6, were 12 to 40%, 12 to 29%, and 11 to 25%, respectively, as found in a field survey with a five-plot sampling method in 2010. A total of 112 leaf samples showing mosaic or mottling or leaves without symptoms were collected from the cvs. GH2, GH5, GH6, and seven additional cultivars (D. alata cvs. GY2, GY23, GY47, GY69, GY62, GY72, and D. batatas cv. Tiegun). To determine if the symptoms were caused by Yam mild mosaic virus (YMMV; genus Potyvirus, family Potyviridae), total RNA was extracted from leaves with a commercial RNA purification kit (TIANGEN, Beijing, China), and reverse-transcription (RT)-PCR was conducted with a YMMV-specific primer pair (4) that amplifies the 3′-terminal portion of the viral genome. A PCR product with the predicted size of 262 bp was obtained from samples of GH5 (number testing positive of total number of leaves = 5 of 12), GH6 (24 of 42), and GY72 (1 of 1), but not from asymptomatic leaves. PCR products from a GH5 sample (YMMV-Nanning) and a GH6 sample (YMMV-Luzhai) were cloned and sequenced using an ABI PRISM 3770 DNA Sequencer. The two PCR products were 97% identical at nucleotide (nt) level and with the highest homology (89% identity) to a YMMV isolate (GenBank Accession No. AJ305466). To further characterize the isolates, degenerate primers (2) were used to amplify viral genome sequence corresponding to the C-terminal region of the nuclear inclusion protein b (NIb) and the N-terminal region of the coat protein (CP). These 781-nt fragments were sequenced and a new primer, YMMV For1 (5′-TTCATGTCGCACAAAGCAGTTAAG-3′) corresponding to the NIb region, was designed and used together with primer YMMV UTR 1R to amplify a fragment that covers the complete CP region of YMMV by RT-PCR. These 1,278-nt fragments were sequenced (GenBank Accession Nos. JF357962 and JF357963). CP nucleotide sequences of the YMMV-Nanning and YMMV-Luzhai isolates were 94% similar, while amino acid sequences were 99% similar. BLAST searches revealed a nucleotide identity of 82 to 89% and a similarity of 88 to 97% for amino acids to sequences of YMMV isolates (AF548499 and AF548519 and AAQ12304 and BAA82070, respectively) in GenBank. YMMV is known to be prevalent on D. alata in Africa and the South Pacific, and has recently been identified in the Caribbean (1) and Colombia (3). To our knowledge, this is the first report of the natural occurrence of YMMV in China and it may have implications for yam production and germplasm exchange within China. References: (1) M. Bousalem and S. Dallot. Plant Dis. 84:200, 2000. (2) D. Colinet et al. Phytopathology 84:65, 1994. (3) S. Dallot et al. Plant Dis. 85:803, 2001. (4) R. A. Mumford and S. E. Seal. J. Virol. Methods 69:73, 1997.

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First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia

Plant Disease ◽

10.1094/pdis-05-21-1065-pdn ◽

2021 ◽

Author(s):

Ahmed Sabra ◽

Mohammed Ali Al Saleh ◽

I. M. Alshahwan ◽

Mahmoud A. Amer

Keyword(s):

Saudi Arabia ◽

Mosaic Virus ◽

Solanum Lycopersicum ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Pcr Products ◽

Dark Green ◽

Leaf Symptoms

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia References: Luria N, et al., 2017. PLoS ONE 12(1): 1-19. Salem N, et al., 2015. Archives of Virology 161(2): 503-506. Fig. 1. Symptoms caused by ToBRFV showing irregular brown spots, deformation, yellowing spots on fruits (A, B, C) and bubbling and mottling, mosaic with dark green wrinkled and narrowing on leaf (D).

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First Report of Grapevine Pinot gris virus (GPGV) in grapevine in France

Plant Disease ◽

10.1094/pdis-10-14-1008-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 293-293 ◽

Cited By ~ 25

Author(s):

M. Beuve ◽

T. Candresse ◽

M. Tannières ◽

O. Lemaire

Keyword(s):

Large Scale ◽

De Novo ◽

Pinot Noir ◽

Rt Pcr ◽

Wine Quality ◽

First Report ◽

Italian Isolate ◽

Pcr Products ◽

Grapevine Pinot Gris Virus ◽

Plant Pathol

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate “Mer” (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5′ extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5′-GGAGTTGCCTTCGTTTACGA-3′) and Pg-Mer-R1 (5′-GTACTTGATTCGCCTC GCTCA-3′), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.

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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 Grapevine Kizil Sapak Virus in Grapevine in China

Plant Disease ◽

10.1094/pdis-02-21-0340-pdn ◽

2021 ◽

Author(s):

Fang Ren ◽

Zunping Zhang ◽

Xudong Fan ◽

Guojun Hu ◽

Yafeng Dong

Keyword(s):

Complete Genome ◽

High Throughput Sequencing ◽

Pairwise Comparison ◽

Bioinformatic Analysis ◽

Rt Pcr ◽

Illumina Hiseq ◽

First Report ◽

Hop Stunt Viroid ◽

Pcr Products ◽

Rapid Amplification

Grapevine Kizil Sapak virus (GKSV) is a novel member of the family Betaflexiviridae classified into the proposed genus Fivivirus within the subfamily Trivirinae. It was first discovered in USA from a grapevine originating from Turkmenistan (Al Rwahnih et al. 2019) and later in France from a grapevine accession from Iran (Marais et al. 2020). In October 2019, an asymptomatic grapevine cv. ‘Crimson Seedless’ (native to USA) was collected from Xinjiang province in China and analyzed by high-throughput sequencing (HTS). Ribosome-depleted RNA preparations were used for library synthesis followed by HTS on an Illumina HiSeq X-ten platform. A total of 29,141,024 cleaned reads were obtained, and 7,878 contigs were generated using CLC Genomics Workbench 9.5 (QIAGEN). One long contig (7,328 bp) showed 88.2% nucleotide (nt) identity with the sequence of GKSV-127 (MN172165) via Blastx, with an average coverage of 284-X. Bioinformatic analysis of the remaining contigs showed the presence of Grapevine leafroll-associated virus 4, Grapevine rupestris vein feathering virus, Grapevine fabavirus, grapevine yellow speckle viroid-1 (GYSVd-1), GYSVd-2 and Hop stunt viroid in the sample. The presence of GKSV was checked by RT-PCR using the primer GKSV-F/R (Al Rwahnih et al. 2019); the 1,240 bp PCR product was cloned using a pTOPO-T vector (Aidlab, China) and sequenced. In pairwise comparison, the obtained nt sequences shared 92.6 to 95.2% identity to the corresponding HTS sequence, confirming the presence of GKSV in the sample. The complete GKSV genome sequence was obtained as two pieces of overlapping DNA sequence using primers GKSV-20A/20B (5’-TAGTCTGGATTTCCCTACCT/5’-CTCCCTAAACTGATTTGATG) and GKSV-25A/25B (5’-GCCACTGGTGAATGAAAAGA/5’-CTAAATGAATGGGCAGGTAT) designed based on the HTS-generated sequence. The 5’ and 3’ termini were determined by rapid amplification of cDNA ends using SMARTer RACE 5’/3’ Kit (Takara, Dalian, China). The complete genome of GKSV isolate CS (MW582898) comprised 7,604 nt (without the polyA tail) and shared 77.8 to 89.2% identities with the other nine reported GKSV isolates, among which it shared the highest nt identity (89.2%) with GKSV-127. In phylogenetic analysis based on complete or nearly complete genome sequences of representative members of Betaflexiviridae, GKSV-CS clustered with the nine known GKSV isolates, forming a subclade with GKSV-127 (Supplementary Fig. 1). To determine the incidence and distribution of GKSV in China, 476 grapevine samples of 75 cultivars were collected from 20 provinces and tested by RT-PCR using primers GKSV-F/R (Al Rwahnih et al. 2019) and Vini-F1/R1 (Marais et al. 2020). The results showed that 0.42% (2 of 476) of the samples tested positive with both primers, including samples GKSV-CS and a ‘Black Monukka’ grape (native to India) also sampled from Xinjiang. Both PCR products of ‘Black Monukka’ were cloned and sequenced (MZ311588 to MZ311602) and they showed 85.1 to 88.9% nt identities to the GKSV-CS sequence. This is the first report of GKSV infecting grapevine in China. Although the pathogenicity of GKSV is yet to be determined, it has been found in several countries such as USA (Al Rwahnih et al. 2019), France (Marais et al. 2020) and China (this study). Both positive samples in this study were collected from Nanjiang region in Xinjiang province, indicating the sporadic occurrence of GKSV in this area.

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First Report of Tomato chlorosis virus in Tomato in Costa Rica

Plant Disease ◽

10.1094/pdis-93-9-0970a ◽

2009 ◽

Vol 93 (9) ◽

pp. 970-970 ◽

Cited By ~ 4

Author(s):

R. M. Castro ◽

E. Hernandez ◽

F. Mora ◽

P. Ramirez ◽

R. W. Hammond

Keyword(s):

Costa Rica ◽

Sequence Analysis ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

Greenhouse Whitefly ◽

Specific Primers ◽

First Report ◽

Sequence Identity ◽

Pcr Products ◽

Tomato Chlorosis Virus

In early 2007, severe yellowing and chlorosis symptoms were observed in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants in Costa Rica. Symptoms resembled those of the genus Crinivirus (family Closteroviridae), and large populations of whiteflies, including the greenhouse whitefly Trialeurodes vaporariorum (Westwood), were observed in the fields and on symptomatic plants. Total RNA was extracted from silica gel-dried tomato leaf tissue of 47 representative samples (all were from symptomatic plants) using TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed separately with each of the four primer sets with the Titan One-Tube RT-PCR Kit (Roche Diagnostics Corp., Chicago IL). Specific primers used for the detection of the criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), were primer pair ToCV-p22-F (5′-ATGGATCTCACTGGTTGCTTGC-3′) and ToCV-p22-R (5′-TTATATATCACTCCCAAAGAAA-3′) specific for the p22 gene of ToCV RNA1 (1), primer pair ToCVCPmF (5′-TCTGGCAGTACCCGTTCGTGA-3′) and ToCVCPmR (5′-TACCGGCAGTCGTCCCATACC-3′) designed to be specific for the ToCV CPm gene of ToCV RNA2 (GenBank Accession No. AY903448) (2), primer pair ToCVHSP70F (5′-GGCGGTACTTTCGACACTTCTT-3′) and ToCVHSP70R (5′-ATTAACGCGCAAAACCATCTG-3′) designed to be specific for the Hsp70 gene of RNA2 of ToCV (GenBank Accession No. EU284744) (1), and primer pair TICV-CP-F and TICV-CP-R specific for the coat protein gene of TICV (1). Amplified DNA fragments (582 bp) were obtained from nine samples, four from the greenhouse and five from the open field, with the ToCV-p22 specific primers and were cloned into the pCRII TOPO cloning vector (Invitrogen, Carlsbad, CA). Nucleotide sequence analysis of all purified RT-PCR products verified their identity as ToCV, sharing 99.5 to 100% sequence identity among themselves and 96% to 98% sequence identity with previously reported ToCV p22 sequences from Florida (Accession No. AY903447), Spain (Accession No. DQ983480), and Greece (Accession No. EU284745). The presence of ToCV in the samples was confirmed by additional amplification and sequence analysis of the CPm (449-bp fragment) and Hsp70 (420-bp fragment) genes of ToCV RNA2 and sharing 98 to 99% sequence homology to Accession Nos. AY903448 and EU284774, respectively. One representative sequence of the p22 gene of the Costa Rican isolate was deposited at GenBank (Accession No. FJ809714). No PCR products were obtained using either the TICV-specific primers nor from healthy tomato tissue. The ToCV-positive samples were collected from a region in the Central Valley around Cartago, Costa Rica. To our knowledge, this is the first report of ToCV in Costa Rica. The economic impact on tomato has not yet been determined. Studies are underway to determine the incidence of ToCV in Costa Rica field-grown and greenhouse tomatoes. References: (1) A. R. A. Kataya et al. Plant Pathol. 57:819, 2008. (2) W. M. Wintermantel et al. Arch. Virol. 150:2287, 2005.

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First Report of Potato Virus H on Solanum muricatum in China

Plant Disease ◽

10.1094/pdis-01-14-0024-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1016-1016 ◽

Cited By ~ 8

Author(s):

H. Abouelnasr ◽

Y.-Y. Li ◽

Z.-Y. Zhang ◽

J.-Y. Liu ◽

S.-F. Li ◽

...

Keyword(s):

Amino Acid ◽

Potato Virus ◽

Protein Gene ◽

Triple Gene Block ◽

Polyclonal Antiserum ◽

Rt Pcr ◽

Capsid Protein Gene ◽

First Report ◽

Gene Block ◽

Solanum Muricatum

Solanum muricatum, commonly known as pepino, pepino dulce, or tree melon, is a perennial shrub well known for its attractive, sweet, flavorful fruits and is frequently cultivated as an annual. It has gained increasing popularity in China and is grown as a cash crop in many provinces. S. muricatum belongs to the family Solanaceae and is closely related to tomato, eggplant, and potato. In 2012, during a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. PVH is a recently discovered carlavirus reported from potato plants from Huhhot, Inner Mongolia Autonomous Region. Since then, it was found on potatoes in Yunnan, Hebei, Liaoning, Heilongjiang, and Xinjiang provinces. PVH induces mild symptoms with a slight leaf curl in systemic leaves, but most often it is almost symptomless or latent on potatoes (2). To confirm the presence of PVH on S. muricatum, surveys were conducted in 2012 and 2013 in Gansu, Yunnan, and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR (2), and extracted samples were probed by Western blotting with the specific polyclonal antiserum against PVH (2). Among the 50 plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6 kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair (PVHB1F 5′-TGATGGAATTTACAAAAAC-3′ and PVHUR 5′-CTTATGCGCATCTATCAATC-3′), and then cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced (PVH-Pepino with GenBank Accession No. KF546312). Further sequence comparison showed that PVH-Pepino shared 91 to 98% nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN (HM584819 and JQ904630, respectively). PVH-Pepino shared deduced amino acid identity of 98 to 99% in CP gene with PVH-Ho and PVH-YN, respectively, but only shared 57 to 67% amino acid identities with other reported carlaviruses (1,2). Thus, latent infection of PVH on S. muricatum was confirmed. To our knowledge, this is the first report of S. muricatum as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepinos are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult. Although this finding does not show that PVH is economically important to pepino, this cannot be excluded in the presence of other viruses (2). References: (1) A. King et al. Page 881 in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, London, 2011. (2) Y. Y. Li et al. PLOS ONE 8(6):e69255, 2013.

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