Detection of a Region of the Coat Protein Gene of Grapevine Fanleaf Virus by RT-PCR in the Nematode VectorXiphinema index

Cherry virus A (CVA), a member of the genus Capillovirus, has been reported in sweet cherry in Germany, Canada, and Great Britain. No data are available on the effects of CVA on fruit quality and yield of infected trees. Little cherry disease (LChD) occurs in most cherry growing areas of the world. Symptoms on sensitive cultivars include discolored fruit that remain small, pointed in shape, and tasteless. Three Closterovirus spp. associated with LChD have been described (Little cherry virus-1 [LChV-1], LChV-2, and LChV-3). Diseased local and commercial cultivars of sour cherry trees were found in a Prunus sp. germplasm collection and orchards in Poland during the 2003 growing season. The foliar symptoms included irregular, chlorotic mottling, distortion, and premature falling of leaves. Some of the diseased trees developed rosette as a result of decreased growth and shortened internodes. Severely infected branches exhibited dieback symptoms. Because the symptoms were suggestive of a possible virus infection, leaf samples were collected from 38 trees and assayed for Prune dwarf virus and Prunus necrotic ringspot virus using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). RNA extracted from leaves was used in a reverse transcription-polymerase chain reaction (RT-PCR) with the One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies) and primer sets specific for CVA (1), LChV-1 (3), and LChV-2 (3). The RNA samples were also tested using RT-PCR for detection of Cherry mottle leaf virus (CMLV), Cherry necrotic rusty mottle virus (CNRMV), and Cherry green ring mottle virus (CGRMV) with specific primer sets (2). Amplification of a 397-bp coat protein gene product confirmed infection of 15 trees with CVA. A 419-bp fragment corresponding to the coat protein gene of LChV-1 was amplified from cv. Gisela rootstock and local cv. WVIII/1. To confirm RT-PCR results, CVA amplification products from local cv. WX/5 and LChV-1 from cvs. Gisela and WVIII/1 were cloned in bacterial vector pCR 2.1-TOPO and then sequenced. The sequences were analyzed with the Lasergene (DNASTAR, Madison, WI) computer program. The alignment indicated that the nucleotide sequence of cv. WX/5 was closely related to the published sequences of CVA (Genbank Accession No. NC_003689) and had an 89% homology to the corresponding region. The nucleotide sequence similarity between the 419-bp fragment obtained from cvs. Gisela and WVIII/1 was 87% and 91%, respectively, compared with the reference isolate of LChV-1 (Genbank Accession No. NC_001836). The sampled trees tested negative for LChV-2, CGRMV, CMLV, and CNRMV using RT-PCR. Some trees tested positive for PNRSV and PDV. To our knowledge, this is the first report of CVA and LChV-1 in Poland. References: (1) D. James and W. Jelkmann. Acta Hortic. 472:299, 1998. (2) M. E. Rott and W. Jelkmann. Eur. J. Plant Pathol. 107:411,2001. (3) M. E. Rott and W. Jelkmann. Phytopathology. 91:61, 2001.

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Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada

Plant Disease ◽

10.1094/pdis.2004.88.4.363 ◽

2004 ◽

Vol 88 (4) ◽

pp. 363-367 ◽

Cited By ~ 40

Author(s):

H. Xu ◽

T.-L. DeHaan ◽

S. H. De Boer

Keyword(s):

United States ◽

Coat Protein ◽

Coat Protein Gene ◽

Protein Gene ◽

The United States ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

The North ◽

Potato Mop Top Virus ◽

Pcr Targeting

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

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First Report of Cucurbit yellow stunting disorder virus on Melon in China

Plant Disease ◽

10.1094/pdis-94-4-0485a ◽

2010 ◽

Vol 94 (4) ◽

pp. 485-485 ◽

Cited By ~ 6

Author(s):

L. Z. Liu ◽

Y. Y. Chen ◽

W. M. Zhu

Keyword(s):

Coat Protein ◽

Coat Protein Gene ◽

Fragment Length ◽

Protein Gene ◽

Disease Incidence ◽

Rt Pcr ◽

First Report ◽

Genes Encoding ◽

Pcr Products ◽

Symptomatic Leaf

Melon (Cucumis melo L.) plants in commercial fields in Shanghai, Jiangsu, and Zhejiang exhibited stunting, deformation, interveinal chlorosis, and leaf mottling in the spring of 2008. In addition, adult and immature whiteflies (Bemisia tabaci biotype B) were present in these melon fields. Thirty-two symptomatic leaf samples were collected from these fields for further analysis (9 from Nanhui County in Shanghai, 11 from Fengxian County in Shanghai, 6 from Kunshan County of Jiangsu, and 6 from Jiashan County of Zhejiang). Total RNA was extracted from these samples along with asymptomatic control plants and screened for the presence of Cucurbit yellow stunting disorder virus (CYSDV) by using primers specific to genes encoding coat protein (2) and HSP70h (1) of CYSDV through reverse transcription (RT)-PCR methods. RNA was successfully extracted from 31 of 32 symptomatic samples. All 31 symptomatic leaf samples tested with coat protein primers were positive for CYSDV and yielded the expected fragment length of 394 bp. The RT-PCR products of the coat protein gene from all 31 isolates were cloned and found to be identical in sequence. Thus, only one was deposited in GenBank (No. GU189240). The submitted sequence of the amplified part of the coat protein gene was 99% identical to the sequence of coat protein gene of CYSDV from Jordan, France, and Florida (GenBank Accession Nos. DQ903107, AY204220, and EU596528, respectively) and 98% identical to that of an isolate from Spain (GenBank Accession No. AJ243000). Similarly, all 31 samples were also positive for CYSDV with the primers specific to HSP70h and yielded the expected fragment length of 175 bp. The RT-PCR products of the HSP70h gene from these isolates were also cloned and found to be identical in sequence. The sequence of the amplified portion of the HSP70h gene was found to be identical to the sequence of HSP70h of CYSDV deposited in GenBank (No. AJ439690.2). CYSDV was noticed in all three surveyed regions and the percentage of disease incidence was approximately 68% in all these regions. The occurrence of CYSDV has been previously reported in Europe (Spain and France), southern Asia (Iran and Jordan), North America (United States and Mexico), and other countries (1). To our knowledge, this is first report of CYSDV in China. References: (1) Y.-W. Kuo et al. Plant Dis. 91:330, 2007. (2) J. E. Polston et al. Plant Dis. 92:1251, 2008.

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