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

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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First Report of Sharka Disease Caused by Plum Pox Virus in Lithuania

Plant Disease ◽

10.1094/pdis.1998.82.12.1405c ◽

1998 ◽

Vol 82 (12) ◽

pp. 1405-1405 ◽

Cited By ~ 1

Author(s):

J. Staniulis ◽

J. Stankiene ◽

K. Sasnauskas ◽

A. Dargeviciute

Keyword(s):

Coat Protein ◽

Plant Protection ◽

Virus Disease ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

First Report ◽

Das Elisa ◽

Sharka Disease

Plum pox (sharka) disease caused by plum pox potyvirus (PPV) is considered the most important virus disease of stone fruit trees in Europe and the Mediterranean region. Nearly all those countries that produce stone fruits are affected (3). The causal virus of the disease is a European Plant Protection Organization A2 quarantine pathogen. Symptoms of leaf mottling, diffuse chlorotic spots, rings, and vein banding of varied intensity characteristic for plum pox virus infection were observed in the plum (Prunus domestica) orchard tree collection of the Lithuanian Institute of Horticulture in Babtai in 1996. Presence of this virus in the diseased trees was confirmed by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with kits from BIOREBA (Reinach, Switzerland) and by polyclonal antibodies raised against a Moldavian isolate of PPV courtesy of T. D. Verderevskaya (Institute of Horticulture, Kishinev, Moldova). ELISAs with both sources of antiserum were positive for presence of PPV. Electron microscopy revealed the presence of potyvirus-like particles averaging 770 nm in extracts of mechanically inoculated plants of Chenopodium foetidum (chlorotic LL [local lesions]) and Pisum sativum cvs. Rainiai and Citron (mottling). For molecular diagnosis and characterization of this isolate, PPV-971, reverse transcription-polymerase chain reaction (RT-PCR) was employed. Total RNA from the leaves of infected pea was isolated as described (2). High molecular weight RNA selectively precipitated with 2 M lithium chloride was used for RT-PCR amplification of the coat protein encoding sequence by use of specific primers complementary to 5′ and 3′ parts of PPV coat protein L1 (GenBank accession no. X81081). Amino acid sequence comparison with GenBank data indicated 98.2% similarity with coat protein of PPV potyvirus isolated by E. Mais et al. (accession no. X81083) and 97.3% with PPV strain Rankovic (1).The specific DNA fragment, corresponding to predicted coat protein sequence size, was cloned into Escherichia coli pUC57 for DNA sequencing. Expression of the cloned sequence in bacteria and yeast expression systems is under investigation. The presence of PPV in plum trees in the 9-year-old collection at Babtai was confirmed by DAS-ELISA in 1997 and again in 1998. PPV was then detected in 20% of symptomatic trees of three cultivars. The Lithuanian PPV isolate reacted positively with “universal” Mab.5b and with a Mab (Mab.4DG5) specific for PPV-D. No reaction was observed with Mabs specific for PPV-M (Mab.AL), PPV-C (Mab.AC and Mab.TUV), and PPV-El Amar (Mab.EA24). PPV-971 seems to be a typical member of the less aggressive Dideron strain cluster of PPV (D. Boscia, personal communication). This is the first report of PPV in Lithuania and confirms the necessity for continuing the precautionary measures established in this country for indexing of nursery plum trees used for graft propagation. References: (1) S. Lain et al. Virus Res. 13:157, 1989. (2) J. Logemann et al. Anal. Biochem. 163:16, 1987. (3) M. Nemeth. OEPP/EPPO Bull. 24:525, 1994.

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First Incidence of Plum Pox Virus on Apricot Trees in China

Plant Disease ◽

10.1094/pd-89-0338c ◽

2005 ◽

Vol 89 (3) ◽

pp. 338-338 ◽

Cited By ~ 21

Author(s):

M. Navratil ◽

D. Safarova ◽

R. Karesova ◽

K. Petrzik

Keyword(s):

Virus Disease ◽

Pcr Amplification ◽

The United States ◽

Prunus Armeniaca ◽

Hunan Province ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

Pcr Product ◽

Multiple Sequences

Plum pox disease, caused by Plum pox virus (PPV), is the most severe virus disease of plums, apricots, and peaches. The disease causes heavy losses for fruit growers and the international trade of propagation materials and fresh fruits. PPV was first reported in Bulgaria in 1917 (1). It is now widespread in Europe and has been reported from Cyprus, Syria, Egypt, India, Kazakhstan, Chile, the United States, and Canada. Leaves on symptomatic apricot trees (Prunus armeniaca cvs. Hong Mei and Bai Mei and a selected genotype) in the Hunan Province of China showed typical yellow rings and diffused chlorotic spots. Samples from three suspected trees were repeatedly analyzed using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) in the summers of 2001-2003. PPV was detected in leaves, bark, and leaf buds of all three trees using ELISA with polyclonal and monoclonal antibodies provided by M. Navratil, Palacky University, Olomouc, Czech Republic (3). The results were confirmed using RT-PCR amplification of a 243-bp of the coat protein gene with a PPV-specific primer pair (2). BLAST analysis of two RT-PCR product sequences (GenBank Accession Nos. AY750961 and AY795603) showed 100% homology to multiple sequences of the PPV-D strain (GenBank Accession Nos. X81080, AF440743, and AF401295). The third sequence (GenBank Accession No. AY795602) had a C at position 112 rather than the T found in the other sequences. The ELISA, RT-PCR, and sequence results indicate that PPV-D was present in the apricot trees. To our knowledge, this is the first indication of PPV occurrence in China. This sporadic incidence of PPV on apricot trees requires addressing problems with the occurrence and spread of plum pox diseases in China and starting an eradication program. References: (1) D. Atanasoff. Annu. Univ. Sofia Fac. Agron. et Sylvic. 11:49, 1932. (2) T. Candresse et al. Phytopathology. 88:198, 1998. (3) I. Hilgert et al. Hybridoma. 12:215, 1993.

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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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Prunus Host Range of Plum pox virus (PPV) in the United States by Aphid and Graft Inoculation

Plant Disease ◽

10.1094/pd-91-0018 ◽

2007 ◽

Vol 91 (1) ◽

pp. 18-23 ◽

Cited By ~ 22

Author(s):

V. D. Damsteegt ◽

R. Scorza ◽

A. L. Stone ◽

W. L. Schneider ◽

K. Webb ◽

...

Keyword(s):

Virus Disease ◽

Systemic Infection ◽

Sour Cherry ◽

Aphid Transmission ◽

The United States ◽

Enzyme Linked Immunosorbent Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

Prunus Species ◽

Wide Range

Plum pox (Sharka) is a serious virus disease of stone fruits caused by the Plum pox virus (PPV). To determine which species could function as potential hosts and virus reservoirs, we used aphid transmission and bud or chip grafting to evaluate the susceptibility of commercial, ornamental, and wild Prunus species to isolates of PPV found in Pennsylvania, USA. Following inoculation, test trees were observed for symptoms, analyzed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), back-assayed to healthy peach, and followed through at least four cold-induced dormancy (CID) cycles over 4 years. Thirty-one of 33 Prunus species and cultivars were systemically infected following aphid transmission. Systemic infection could not be detected in P. cerasus (sour cherry) and P. × ‘Snofozam’ (Snow Fountains) despite repeated aphid inoculation attempts. Following grafting of PPV-infected budwood, all 40 species and varieties became infected, although species differed in their susceptibility. Within most species, some individual plants remained PPV negative throughout the study despite repeated inoculations. Infection in some species could be detected only through quantitative reverse transcription (RT)-PCR. Most species displayed clear symptoms, were highly positive by ELISA and RT-PCR, and could be back-inoculated into peach seedlings following CID. Our results indicate that a wide range of native and ornamental Prunus species are susceptible to U.S. isolates of PPV-D.

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First Report of Cucurbit aphid-borne yellows virus in Tunisia Causing Yellows on Five Cucurbitacious Species

Plant Disease ◽

10.1094/pd-89-0776b ◽

2005 ◽

Vol 89 (7) ◽

pp. 776-776 ◽

Cited By ~ 18

Author(s):

M. Mnari Hattab ◽

J. Kummert ◽

S. Roussel ◽

K. Ezzaier ◽

A. Zouba ◽

...

Keyword(s):

Citrullus Lanatus ◽

Aphis Gossypii ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Fresh Leaf ◽

First Report ◽

Reference Isolate ◽

Leaf Tissues ◽

Beet Pseudo Yellows Virus

Viruses, distributed worldwide on cucurbits, cause severe damage to crops. Virus surveys in 2003 and 2004 were made in all the major cucurbit-growing areas in Tunisia. Large populations of aphids (Aphis gossypii Glover) and severe yellowing symptoms of older leaves of cucurbits were observed in outdoor and under plastic-tunnel cultivation, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae). Leaf samples collected from symptomatic and asymptomatic plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita pepo L.), watermelon (Citrullus lanatus L.), and ware cucurbit (Ecballium elaterium L. T. Richard) were screened for the presence of CABYV using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Reference isolate, CABYV-N (GenBank Accession No. X76931) was provided by H. Lecoq (INRA-Monfavet Cedex, France). Sample extracts from fresh leaf tissues were tested using ELISA with an antiserum prepared against this isolate. In addition, total RNA was extracted from fresh leaf tissues according to the technique of Celix et al. (2) using the Titan RT-PCR kit from Roche Diagnostics (Penzberg, Germany). Forward primer (5′-GAGGCGAAGGCGAAGAAATC-3′) and reverse primer (5′-TCTGGACCTGGCACTTGATG-3′) were designed with the available sequence of the reference isolate. ELISA tests demonstrated that 91 plants were positive among 160 plants tested with severe yellowing symptoms. All asymptomatic plants were negative. RT-PCR results yielded an expected 550-bp product that was amplified from the reference isolate. Of the 160 plants tested using ELISA, 106 plants were screened with RT-PCR including the 91 plants that were positive in ELISA. These 91 plants also were positive after RT-PCR amplification as were 12 more plants. This demonstrated that the RT-PCR test is more sensitive. No amplicons were produced from extracts of asymptomatic plants, RNA preparations of Cucurbit yellow stunting disorder virus (CYSDV), or Beet pseudo yellows virus (BPYV) positive controls provided by B. Falk (University of California, Davis). CYSDV and BPYV can induce similar yellowing symptoms in cucurbits. The results of the ELISA and RT-PCR tests showed that CABYV is widely distributed on five cucurbit species in the major growing areas of Tunisia including the northern, Sahel, central, and southern regions where it was detected, respectively, in 10 of 25, 11 of 21, 24 of 37, and 58 of 77 samples tested. CABYV was detected at the rates of 63 of 72 on melon, 10 of 21 on cucumber, 17 of 24 on squash, 10 of 25 on watermelon, and 3 of 18 on ware cucurbit. CABYV also seems to be widespread throughout the Mediterranean Basin (1,3,4), but to our knowledge, this is the first report of the occurrence of CABYV in Tunisia on different species of cucurbit and ware cucurbit. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) A. Celix et al. Phytopathology 86:1370, 1996. (3) M. Juarez et al. Plant Dis. 88:907, 2004. (4) H. Lecoq et al. Plant Pathol. 41:749, 1992.

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First Report of Plum pox virus on Plum in New York State

Plant Disease ◽

10.1094/pdis-91-11-1512c ◽

2007 ◽

Vol 91 (11) ◽

pp. 1512-1512 ◽

Cited By ~ 6

Author(s):

K. L. Snover-Clift ◽

P. A. Clement ◽

R. Jablonski ◽

R. J. Mungari ◽

V. A. Mavrodieva ◽

...

Keyword(s):

New York ◽

Real Time ◽

New York State ◽

The United States ◽

Taqman Assay ◽

Plum Pox Virus ◽

Rt Pcr ◽

Surrounding Area ◽

First Report ◽

Nontranslated Region

Plum pox, also known as Sharka, is one of the more significant viral diseases of stone fruit trees such as plum, peach, and apricot. It was first reported in Europe in the early 1900s and more recently in Chile in 1992, the United States (Pennsylvania) in 1999, Canada (Ontario and Nova Scotia) in 2000, China in 2001, and Argentina in 2004. Plum pox virus (PPV) was recently detected in two plum (Prunus domestica) trees in an orchard in Niagara County, NY, located within 5 miles from a Canadian plum pox eradication zone. Typical symptoms of chlorotic rings and spots were observed on some of the leaves from these trees. No symptoms were reported prior to the survey collection in July 2006. Survey samples were screened for the presence of PPV by ELISA using the Agdia PPV (Agdia, Elkhart, IN) specific kit that recognizes all strains but C of PPV. Approximately 5% of the survey samples were additionally analyzed by a validated immunocapture reverse transcription (IC-RT)-PCR TaqMan assay in a Cepheid SmartCycler (Cepheid, Sunnyvale, CA). Both replicates of the two New York plum trees produced a positive ELISA reaction in two consecutive tests. The ELISA-positive samples also produced positive results when subjected to the real-time IC-RT-PCR test. The PPV-positive trees were sampled again and an additional 53 samples were collected from trees in the surrounding area. Suspect trees again tested positive, while all the trees in the surrounding area tested negative. The methods used for confirmation included two ELISA tests (Durviz [Valencia, Spain] DAS indirect monoclonal ELISA and Agdia DAS polyclonal ELISA). Confirmatory real-time IC-RT-PCR was performed using universal 3′ nontranslated region (NTR) primers (2,3) in a SYBR Green assay format and a coat protein (CP) primers/probe TaqMan assay (3,4). Further, the New York PPV isolate was identified as PPV D group using a subgroup specific conventional IC-RT-PCR (1). A 1.4-kb sequence fragment from the 3′ end of the New York PPV was sequenced (GenBank Accession No. DG 883816). Comparison of the sequence with the database confirmed this isolate as subgroup D and exhibited a high degree of identity with other PPV D accessions (PPV D Teycheney [Accession No. X16415]; Penn4 [Accession No. DQ465243] Cnd 123-1 [Accession No. AY9553267]; and Cnd 3 [Accession No. AY953262]). To our knowledge, this is the first report of PPV in New York. References: (1) T. Candresse et al. Phytopathology. 88:198, 1998. (2) L. Levy et al. J. Virol. Methods. 49:295, 1994. (3) V. Mavrodieva and L. Levy. Acta Hortic. 657:141, 2004. (4) T. Wetzel et al. J.Virol. Methods 33:355, 1991.

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First Report of Prunus necrotic ringspot virus in Peach in Mexico

Plant Disease ◽

10.1094/pdis-92-3-0482b ◽

2008 ◽

Vol 92 (3) ◽

pp. 482-482 ◽

Cited By ~ 1

Author(s):

R. De La Torre-Almaraz ◽

J. V. Montoya-Piña ◽

S. Alcacio-Rangel ◽

G. Camarena-Gutiérrez ◽

M. Salazar-Segura

Keyword(s):

Prunus Persica ◽

Rna Extraction ◽

Ringspot Virus ◽

Plum Pox Virus ◽

Rt Pcr ◽

First Report ◽

Field Samples ◽

Prunus Necrotic Ringspot Virus ◽

San Martín ◽

Das Elisa

Peach (Prunus persica (L.) Batsch) is one of the most important fruit crops in the temperate regions of Mexico. In 2006, during a survey conducted in commercial peach orchards in Puebla, Mexico for viral diseases, many trees were observed with foliar symptoms that included yellow mottle, ringspot, line patterns, and mosaic. Samples (flowers, young shoot tips, and leaves) were collected from 120 symptomatic trees in three locations (San Martin Texmelucan, Domingo Arenas, and Tepetzala). All samples were tested using double-antibody sandwich (DAS)-ELISA kits (Agdia, Inc., Elkhart, IN) for the presence of the following viruses: Apple mosaic virus, Plum pox virus, Prune dwarf virus, and Prunus necrotic ringspot virus (PNRSV). Sap extracts from young symptomatic leaves and shoots were used to mechanically inoculate Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Nicotiana tabacum cv. Xanthi, N. glutinosa, N. clevelandii, N. benthamiana, Datura stramonium, Capsicum annuum, and Solanum lycopersicum. Plants were kept in a greenhouse with approximate temperatures of 25 to 35°C, humidity of 70%, and 12 h of light. Sap extracts were also used for dsRNA extraction and analyses (2) and RNA extraction for use in reverse transcription (RT)-PCR with the Access RT-PCR system (Promega, Madison, WI) and primers that annealed to a conserved region in the PNRSV coat protein gene (1). The expected size amplicons of approximately 450 bp were generated from all field-collected samples. The PCR products from three geographically distinct PNRSV isolates (Domingo Arenas [Accession No. DQ979004], Tepetzala [Accession No. DQ979005], and San Martin Texmelucan [Accession No. EF456771]) were directly sequenced with a Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA) and their nucleotide and deduced amino acids sequences were more than 93% identical to corresponding sequences of PNRSV available in the NCBI/GenBank database. PNRSV was the only virus detected by DAS-ELISA in flowers and young shoots from 60 of the symptomatic field samples tested from the three locations. DsRNA banding patterns were obtained from 40 field-collected symptomatic samples; all showed three bands of approximately 3.6, 2.5, and 1.8 kb, the expected sizes for RNAs 1, 2, and 3 of PNRSV, respectively. DsRNAs were not detected in asymptomatic plants. PNRSV transmission by mechanical inoculation induced mosaic symptoms in N. tabacum cv. Xanthi and necrotic local lesions in G. globosa. Although G. globosa is reported to be a systemic host of PNRSV and N. tabacum is not reported to be a host, symptomatic plants were positive for PNRSV in DAS-ELISA tests. It is possible that there was an additional virus not detected in our assays that was responsible for the unexpected reactions in the host range studies. To our knowledge, this is the first report of PNRSV in peach in Mexico. References: (1) D. J. MacKenzie et al. Plant Dis. 81:222, 1997. (2) R. A. Valverde et al. Plant Dis. 74:255,1990.

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First Report of Garlic virus B infecting Garlic in India

Plant Disease ◽

10.1094/pdis-01-20-0156-pdn ◽

2020 ◽

Author(s):

Praveen Baliram Roylawar ◽

Kiran S Khandagale ◽

Pragati Randive ◽

Gorakshnath E. Atre ◽

Suresh Janardhan Gawande ◽

...

Keyword(s):

Consensus Sequence ◽

Sandwich Elisa ◽

Rna Extraction ◽

Enzyme Linked Immunosorbent Assay ◽

Experimental Plot ◽

Rt Pcr ◽

First Report ◽

The Impact ◽

Garlic Virus ◽

Germplasm Accessions

Garlic (Allium sativum L.) is an economically important spice and vegetable crop grown throughout the world. Garlic viral disease complex caused by multiple virus infections is an important constraint in exploiting the potential yield of garlic. Among these viral pathogens, allexivirus (family Alphaflexiviridae) is the genus of viruses known for their degenerative effect on garlic yield. Their coexistence with other viruses, particularly potyviruses, has an adverse effect on garlic yield and quality (Perotto et al. 2010). During Sept 2018, while screening garlic germplasm accessions for the presence of allexiviruses, symptoms like foliar mosaic and curling were observed on accession G-204, planted at an experimental plot of ICAR-DOGR, Pune, India. A total of five samples comprised of five randomly selected G-204 garlic plants were collected from the experimental plot. Each sample contained leaves from the top, middle, and bottom portion of the individual garlic plants. These samples were subjected to RNA extraction using the RNeasy Plant Mini Kit (Qiagen, Germany) followed by reverse transcription (RT) using the Transcriptor cDNA synthesis kit (Roche Diagnostics, GmbH, Germany). The extracted RNA was then tested for allexiviruses such as garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), garlic virus D (GarV-D), and garlic virus X (GarV-X) by polymerase chain reaction (PCR) (Gawande et al. 2015; Roylawar et al. 2019; Baranwal et al. 2011; Gieck et al. 2009). Leaf samples tested through RT-PCR were found positive for garlic viruses GarV-A, GarV-B, GarV-C, GarV-D, and GarV-X. Allexiviruses other than GarV-B had been previously reported in India and hence further tests were conducted to confirm GarV-B infection. RT-PCR using primers, CF 5’- ATGGGAGACAGGTCGCAA-3’ and CR5’- CTAAAATGTAAGCATGAGCGGT-3’ designed specific to the coat protein yielded a 735-bp amplicon from all five G-204 plants. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen, Germany) and cloned in pJET1.2 vector (Thermo Scientific, Lithuania). Two clones containing the CP gene were bidirectionally sequenced, and a consensus sequence was submitted to GenBank (MN650206). BLASTn results indicated that this consensus sequence showed 97.96% nucleotide (KP657919.1) and 100% amino acid sequence (AKN19940.1) identity with the CP sequence of GarV-B isolate from Poland. The presence of GarV-B was confirmed by enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich ELISA kit (Arsh Biotech, Delhi, India) as per the manufacturer’s protocol. An absorbance of reaction was read using a microplate reader at 405 nm. The mean OD values of negative and positive controls were 0.034 and 0.373, respectively. The OD values of five samples tested ranged from 0.210 to 0.296 indicating a positive reaction for GarV-B. To assess the presence of GarV-B in the available genetic stock, we tested 30 garlic germplasm accessions for GarV-B using RT-PCR. Out of these, 17 accessions were found positive for GarV-B. GarV-B has been reported from many countries (Gieck et al. 2009). This is the first report of GarV-B from India. Globally, allexiviruses are known for their adverse impact on garlic production (Oliveira et al. 2014). GarV-B together with other viruses can be a potential threat to garlic production in India. Further, detailed evaluations are needed to study the impact of GarV-B on garlic production in India.

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First Report of Orchid fleck virus in Costa Rica

Plant Disease ◽

10.1094/pdis.2002.86.12.1402d ◽

2002 ◽

Vol 86 (12) ◽

pp. 1402-1402 ◽

Cited By ~ 10

Author(s):

Juliana Freitas-Astúa ◽

Lisela Moreira ◽

Carmen Rivera ◽

Carlos M. Rodríguez ◽

Elliot W. Kitajima

Keyword(s):

Costa Rica ◽

Consensus Sequence ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Thin Sections ◽

Orchid Fleck Virus ◽

First Report ◽

Nucleocapsid Gene ◽

Pcr Product

Orchid fleck virus (OFV), a tentative member of the family Rhabdoviridae, infects orchids in several countries. The virus is vectored worldwide by the mite Brevipalpus californicus (Banks) (Acari: Tenuipalpidae). Eleven plants of Oncidium spp. and one plant each of the genera Cymbidium and Maxillaria exhibiting numerous yellow flecks and necrotic ringspot lesions on leaves were collected in two private orchid collections in Costa Rica. Presence of OFV was assessed by plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using an antiserum developed against an OFV isolate in Japan (2), analyses of ultrathin sections of the host cell with transmission electron microscopy (TEM), and reverse transcription-polymerase chain reaction (RT-PCR) amplification using specific primers for the viral nucleocapsid gene (1). Eight of eleven Oncidium samples, and both Cymbidium and Maxillaria samples tested positive for OFV with PTA-ELISA having A405 values ranging from 3.9 to 14.6 times higher than negative controls. Thin sections from individual samples of Cymbidium, Oncidium, and Maxillaria revealed electron-lucent intranuclear viroplasm and short, rodlike particles (40 to 50 × 100 nm) in the nucleus or cytoplasm typical of OFV-infected cells. RT-PCR amplifications from one sample of each genera resulted in PCR-product bands of approximately 800 bp. The Cymbidium RT-PCR product was cloned into a pGEM-T-Easy expression vector and sequenced using an ABI 3700 sequencer. The 619-bp nucleocapsid gene consensus sequence had 98% homology with the OFV isolate 0023 identified in Germany (GenBank Accession No. AF343870) (1). However, it had only approximately 85% nucleocapsid gene homology with other OFV isolates available through GenBank, including those from countries geographically closer to Costa Rica, such as Brazil (1). To our knowledge, this is the first report of OFV infecting orchids in Costa Rica. References: (1) A. L. Blanchfield et al. J. Phytopathol. 149:713, 2001. (2) H. Kondo et al. Bull. Res. Inst. Bioresour. Okayama Univ. 4:149, 1996.

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First Report of Peach latent mosaic viroid in Peach Trees in Montenegro

Plant Disease ◽

10.1094/pdis-06-11-0487 ◽

2012 ◽

Vol 96 (1) ◽

pp. 150-150 ◽

Cited By ~ 2

Author(s):

I. Mavric Pleško ◽

M. Viršcek Marn ◽

Z. Miladinovic ◽

J. Zindovic

Keyword(s):

Prunus Persica ◽

Stone Fruit ◽

Plum Pox Virus ◽

Rt Pcr ◽

First Report ◽

Fruit Species ◽

Hop Stunt Viroid ◽

Viroid Infection ◽

Fruit Samples ◽

Peach Trees

Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) are known to infect stone fruit species worldwide. The viroid infection can be latent or induce a variety of disease symptoms. Stone fruit samples were collected in Montenegro for a Plum pox virus (PPV) survey in 2007. Thirteen samples infected with PPV, taken from 12-year-old peach trees (Prunus persica (L.) Batsch, cv. Elegant Lady) in the area of Cemovsko field, were tested for the presence of PLMVd and HSVd by reverse transcription (RT)-PCR. Mild or severe mosaic, chlorotic rings, and fruit deformations were observed on some trees. Total RNA was extracted from all samples with a RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) and RT-PCR was performed. Samples were tested for HSVd and PLMVd infection using primer pairs RF-43/RF-44 for PLMVd (1) and VP-19/VP-20 for HSVd (2). Amplification products of approximately 348 bp were obtained from nine samples with PLMVd primers. Amplification products from seven samples were successfully cloned into pGEM-T Easy Vector (Promega, Madison, WI) and used for transformation of Escherichia coli. At least four clones of each sample were sequenced. Obtained sequences were 337 and 338 nucleotides long and shared 90.3 to 100% identity. Consensus sequences of each sample were deposited in GenBank under Accession Nos. JF927892–JF927898. They showed 92.6 to 97.9% identity among each other, 94 to 98% identity with the PLMVd isolate G sequence (Accession No. EF591868) and 91.8 to 94.4% identity with PLMVd sequence M83545. HSVd was not detected in analyzed samples. PLMVd infections were found on peach trees in an area where approximately 40% of the peach production is located. Therefore, PLMVd infections can pose a threat to peach production in Montenegro. To our knowledge this is the first report of PLMVd infection of peach in Montenegro. References: (1) S. Ambrós et al. J. Virol. 72:7397, 1998. (2) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997.

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