Search results for: FTA PLANT KIT
#31838746 
2020/01/21
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Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest.
Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTA filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTA technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.S Latvala, M Haapalainen, P Kivijärvi, T Suojala-Ahlfors, S Iivonen, A Hannukkala
1398 related Products with: Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest.
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#30699584 //
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First Report of Cucumber mosaic virus from Eggplant (Solanum melongena) in Bangladesh.
In Bangladesh, eggplant (Solanum melongena L.) is largely cultivated by subsistence farmers for domestic consumption and generating family income. During a survey of family-owned farms in April of 2014 in Barisal region of Bangladesh, we observed a farmer's field (0.25 acres) of 3-month-old eggplants with nearly 90% of plants showing mild mosaic and mottling of leaves. Symptomatic plants showed reduced growth, with nearly 50% fewer fruits than from healthy plants. Symptomatic leaves tested positive for Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) by immunostrip diagnostic kit (Agdia, Elkhart, IN). For confirmation of the virus identity, leaf samples were pressed on FTA Plant Cards (Whatman International, Maidstone, UK) and air-dried at room temperature. For eluting total nucleic acids, four to eight disks were punched from the spotted circles of each FTA card using a Harris micropunch (2-mm diameter, Sigma-Aldrich, USA) and soaked for 1 h in 300 μl of extraction buffer (15 mM NaCO, 35 mM NaHCO, 2% [w/v] PVP40, 0.2% [w/v] BSA, 0.05% [v/v] Tween 20, pH 9.6). After vortexing followed by a brief centrifugation, 10 μl of the supernatant was mixed with denaturing buffer (0.1M glycine-NaOH, pH 9.0, 50 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% [v/v] Triton X-100) containing 1% β-mercaptoethanol, incubated at 95°C for 10 min, and kept in ice until use. Denatured sample (2 μl) was subsequently used in reverse-transcription (RT)-PCR using primers CMV-RNA3F (5'-GTAGACATCTGTGACGCGA-3') and CMV-RNA3R (5'-GCGCGAAACAAGCTTCTTATC-3') previously reported (2) to amplify a 529-nucleotide (nt) fragment representing the 210-nt intergenic region and the 319-nt partial coat protein (CP) gene of the RNA 3 segment. The amplicons were cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA), and DNA isolated from four independent clones per amplicon was sequenced in both orientations. The derived sequences (GenBank Accession Nos. KM516898 to KM516901) showed close to 100% identity among themselves and 97% identity with the corresponding sequence of CMV isolate BK16 from cucumber in Thailand (FN552546). These results supported immunostrip diagnostic assays in confirming the presence of CMV in symptomatic samples of eggplants from Barisal. For additional confirmation, a second primer pair (CMV-CP-F: 5'-ATGGACAAATCTGAATCAACCAG-3' and CMV-CP-R: 5'-TCAAACTGGGAGCACCCCAGAC-3') was designed using CMV sequences from JN054635 and GU906293 to amplify the full-length CP gene from the same nucleic acid preparations used above. The approximately 657-nt amplicons, representing the full-length CP gene, were cloned, and plasmid DNA from four independent colonies per amplicon wa s sequenced as described above. The derived CP sequences (KM516902 to KM516905) shared 96 and 95% nucleotide and 98.6 and 99.5% amino acid sequence identities with corresponding sequences of CMV isolates from banana (EF178298) and eggplant (GU906293), respectively, from India. Phylogenetic analysis of CP sequences derived from this study with corresponding sequences available in GenBank indicated that CMV from eggplant in Bangladesh aligned closely with CMV subgroup 1B. CMV was previously reported in chili pepper, and tomato from Bangladesh (1) and in eggplant from Israel (4) and India (3). To our knowledge this is the first confirmed report of the occurrence of CMV subgroup 1B in eggplant in Bangladesh. Since no aphids were observed on eggplants, it is likely that CMV was introduced into the farmer's field through seedlings raised from seed carrying the virus. References: (1) A. M. Akanda et al. J. Fac. Agric., Kyushu Univ. 35:151, 1991. (2) C. De Blas et al. J. Phytopathol. 141:323, 1994. (3) S. Kumar et al. Virus Dis. 25:129, 2014. (4) E. Tanne and S. Zimmerman-Gries. Plant Dis. 64:371, 1980.Basavaraj Bagewadi, Md Shahadath Hossain, Amer Fayad, Rayapati A Naidu
1373 related Products with: First Report of Cucumber mosaic virus from Eggplant (Solanum melongena) in Bangladesh.
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First Report of Iris yellow spot virus Infecting Green Onion in Indonesia.
Green onion (Allium fistulosum L.) is an important vegetable crop for small-holder farmers for domestic consumption in Indonesia. Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) transmitted by Thrips tabaci is an economically important viral pathogen of bulb and seed onion crops in many onion-growing areas of the world (1,3). In Asia, IYSV has been reported in India and Sri Lanka (2,4). In April 2013, symptoms suspected to be caused by IYSV were observed on a 1-month-old green onion crop grown for their leaves in a farmer's field in Cipendawa, Pacet, Cianjur District, West Java. Symptoms consisted of elliptical to spindle-shaped, straw colored, irregular, chlorotic lesions with occasional green islands on the leaves. Approximately 25% of the field had plants with these symptoms. The presence of the virus was confirmed with an IYSV-specific Agdia Flash kit. IYSV infection was confirmed by RT-PCR with primers specific to the nucleoprotein (N) gene of IYSV. Primers 465c: 5'-AGCAAAGTGAGAGGACCACC-3' and IYSV-239f: 5' TGAGCCCCAATCAAGACG3' (3) were used as forward and reverse primers, respectively, using total nucleic acids eluted from FTA cards that were previously coated with freshly prepared sap extracts from field samples. Amplicons of approximately 240 bp were obtained from four symptomatic plants tested but not from healthy and water controls. The amplicons were cloned and sequenced. Consensus sequence was derived from three clones. Comparison with IYSV N gene sequences available in GenBank showed sequence identity of 95 to 99% confirming the identity of the virus as IYSV. To our knowledge, this is the first report of IYSV infecting onion in Indonesia. The finding in Java underscores the need for conducting surveys in Java as well as other onion-growing regions of Indonesia to gain a better understanding of its incidence, distribution, and potential impact. References: (1) D. H. Gent et al. Plant Dis. 88:446, 2004. (2) B. Mandal et al. Plant Dis. 96:468, 2012. (3) H. R. Pappu et al. Virus Res. 141:219, 2009. (4) K. S. Ravi et al. Plant Pathol. 55:288, 2006.H R Pappu, A Rauf
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#30722403 //
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First Report of Recombinant Potato virus Y Strains in Potato in Jalisco, Mexico.
Potato virus Y (PVY) is a serious problem for potato production worldwide. The virus reduces both tuber yield and quality, and recent spread of recombinant strains of PVY in potato production areas is largely credited with the spread of potato tuber necrotic ringspot disease (PTNRD) (1). In Mexico, recombinant strains of PVY were reported in at least two states, Chihuahua (4) and the State of Mexico (3); however, no surveys have been conducted in other potato-producing areas, and the spectrum of PVY isolates circulating in the country has remained uncharacterized. In October 2011, a small-scale survey of seed potato was conducted in the state of Jalisco, Mexico, to identify PVY isolates present in fields. Twelve seed potato fields were inspected visually. These represented various generations of seed potato, from nuclear to G2. Leaf samples were collected from plants displaying mosaic, crinkling, and yellowing symptoms, and were tested for PVY. Fifty samples were collected from cultivars Fabula, Mondial, Fianna, Gigant, Caesar, and Adora. Of the 50 leaf samples collected, seven were PVY-positive using the Immuno-strip Kit (Agdia, Elkhart, IN), and six of these were determined to have a N-serotype according to the typing by the Pocket Diagnostics lateral flow kit (Forsite Diagnostics, Ltd., York, UK). PVY-positive samples came from cultivars Fabula (2 with N serotype), Mondial (4 with N serotype), and Fianna (1 with O serotype). Extracts of the seven PVY-positive leaf samples were applied to Whatman FTA cards (Sigma, St. Louis, MO), dried, and transported to the Plant Virology Laboratory at the University of Idaho for further characterization. All samples immobilized on FTA cards were subjected to RNA extraction and standard reverse transcriptase (RT)-PCR typing using a set of PVY-specific primers (2) to determine the strain type. All PVY isolates were recombinant. The six N-serotype samples were found to contain recombinant PVY isolates and produced characteristic bands of 181 and 452 bp in RT-PCR, which indicated the presence of two recombination junctions in the HC-Pro/P3 and VPg regions typical of European PVY isolates. The one O-serotype sample was identified as a recombinant PVY isolate, and produced 181 and 689 bp bands in RT-PCR, which indicated the presence of one recombination junction in the HC-Pro/P3 region. Sequence analysis of RT-PCR products amplified from five samples with N serotype identified them as PVY isolates, and from the one with O serotype identified it as PVY isolate. Sequence comparisons confirmed that N serotype samples contained PVY isolates most closely related to typical PVY sequences (Accession No. EF026075), while the O serotype sample contained the PVY isolate most closely related to PVY from Europe (HE608963). The data obtained suggest the presence of two different types of PVY recombinants, PVY and PVY, in seed potato in Jalisco. Additional surveillance for these recombinant isolates may be needed, as well as a survey of their effects on tuber quality in production areas. This is the first report of recombinant isolates of PVY often associated with PTNRD circulating in seed potato in Jalisco, Mexico. References: (1) S. M. Gray et al. Plant Dis. 94:1384, 2010. (2) J. H. Lorenzen et al. Plant Dis. 90:935, 2006. (3) V. R. Ramirez-Rodriguez et al. Virol. J. 6:48, 2009. (4) L. Robles-Hernandez et al. Plant Dis. 94:1262, 2010.A Quintero-Ferrer, A V Karasev
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#30780619 //
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Detection of Tomato spotted wilt virus in Tomato in the Baja California Peninsula of Mexico.
In 2005, serological screening by ELISA of 24 tomato (Solanum lycopersicon Mill.) plants with virus-like foliar symptoms (locally known as "marchitez manchada" [spotted wilt] disease) was done for a variety of RNA viruses: Tomato spotted wilt virus (TSWV; family Bunyaviridae, genus Tospovirus) was specifically detected. The symptomatic plants testing positive were from the most important tomato areas in San Quintin, in the north of Baja California. Symptoms characteristic of TSWV (4), including chlorosis, malformation of apical leaves, stunting, and ringspot lesions, were observed in this region and throughout the peninsula. In 2006, 42 symptomatic tomato plants from La Paz, in the south of Baja California, were analyzed for TSWV by double-antibody sandwich (DAS)-ELISA with a commercially available kit (TSWV ImmunoStrip Kit; Agdia Inc., Elkhart, IN). Total nucleic acids of the TSWV ELISA-positive samples (16 of 42 = 38%) were extracted and preserved on FTA cards (Whatman, Brentford, U.K.) and processed according to the manufacturer's protocol. The positive TSWV samples were verified by reverse transcription (RT)-PCR with primers specific to the TSWV nucleocapsid protein gene, 5'-ATGTCTAAGGTTAAGCTC-3' and 5'-TTAAGCAAGTTCTGTGAG-3' (2). Amplicons of the expected size (approximately 800 bp) were obtained from all 16 positive samples but not in the ELISA-negative samples. The spotted wilt disease was mechanically transmitted to tomato (cv. Rutgers) and Nicotiana glauca seedlings. Symptoms on leaves consisting of chlorotic ring patterns and necrotic lesions were observed in tomato, and slightly concentric chlorotic lesions were observed in N. glauca. All symptomatic plants from San Quintin and La Paz were positive for TSWV in the DAS-ELISA and RT-PCR tests and none were positive for the tobamoviruses, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV). TSWV was not detected in symptomless tomato plants used as negative controls. TSWV was detected in Mexico in tomatillo (Physalis ixocarpa), tobacco (Nicotiana tabacum), jimsonweed (Datura stramonium) (3), and recently, in tomato and pepper in the Central Plateau of Mexico (1). Although spotted wilt disease has been previously observed in San Quintin tomato-producing areas, to our knowledge, this is the first confirmation of TSWV in the Baja Peninsula. The role of weed hosts as a natural reservoir and the role of species of thrips in the epidemiology of the disease are currently unknown, although the incidence of the virus in these regions has risen to destructive levels in tomato. References: (1) R. De La Torre-Almaráz et al. Agrociencia 36:211, 2002. (2). R. K. Jain et al. Plant Dis. 82:900, 1998. (3) M. E. Llamas-Llamas et al. Plant Pathol. 47:341, 1998. (4) G. Marchoux et al. Plant Pathol. 40:347, 1991.R J Holguín-Peña, E O Rueda-Puente