Detection Of Potato Leafroll Virus Research Articles

Duplex RT-PCR: reagent concentrations at reverse transcription stage affect the PCR performance

Test conditions for the simultaneous detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription-polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. In the simplex RT-PCR a onefold dNTPs concentration (0.5 mM) was sufficient in yielding a PLRV or PVY band. In contrast, the duplex RT-PCR required a minimum twofold dNTPs concentration (1.0 mM) during RT to produce distinct bands in PCR. Similarly, various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49 μM of PLRV:PVY antisense primers. An interaction of dNTPs and RNA template concentration was observed. A higher concentration of RNA was required at onefold dNTPs concentration than at twofold dNTPs. Dilutions down to 1:300 of RNA template yielded distinct bands of both viruses at twofold dNTPs concentration. At optimized conditions of the duplex RT-PCR both viruses were reliably detected in composite samples at a ratio of one part infected sap mixed with 399 parts of sap from healthy tubers. Application of optimized conditions to singly- and doubly-infected tubers detected both viruses from naturally infected field-grown tubers. A nearly perfect correlation ( r 2=0.99) was observed between visible plant symptoms and the virus detection from leaves and tubers by the duplex RT-PCR.

Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The NASBA reaction products were visualized after electrophoresis by ethidium bromide or acridine orange staining. The specificity of the amplification products was validated by Northern blot analysis with a PLRV-specific 32P-labelled oligonucleotide probe. The procedure was coupled to immunocapture of PLRV virions from tuber extracts by immobilized antibodies in microtubes. It was possible to discriminate readily by this method between uninfected and primarily PLRV-infected potato tubers. NASBA is suitable for the direct detection of PLRV in potato tubers from primarily infected plants, offering the potential to considerably simplify the inspection of seed-potatoes for virus infection.

Detection of Potato Leafroll Virus in Dormant Potato Tubers by Immunocapture and a Fluorogenic 5 prime Nuclease RT-PCR Assay

A gelfree, reverse-transcription polymerase chain reaction-based fluorogenic detection method for potato leafroll virus (PLRV) in dormant potato tubers has been designed. A PLRV sequence-specific oligonucleotide (TaqMan probe, Roche Molecular Systems, Inc., Roche Molecular Systems, Inc., Alameda, CA), containing a 5'-terminal 'reporter' fluorescein and a 3'-terminal rhodamine 'quencher,' was specifically degraded during amplification, resulting in a relative increase in reporter-associated fluorescence. The system was coupled to immunocapture of PLRV from tuber sap by paramagnetic beads coated with an antiserum against the virus. Addition of cell wall-degrading enzymes, cellulase, and macerozyme to tuber sap was essential to make detection of PLRV possible on tubers after a storage period. The potential for application in routine detection of PLRV in seed potatoes was investigated by testing the method on primarily infected, dormant tubers of four potato cultivars. Results were validated both by gel electrophoresis and enzyme-linked immunosorbent assay of shoots from sprouted tubers, which is the current assay for postharvest control of PLRV. All methods showed complete agreement in discriminating between PLRV-infected and noninfected samples. Detection took place in microtiter plates and was rapid, reproducible, semi-quantitative, and amenable to automation. The system offers the possibility of reducing the inspection time of seed potatoes for PLRV infection from 5 weeks to 1 day.

Detection of potato leafroll virus in single aphids by the reverse transcription polymerase chain reaction and its potential epidemiological application.

A reverse transcription and polymerase chain reaction (RT-PCR) system was developed using two 20-mer primers located in the potato leafroll virus (PLRV) capsid gene. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined by Southern blots and detection by a PLRV-specific probe. As little as 5 min exposure of aphids to PLRV-infected leaves resulted in the presence of PLRV-specific bands in 13% of aphids. However, the percentage of PLRV-positive aphids increased with longer exposure to infected sources and reached 90% after 3-4 days of feeding. PLRV can be detected from a single viruliferous aphid or a single viruliferous aphid combined with up to 29 non-viruliferous aphids. PLRV can be detected from freshly collected aphids, those stored at -70 degrees C, or those stored in 70% ethanol at room temperature for extended periods. This method is applicable to assess the viruliferous nature of aphids caught in yellow-pan traps during the growing season or stored for over a year.

Improved detection of potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA

SummaryThe polymerase chain reaction (PCR) method was reliably used for detection of potato leafroll virus (PLRV) RNA in dormant tubers from field‐grown plants and in vitro‐propagated microtubers. A simple RNA extraction from tuber and microtuber tissue allowed reverse transcription and PCR amplification of viral sequences. It was also demonstrated that ELISA could be used reliably for detection of PLRV in crude sap extracts of dormant microtubers produced in vitro but not in tubers from field‐grown plants.

Detection of potato leafroll virus by visual inspection, direct tissue blotting and ELISA techniques

Greenhouse-grown Russet Burbank and Russet Norkotah plants were tested for potato leafroll virus (PLRV) by visual examination, enzyme-linked immunosorbent assay (ELISA) and direct tissue-blotting assay (DTBA). Visibly infected and healthy plants were obtained from excised eyes of tubers submitted for winter certification tests. Each plant was sectioned into leaf, petiole, stem, root, seed piece, and tuber portions. Tissues were blotted on a nitrocellulose membrane for DTBA and then homogenized for use in a double-antibody sandwich ELISA system. Agreement between the two serological detection methods and with visual readings was high for petioles and stems, but lower for leaf, tuber and root tissues. Comparison of DTBA with ELISA and with visual plant symptoms suggest that DTBA can be used with the same accuracy as ELISA for detecting PLRV in stems and petioles

Detection of Potato Leafroll Virus in Leaf and Aphid Extracts by Dot-Blot Hybridization

Cloned cDNA to potato leafroll virus (PLRV) RNA was evaluated for detection of PLRV in virus-infected samples from four states and the countries of Colombia and Russia. The use of formaldehyde was 32 times more effective than formamide for denaturing leaf tissue extracts in the dot-blot assay, and the sensitivity of the technique was estimated to equal that of the enzyme-linked immunosorbent assay (ELISA) for PLRV detection. These results, use of a commercial leaf sap extractor, detection of virus in bulk aphid samples, and use of the assay in a nonradioactive format demonstrate that the technology has potential for large-scale disease screening programs

Improved Detection of Potato Leafroll Virus in Plant Material and in Aphids

To improve detection of potato leafroll virus (PLRV) in plant material and viruliferous aphids, the enzyme-linked immunosorbent assay (ELISA) was modified by simultaneous incubation of sample and conjugate (cocktail ELISA) and by amplification of the enzyme reaction. Using this technique, 50-100 pg of PLRV could be detected per sample, and the virus could be detected in highly diluted leaf sap and in single Myzus persicae nymphs after a 12-hr acquisition access period on PLRV-infected Physalis floridana plants. In addition, when coated plates were used, the total assay time for PLRV detection in plant material could be reduced to 20 min

Comparison of three potato leafroll virus antisera and a single Beet western yellows virus antiserum for luteovirus detection in potato

Three potato leafroll virus (PLRV) antisera, representing European, British Columbian, and Californian isolates, performed similarly in detection of PLRV in ELISA tests of samples collected in three successive years at the Florida certification test plots and in tests of other samples collected in New York State. Although a range of absorbance values occurred, this was probably due to random variation in virus titers of samples rather than the occurrence of different virus strains or differential serological reactions by the antisera. Beet western yellows virus (BWYV) was detected in potato leafroll samples from nine states and provinces in North America. The BWYV-positive samples represented 40% in 1983 and 62.5% in 1984 of the total number of samples tested. These results confirm previous reports on the widespread occurrence of BWYV in potato with symptoms of leafroll.

Serological reaction of beet western yellows and potato leafroll viruses in ELISA

The detection of potato leafroll virus (PLRV) and beet western yellows virus (BWYV) and their serological relatedness were investigated by the double sandwich ELISA (DS). In both pure preparations and crude plant extracts, an unequivocal detection of each of these viruses was obtained by DS, provided the homologous antisera were used. No detection was achieved when the heterologous antisera were used, although purified virus could be detected when using the heterologous antisera for coating, providing the homologous antisera were used as conjugates. Therefore, for routine screening of BWYV or PLRV infection in plants, the DS method with homologous antisera can be used. However, it does not seem that BWYV infection in potato could be detected by the routine DS aimed for PLRV screening. In immunosorbent electron microscopy, PLRV and BWYV could be easily detected by coating grids with either homologous or heterologous antiserum. This and the possibility of trapping each virus in ELISA plates with the heterologous antiserum indicate serological cross-reactivity between PLRV and BWYV.

Detection of Potato Leafroll Virus in Primarily Infected Tubers by Enzyme‐Linked Immunosorbent Assay

The effects of artificially breaking dormancy on the detection of potato leafroll virus (PLRV) in tubers of four field‐grown potato cultivars with primary infection were studied by ELISA. Treatment with Rindite increased the concentration of PLRV at the rose end of tubers to a level which exceeded in the susceptible cv. Sieglinde that of the heel end and was in cultivars Sirco and Dirus almost as high as that of the heel end. PLRV concentration was highest at four weeks after Rindite treatment and remained on a much higher level in treated tubers from August till November than in untreated ones. At 14 weeks after breaking dormancy, PLRV dropped in treated tubers to a very low level which was similar to that of untreated ones. With sprouting tubers of cv. Jetta, PLRV occurred at significantly higher concentrations in samples from rose‐end eye tissue than in those from heel‐end tissue next to an eye. The reliability of detecting PLRV was higher in treated tubers than in untreated ones and did not depend upon the tuber end tested. With the exception of cv. Sieglinde, all infected tubers which could not be detected by ELISA in the tuber test originated from late‐inoculated plants. PLRV was reliably detected in sprouts. ELISA on leaves of field‐grown plants with primary infection gave very unsatisfactory results, irrespective of the time of inoculation.

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato leafroll virus and potato virus Y in potato tubers after artificial break of dormancy

Conditions necessary for the detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in tubers from primary and secondary infected plants were investigated. Tubers were analysed before and after breaking dormancy by rindite treatment. PLRV was reliably detected indormant tubers whereas PVY was readily detected only when tubers had been rindite-treated and held for two to three weeks at 22°C and high humidity in the dark. PLRV occurred in higher concentration at the heel end than at the rose end of infected tubers and the concentration remained nearly unchanged during the experimental period of 35 days, whereas PVY was found to be more concentrated at the rose end and was rapidly accumulating in the tubers after the break of dormancy. In dormant tubers PVY concentration dropped during storage at 22°C. The use of ELISA for tuber indexing is discussed.

Application of enzyme‐linked immunosorbent assay to the detection of potato leafroll virus in potato tubers

SUMMARYFactors affecting the detection of potato leafroll virus (PLRV) by enzyme‐linked immunosorbent assay (ELISA) in tubers of field‐grown potato plants with primary or secondary infection were studied. The reactions of extracts of virus‐free potato tubers were minimised by pre‐incubating the extracts at room temperature and by careful choice of the dilution of enzyme‐conjugated globulin. PLRV was reliably detected in tubers produced by secondarily infected plants of all six cultivars tested. PLRV concentration was greater in heel‐end than in rose‐end vascular tissue of recently harvested tubers but increased in rose‐end tissue when tubers stored at 4°C for at least 5 months were placed at 15–24°C for 2 wk.PLRV occurred at greater concentration in tubers from plants of cv. Maris Piper with natural or experimentally induced primary infection than in tubers from secondarily infected plants; again PLRV concentration was greater in heel‐end than in rose‐end vascular tissue. Plants whose shoots were infected earliest in the growing season were invaded systemically and produced the greatest proportion of infected tubers; plants infected late in the season also produced infected tubers but PLRV was not detected in their shoot tops. PLRV concentration in tubers from the earliest‐infected plants was less than in tubers from later‐infected plants. PLRV was detected reliably by ELISA in tubers from progenies that were totally infected but was not detected in all infected tubers from partially infected progenies.ELISA is suitable as a routine method of indexing tubers for PLRV, although the virus will not be detected in all infected tubers produced by plants to which it is transmitted late in the growing season.

Factors affecting the detection of potato leafroll virus in potato foliage by enzyme‐linked immunosorbent assay

SUMMARYUsing antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme‐linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10‐1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme.The detection end‐point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10‐2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus‐free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested.PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field‐grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse‐grown plants. In field‐grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.

Detection of potato leafroll virus in the potato leaves and sprouts by ELISA.

Detection of potato leafroll virus in the potato leaves and sprouts by ELISA.

Detection of Potato Leafroll Virus in Potato and in Physalis jloridana by Enzyme‐Linked Immunosorbent Assay (ELISA)

Journal of PhytopathologyVolume 90, Issue 4 p. 364-368 Detection of Potato Leafroll Virus in Potato and in Physalis jloridana by Enzyme-Linked Immunosorbent Assay (ELISA) R. Casper, Corresponding Author R. Casper Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Viruskrankheiten der Pflanzen, BraunschweigAuthor's address: Dr. R. Casper, Institut für Viruskrankheiten der Pflanzen, Biologische Bundesanstalt, Messeweg 11/12, D-3300 Braunschweig.Search for more papers by this author R. Casper, Corresponding Author R. Casper Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Viruskrankheiten der Pflanzen, BraunschweigAuthor's address: Dr. R. Casper, Institut für Viruskrankheiten der Pflanzen, Biologische Bundesanstalt, Messeweg 11/12, D-3300 Braunschweig.Search for more papers by this author First published: December 1977 https://doi.org/10.1111/j.1439-0434.1977.tb03257.xCitations: 53AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume90, Issue4December 1977Pages 364-368 RelatedInformation