Potato leafroll virus concentration in the vascular region of potato tubers examined by enzyme-linked immunosorbent assay (ELISA)

Summary Enzyme-linked immunosorbent assay (ELISA), used in conjunction with a new rapid extraction method, showed that potato leafroll virus (PLRV) concentration in the vascular region of infected potato tubers decreases from the heel to the rose end. Lower virus concentration at the rose than at the heel end was found not only in dormant tubers but also in tubers three weeks after breaking dormancy although the difference was then less pronounced. These results were obtained from plants with both primary and secondary infection by one of two French virus isolates which behave differently in respect of either accumulation in the ants or in their serological properties or both.     

Oxygen and carbon dioxide treatment to break potato tuber dormancy for reliable detection of potato virus Y (PVY) by enzyme-linked immunosorbent assay (ELISA)

Summary Dormant tubers of the potato cv. Bintje were treated for 7 or 14 days in an atmosphere enriched to either 40% O₂ plus 1% CO₂, or 40% O₂ plus 20% CO₂, or they were stored in closed plastic bags for identical periods. Rindite-treated and untreated tubers served as references. Treatment with 40% O₂ plus 20% CO₂ for 7 days was nearly as efficient as rindite for inducing sprouting. Both the O₂ plus CO₂ treatments, for 7 and 14 days, considerably increased virus concentration in tubers and had an effect similar to that of rindite 40 days after treatment, but the plastic bag treatment was not as efficient. It is concluded that O₂ plus CO₂ enriched atmospheres could be used for breaking tuber dormancy in order to detect reliably PVY in tuber extracts.     

Detection of potato virus X in tubers with the enzymelinked immunosorbent assay (ELISA)

Summary Tubers of field-grown plants of ten Dutch potato cultivars, secondarily infected with potato virus X (PVX) or free from this virus were submitted to ELISA after storage at 4°C. About 40 weeks after lifting PVX could be detected reliably. Extinction values with the apical parts of the tubers were slightly higher than those with the basal parts.     

Purification of potato virus A and its detection in potato by enzyme-linked immunosorbent assay (ELISA)

Potato virus A (PVA) was purified fromNicandra physaloides by a simple method that omitted organic solvent clarification and consisted of differential centrifugation followed by equilibrium centrifugation in CsCl. An antiserum was produced that specifically detected PVA in potato leaf sap using either the SDS-agar test or enzyme-linked immunosorbent assay (ELISA). No heterlogous reaction of the antiserum with potato virus Y was detected. Purified PVA was highly infectious; it had an A 258/280 nm absorbance ratio of 1.28. The particles had a normal intact appearance in the electron microscope. Detection of PVA in potato sprouts and foliage by ELISA was compared with the local lesion assay onPhysalis angulata L. plants. ELISA was superior over an indicator plant test when sprout tissue was used. PVA antiserum reacted similarly with mild and crinkle isolates.     

The callose test for the detection of leafroll virus in potato tubers

Summary Formation of abnormal callose in the sieve tubes is the basis of a practical test for leafroll virus infection in potato tubers. However, as it has often been stated that the test is not consistent enough, the following features were examined, with standardisation in mind: distribution of affected phloem in the tuber, detectability with different stains, the effect of the ‘Rindite’ treatment for breaking dormancy, and the effects of time and temperature of storage. In early-harvested tubers infected with leafroll virus, sieve tubes near the heel end are the most likely to contain abnormal callose but elements located in the cortex and medulla, as well as those near the cambium, can also be affected. Callose continues to form in early-harvested tubers during at least the first month of storage, but does not appear in tubers infected within a few weeks of harvest. Relatively less callose is formed at 28 C than within the range 4–18 C. The callose test may help in judging the health of a crop but it cannot be made precise enough for more critical purposes.

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Zusammenfassung Obwohl verschiedene Methoden zur Ermittlung von Blattrollvirusinfektion bei Kartoffeln entwickelt worden sind, wird zu ihrem Nachweis im allgemeinen noch immer die Augenstecklingsprüfung angewendet. Im Jahre 1955 wurde der Kallosetest von einigen Forschern eingeführt. Er beruht auf der Tatsache, dass das Blattrollvirus in den Siebr?hren des Phloems der Kartoffelknollen eine abnormale Kallosebildung hervorrufen kann. Wenn Schnittstücke von befallenen Knollen mit einem die Kallose f?rbenden Farbstoff behandelt werden, wird die Kallose sichtbar. Der Vorteil dieser Methode besteht darin, dass die vorhandene Kallose rasch festgestellt werden kann. Aus diesem Grunde wird der Test in verschiedenen L?ndern Europas im Anerkennungsverfahren für Saatkartoffeln angewendet. Ein Nachteil dieser Methode liegt darin, dass nicht das Virus selbst entdeckt wird, sondern nur eine seiner Sekund?rwirkungen. Da die Beurteilung der Testergebnisse weitgehend von den pers?nlichen F?higkeiten des Bearbeiters abh?ngt, k?nnte die gestellte Diagnose angezweifelt werden. Wir haben Versuche durchgeführt, um zu sehen, ob die Zuverl?ssigkeit des Teste verbessert werden k?nnte. Verschiedene Kallose-Farbstoffe, die Stelle der Kallose in der Knolle und Methoden zur F?rderung der Kallosebildung waren Gegenstand unserer Untersuchungen. Es wurden auch Versuche unternommen, um die Zeit zwischen der Infektion der Kartoffelpflanze mit Blattrollvirus und der Kallosebildung in der Knolle zu bestimmen. Bis jetzt ergab Resorzinblau die besten Resultate als F?rbemittel für die Kallose. Kein anderer der untersuchten Farbstoffe erh?hte die Zuverl?ssigkeit des Testes. Tabelle 1 zeigt, dass die Verteilung der Kallose in der Knolle sehr unregelm?ssig ist und dass sie sich haupts?chlich in der N?he des Nabelendes befindet. In Knollen von früh infizierten pflanzen findet man die Kallose auch am Kronenende. In früh geernteten Knollen wird die Kallosebildung durch Lagerung w?hrend 4 Wochen bei einer Temperatur von 10 bis 18 C erh?ht (Tabelle 2). Behandlung mit Rindite regte die Kallosebildung in Knollen der SorteBintje nicht an. Eine eigens aufgestellte Skala wurde verwendet um die beobachtete Kallosemenge zu vermerken. Die gef?rbten Siebr?hren zwischen den Schwarzen Linien in Abb. 1 wurden gez?hlt und die Zahl der gef?rbten Siebr?hren in Cortex und Medulla gesch?tzt. Auf Grund der Ergebnisse in Tabelle 4 wurde beschlossen zu empfehlen, dass auch die in Cortex und Medulla vorhandene Kallose, entgegen den Feststellungen vonWeller undArenz (1957) sowieSchuster undByhan (1958) in Betracht gezogen werden sollte. Dies bedeutet, dass wenn in Cortex und Medulla viel Kallose und in den Siebr?hren nahe beim Xylem keine gefunden wird, die Knolle trotzdem als infiziert betrachtet werden muss. Knollen, die eine Minimalmenge (1T) an Kallose aufweisen, n?mlich eine vollst?ndig mit Kallose gefüllte Siebr?hrenl?nge in der N?he des Kambiums, werden als krank bezeichnet. Die Ergebnisse in Tabelle 3 zeigen, dass Knollen von Pflanzen, die in einem sp?ten Entwicklungsstadium mit Blattrollvirus infiziert werden, nicht mehr Kallose bilden als gesunde Knollen. Daraus wird geschlossen, dass der Kallosetest nicht genügend zuverl?ssig ist, um im Studium des Viruswanderungsproblems von irgendwelchem Wert zu sein.

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Résumé Bien que différentes méthodes de diagnostic du virus de l’enroulement sur pomme de terre se soient développées, la méthode du tuber-test est ordinairement encore utilisée pour cette recherche. Le test de callose a été adopté en 1955 par un nombre de chercheurs. Il est basé sur le fait que le virus de l’enroulement peut produire une callose anormale dans les tubes criblés de phloème du tubercule de pomme de terre. Le trempage dans un colorant de sections de tubercules infectés met en évidence, par coloration, la présence de cals. L’avantage de cette méthode est la détection rapide de la présence de callose. Pour cette raison, ce test figure au programme de production de plants de différents pays d’Europe. Cette méthode a l’inconvénient de mettre en évidence non le virus lui-même, mais un de ses effets secondaires. Comme l’appréciation des résultats issus de ce test dépend pour beaucoup du jugement personnel de l’examinateur, le diagnostic posé peut prêter à discussion. Nous avons effectué des expériences pour voir comment on pourrait augmenter la sécurité du test. Nous avons étudié la variation de la coloration des cals, la localisation des cals dans le tubercule et les moyens de stimuler leur formation. Des essais ont également porté sur la détermination de la période s’écoulant entre l’infection de la plante par le virus de l’enroulement et la formation des cals dans le tubercule. Jusqu’à présent, le bleu de résorcine donne le meilleur résultat comme colorant des cals. Aucun autre colorant n’augmente la sécurité du test. Tableau 1 montre l’irrégularité de la répartition des cals dans le tubercule et leur localisation fréquente piés du talon. Dans des tubercules de plantes infectées précocement, les cals se trouvent également prés de la couronne. La production de cals chez des tubercules récoltés précocement est favorisée par conservation pendant 4 semaines à une température de 10 à 18 C (Tableau 2). Le traitement à la rindite ne stimule pas la formation des cals dans les tubercules de la variétéBintje. Une échelle arbitraire a été utiliséc pour enregistrer la quantité de cals observéc. Lest tubes criblés colorés entre les lignes noires de Fig. 1 sont comptés et le nombre de tubes criblés colorés dans l’écorce et la mo?lle sont estimés. Selon résultats figurant au Tableau 4, il y a lieu de conseiller, contrairement aux conclusions deWeller etArenz (1957) et deSchuster etByhan (1958), de prendre aussi de considération les cals présents dans ces dernières zones. Ce qui signifie que, si on détecte beaucoup de cals dans l’écorce et la mo?lle et aucun cal dans les tubes criblés près du xylème, le tubercule doit néanmoins être considéré comme infecté. Des tubercules montrant une quantité minimale (1T) de cals, c’est-à-dire une section de tube criblé près du cambium complètement remplie de cals, devront être considérés comme malades. Les résultats figurant au Tableau 3 montrent que des tubercules issus de plantes infectées par le virus de l’enroulement dans le dernier stade de développement ne forment pas plus de cals que des tubercules sains En conclusion, le test de callose n’est pas suffisamment sur pour être valable dans l’étude des problémes de translocation de virus.

     

Reaction to the potato leafroll virus (PLRV) in diploid potatoes

Summary Diploid parents with some resistance to PLRV, were intercrossed to give 3 families with 191 clones which were evaluated for reaction to PLRV and yielding ability. After inoculation with PLRV the clones could be separated into those: 1) resistant, 2) susceptible, 3) intolerant, reacting with low virus concentration, 4) tolerant and 5) intermediate in reaction. Both the ELISA test and the evaluation of external disease symptoms were necessary to separate the clones. No correlation was found between resistance to PLRV and tuber yielding ability.     

Simplified extraction method for ELISA and PCR detection of potato leafroll luteovirus primary infection in dormant potato tubers

This report describes a simple, rapid and inexpensive procedure for sampling large numbers of dormant tubers for analysis of potato leafroll luteovirus (PLRV) infection. The procedure uses a common electric drill to simultaneously remove and macerate tuber-eye samples for detection of PLRV by the enzyme-linked immunosorbant assay (ELISA) and the polymerase chain reaction (PCR). By using these sampling and analysis approaches, 19 of 20 different PLRV isolates were detected in dormant tubers from plants with primary infections. Results from the dormant tuber analysis, were verified by planting the tubers and testing leaf tissue by ELISA and PCR. Similar sampling and testing done on healthy dormant tubers and sprouts from the tubers consistently gave negative results as expected.     

Enzyme-linked immunosorbent assay with single or combined antisera for viruses S and X in potato tubers and plants

Enzyme-linked immunosorbent assay (ELISA), with single or combined antisera was effective for diagnosing potato virus S (PVS), potato virus X (PVX) or both viruses in plants grown in the greenhouse or field. In dormant tubers, stolon, middle and apical end composite sampling with or without eyes and sprouted tubers produced reliable positive assays for PVX. Only tuber pieces with sprouts resulted in consistently reliable assays for PVS. Composite sampling of potato foliage was effective for detecting one PVX infected plant in a total of 100 Kennebec, Norland, or Russet Burbank plants. There were some false negative results and greater variability in composite PVS assays, but on average, one PVS infected plant can be detected in composites of 10 Kennebec, Norland, or Russet Burbank plants. Sodium diethyldithiocarbamate (0.01M NaDIECA) in phosphate buffered saline + 0.5% Tween (PBS-T) added to plant extracts enhanced specific reactions for either virus. Onceor twiceused enzyme conjugate was effective in ELISA of either virus from potato foliage.     

A reevaluation of bromoethane in comparison to rindite for the post-harvest detection of potato virus Y in tubers by ELISA

A reevaluation of breaking tuber dormancy with bromoethane to increase the concentration of potato virus Y in the tuber revealed a positive response, by ELISA testing, to the treatment. The degree of response increased with the maturity of the tuber. Response to treatment with rindite was generally stronger, although differences were slight.     

Environmental factors influencing aphid transmission of potato virus Y and potato leafroll virus

Summary The effect of temperature, relative humidity (RH) and light on aphid transmission of potato virus Y (PVY) and potato leafroll virus (PLRV) was studied using as vectorsMyzus persicae Sulz. andAphis gossypii Glov. Host susceptibility was enhanced by 48 h pre-inoculation exposure at 25°C and by 48 h post-inoculation exposure to 30°C. High RH (80%) in both pre- or postinoculation phases enhanced host susceptibility. Continuous fluorescent light (4000 lux) did not alter the rate of transmission of either virus. High RH (80–90%) and high temperature (25–30°C), when combined, increased virus transmission by 30–35%. Transmission rates were reduced by nearly 50% if RH was maintained at 50% in either of the two phases even if the temperature was 25 or 30°C. Both viruses were acquired by aphids earlier (13–20 days after inoculation) when the source plants were incubated at 25 or 30°C. Most virus was transmitted from plants inoculated with PVY 13 to 16 days and with PLRV 15 to 20 days previously. Transmission rates of PVY were enumerated from symptom expression on test plants and by Enzyme Linked Immunosorbent Assay (ELISA) whereas those of PLRV were enumerated from symptom expression alone.     

Use of enzyme-linked immunosorbent assay to detect potato leafroll virus in field grown potato,cv. Russet Burbank

Use of enzyme-linked immunosorbent assay (ELISA) in detecting potato leafroll infections in field grown potato, cv. Russet Burbank, was studied from 1986 to 1988 at Rosemount, Minnesota. The objective was to determine relative reliability of current season foliage ELISA, tuber tissue ELISA, and tuber progeny foliage ELISA. Serological tests were most accurate when foliage of tuber progenies was tested. ELISA underestimated total leafroll infection when current season foliage from the inoculated plant was used, in those plants inoculated during late tuber bulking stage. Current season foliage ELISA tests using newly expanded terminal leaflets were more reliable than were tests using older leaflets. Leafroll infection was detected in the current season foliage and tuber progenies (tuber tissue as well as tuber progeny foliage) of some plants seven days after inoculation. Most current season foliage infections were detected by day 14–28 depending on year. Differences among years were most likely caused by variation in quality of virus source plants and numbers of vectors used in inoculation. ELISA tests on tuber tissue were almost as effective as ELISA tests on tuber progeny foliage in detecting potato leafroll 20 days after inoculation, but ELISA on tuber tissue substantially underestimated infection if plants were sampled earlier. Maximum percent tuber infection occurred 20 days or more after inoculation. Movement of the virus from the inoculated stem to other stems decreased with increased plant age at inoculation. Percent infected tubers declined with increased plant age at inoculation. Action thresholds developed for aphids in managing potato leafroll virus should take into account the temporal change in percent infected tubers.     

Resistance to powdery dry rot (Fusarium sulphureum) in potato tubers

Summary Fourteen cultivars were tested for resistance to penetration and colonization byF. sulphureum by a point inoculation technique. Differences in resistance to penetration were small when compared with those reported forF. solani var.coeruleum, and were more apparent in one year than another. Tubers grown at different sites gave similar results. The medullary tissue was generally more susceptible than the cortex. There were large differences between cultivars in resistance to colonization and these were consistent between years and sites. A selection derived from a cross between established cultivars was more resistant than its parents.

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Zusammenfassung Um die Resistenz von 14 Kartoffelsorten gegenFusarium sulphureum zu prüfen, wurde eine Punktinokulationsmethode verwendet, die der von Pietkiewicz & Jellis (1975) für Phomaf?ule beschriebenen Methode ?hnelt, das Inokulum bestand aber aus 100 Konidien in 0,01 ml sterilem Wasser und die Inkubation betrug 4 Wochen. Die zu prüfenden Knollen stammten von zwei Herkünften und sieben der Sorten werden in zwei Jahren getestet. Die Eindringungsresistenz wurde durch Bonitur der F?ulen, die mehr als 2 mm über die Verletzungen hinausgingen, erfasst und die Ausbreitungsresistenz in der Rinde durch eine Gesamtbonitur jeder Wiederholung, bei der eine 1–3 Skala verwendet wurde: 1=2–20 mm Durchmesser, 2=20–40 mm, 3=>40 mm. Nicht verwertet wurden F?ulen unter 2 mm im Durchmesser. Insgesamt waren King Edward, Record und Pentland Squire signifikant weniger anf?llig gegenüber der Eindringung als die anderen geprüften Sorten (Tabelle 1) aber keine Sorte besass eine hohe Resistenz. Die Ergebnisse waren zwischen den Herkünften, dem Gewebe und den Jahren vergleichbar, aber die Unterschiede zwischen den Sorten waren in einem Jahr deutlicher als im anderen. Das Markgewebe war im allgemeinen gegen die Eindringung anf?lliger als die Rinde. Die Rangfolge für die Ausbreitungsresistenz war zwischen den Herkünften konstant und wenn auch geringer, zwischen den Jahren (Tabelle 2). Sorten mit Eindringungsresistenz hatten geringe Boniturwerte für die Ausbreitung, aber das umgekehrte stimmte nicht immer. Erste Prüfungen von selektiertem Zuchtmaterial zeigten, dass in der Familie B31, die aus (Pentland Crown x Maris Piper) x Pentland Squire gezüchtet wurde (Tabelle 3a), Resistenz vorhanden sein k?nnte. Ein Wiederholungstest im folgenden Jahr best?tigte, dass der Klon B31/46 eine deutlich h?here Eindringungsresistenz in der Rinde aufwies als King Edward und erfolgreiche Inokulationen ergaben nur kleine Faulzonen (Tabelle 3b). Daraus wurde geschlossen, dass es erfolgversprechend ist, auf Resistenz gegenFusarium sulphureum zu züchten unter Verwendung der in den britischen Sorten vorhandenen genetischen Variabilit?t.

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Résumé Une technique d'inoculation par pointe a été appliquée sur 14 variétés de pommes de terre, afin d'étudier leur résistance àFusarium sulphureum. Cette technique est semblable à celle décrite par Pietkiewicz & Jellis (1975) pour la gangrène, mais la concentration d'inoculum est de 100 conidies par 0,01 ml d'eau stérile et l'incubation est de 4 semaines. Les tubercules testés proviennent de 2 régions et 7 variétés ont été étudiées pendant 2 années. La pénétration du parasite est évaluée en notant la proportion de pourritures se développant sur plus de 2 mm au-delà de la blessure, la surface du sympt?me est notée pour chaque répétition à partir d'une échelle de 1 à 3∶1= diamètre de 2 à 20 mm; 2=20 à 40 mm et 3= supérieur à 40 mm. Les pourritures d'un diamètre inférieur à 2 mm ne sont pas notées. Dans tous les cas, King Edward, Record et Pentland Squire sont significativement moins sensibles à la pénétration que les autres variétés testées (tableau 1), bien qu'aucune variété ne soit hautement résistante. Les résultats sont sensiblement comparables suivant les endroits, les tissus et les années, mais les différences entre les variétés sont plus nettes d'une année sur l'autre. Le tissu médullaire est en général plus sensible à la pénétration que le cortex. L'ordre de résistance au parasite est conforme suivant les endroits et sensiblement le même suivant les années (tableau 2). Les variétés présentant une certaine résistance à la pénétration ont des notes faibles au développement du sympt?me, mais l'inverse n'est pas toujours vrai. Des tests initiaux sous-abri à partir des sélections de plants ont montré une bonne résistance pour la famille B31, croisement de (Pentland Crown x Maris-Piper) x Pentland Squire (tableau 3a). Un test répété l'année suivante a confirmé la forte résistance à la pénétration dans le cortex du cl?ne B31/46 par rapport à King Edward et des inoculations réussies n'ont donné que de petites pourritures (tableau 3b). En conclusion, il est possible de sélectionner, en vue d'une résistance àF. sulphureum, à partir des variabilités génétiques disponibles au sein de variétés anglaises.

     

Simultaneous detection of potato viruses Y,leafroll, X and S by DAS-ELISA technique with artificial polyvalent antibodies (APAs)

Summary A modification of the double antibody sandwich-enzyme linked immunosorbent assay (DASELISA) was used to try to detect simultaneously PVY, PLRV, PVX and PVS in both leaf and sprout samples, using artificial polyvalent antibodies (APAs). Each virus, singly or in a mixture, was detected with similar sensitivity using either specific antibodies or APAs. The APA for all four viruses is suitable for routine leaf testing, but only the APA for PVY+PVX+PVS is suitable for routine sprout testing.     

The infection pressure of potato leafroll virus and potato virus Y in relation to aphid populations in Cyprus

Summary The infection pressure of two viruses, potato leafroll (PLRV) and potato virus Y (PVY), both common in seed potatoes grown in Cyprus, was determined in three experiments in 1982–83. Virus-free bait plants, of potato and four other species, were exposed weekly to field infection during the growing season (March–June), and then returned to an aphid-free glasshouse for symptom expression. Only tobacco plants produced clear symptoms enabling reliable assessment of PVY infection pressure. When assessed with ELISA or by tuber indexing, the potato plants were efficient baits for both viruses whose infection period commenced at emergence (mid March to early April) and ended within 6–7 weeks. The seasonal trend of aphid populations, determined with Moericke traps or 100-leaf counts, correspond to that of virus spread. Correlation and regression analysis of aphid and virus data implicated the alate form ofMyzus persicae as the principal vector of both viruses.     

Potato leafroll virus distribution in potato meristem tips and production of virus-free plants

Summary Uridine-H³ was incorporated into meristem tips of both healthy and PLRV-infected potato plants, of the cultivars, Majestic and Primura. Autoradiograms of tips pretreated with actinomycin D showed that 13 of 14 and 11 of 14 respectively, were virus free in the dome and first 4 leaf primordia, 1 and 3 were infected in the 4th leaf primordium, and 1 of Primura also in its 3rd primordium. The highest plantlet yield from cultures in vitro of meristem tips that included 4 leaf primordia (7 to 9 plantlets per meristem), was obtained by growing them in sequence on 3 sequential media, each based on Murashige and Skoog basic medium complemented with various hormones. The 2nd medium, containing benzylaminopurine (0.5 mg/l) and gibberellic acid (0.5 mg/l), elicited callus and the subsequent formation of adventitious buds. Of the plantlets of the cvs Vivaks, Primura and Majestic, 88, 91 and 100 %, respectively, were PLRV-free. Propagation in vitro, by the single-node cutting technique and tuberlet production, were successful.     

Evaluation of the enzyme-amplified ELISA for the detection of potato viruses A,M, S,X, Y,and leafroll

Various parameters,e.g. types of microtiter plate for DAS-ELISA (double antibody sandwich-enzyme-linked immunosorbent assay), use of fresh or frozen amplifier solutions for enzyme-amplified-ELISA, and use of sodium diethyldithiocarbamate (NaDIECA) in sample buffer in cocktail-ELISA were evaluated for the detection of potato viruses A, M, S, X, Y and leafroll from potato foliage. Dynatech Immulon immunoplates provided higher readings for all viruses. Fresh amplifier solution in amplifed-ELISA was superior to frozen solutions. Amplified ELISA gave only marginal improvement in the sensitivity over the standard DAS-ELISA. Addition of NaDIECA in sample buffer did not improve the detection of viruses in DAS-, amplified-, or cocktail-ELISA. Cocktail-ELISA can reduce antigen incubation time to as short as 15 min for PVA, PVM and PVX; 1 hr for PVY and PLRV; and 2–4 hr for PVS using pre-coated plates. Although amplified-ELISA is slightly more sensitive than DAS-ELISA for certain potato viruses, it is not suitable for large-scale indexing of potato viruses in Seed Certification Laboratories, in view of the additional steps needed in carrying out this procedure.