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1.
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.  相似文献   

2.
Batenburg  L.  Goettsch  H. B. 《Potato Research》1960,3(3):229-235
Potato Research - When tubers of the varietiesEigenheimer, Furore, Bintje and some other varieties were tested, characteristic symptoms were found in the tubers of mosaic-diseased plants, which...  相似文献   

3.
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.  相似文献   

4.
5.
The effectiveness of a tuber incubation method for detection ofErwinia carotovora var.atroseptica andE. carotovora var.carotovora in potato tubers was compared with a lenticel sampling procedure. In the first method, tubers were injured by puncturing lenticels with sterile toothpicks, then wrapped in moist paper towels and polyvinylidene film, and placed in closed chambers flushed with N2. In later experiments, wrapping tubers in two layers of polyvinylidene film and incubation in air was found to be as effective as the single layer of polyvinylidene and incubation in chambers flushed with N2. Isolations were made on a selective crystal violet pectate (CVP) medium from homogenized samples of tissue removed from soft rot lesions developing around injured lenticels. In the second method, 10 lenticels/tuber were aseptically removed with a scalpel and homogenized in distilled water; the suspension was plated on CVP. The first method was less tedious and slightly more effective than the lenticel sampling method. In a preliminary survey, these methods were used to detectErwinia infestations in small samples of certified seed potato tubers from Maine, Minnesota, Montana, New York, North Dakota, and Wisconsin. PectolyticErwinia spp. were detected in at least one sample from each state except Montana. The percentage of tubers withErwinia infestations varied from 0–100% among samples. Characterization ofErwinia isolates showed that bothE. carotovora var.carotovora andE. carotovora var.atroseptica were present. PectolyticErwinia spp. on symptomless potato seed tubers may serve as inoculum sources for blackleg and soft rot diseases.  相似文献   

6.
7.
A simple procedure for detection of potato viruses S and X from dormant tubers by a latex agglutination test was developed. Eyes were pricked with a toothpick after having been covered with 50μ1 of buffer. Approximately 20μ1 of buffer then were drawn into a capillary pipette containing 10μ1 of antibody-sensitized latex and a latex agglutination test was performed. Both viruses were detected in all infected tubers tested. For potato virus S, the mean agglutination value was shown to vary significantly among cultivars. Tests on one eye were sufficient for detection with some cultivars, but tests on three eyes were necessary for detection with other cultivars. Eye location on tubers did not appear to influence test results, but better results were obtained when eyes were tested than when other tuber locations were tested.  相似文献   

8.
Summary Detection of potato virus Y (PVY) in dormant potato tubers using the enzyme-linked immunosorbent assay (ELISA) has been reported not to be accurate and reliable and it requires breaking of the dormancy of tubers prior to testing. We describe a simple, practical and highly sensitive hybridization method based on the use of biotinylated DNA probes for detecting PVY and potato aucuba mosaic virus (PAMV) in dilutions made of crude extracts of infected potato leaves and dormant tubers. As little as 50 fg of RNA can be detected by this method, and the probes are highly specific for their targets, even in crude plant extracts. The presence of one virus did not interfere with the detection of the other.  相似文献   

9.
Summary Using a modified procedure of Return-polyacrylamide gel electrophoresis (R-PAGE) mild strains of potato spindle tuber viroid (M-PSTV) were detected reliably from dormant tubers. The sensitivity of R-PAGE detection of M-PSTV was equivalent to that of nucleic acid hybridization. Both methods detected M-PSTV when infected tissue was mixed with healthy tissue in a ratio of 1 to 100. When extracted nucleic acid was diluted with buffer, R-PAGE detected PSTV at a dilution of 1:256 and nucleic hybridization only up to 1:64 PSTV was readily detected from 18 potato cultivars. In addition, mild, ‘intermediate’ and severe strains were separated by R-PAGE, on the basis of their mobility on the electrophorogram.  相似文献   

10.
Summary A reverse trancription-polymerase chain reaction (RT-PCR) assay was devised and shown to be sensitive and reliable for the detection of potato mop-top virus (PMTV) RNA sequences in the flesh of virus infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferousSpongospora subterranea in agricultural soils, and PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before the virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional baittests and sap inoculation methods for the detection and diagnosis of PMTV.  相似文献   

11.
Summary A PCR-based kit, ProbeliaTM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3×102 to 1.5×103 cells ml−1). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0×101 to 6.2×103 cells ml−1 peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.  相似文献   

12.
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.  相似文献   

13.
Summary 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.  相似文献   

14.
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.
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.

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.
  相似文献   

15.
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.  相似文献   

16.
ELISA (enzyme-linked immunosorbent assay) is a sensitive and reliable method of plant virus detection. It is commonly used in daily research carried out by scientific institutions and laboratories working on the certification of potato tubers. The key stage in this method is a 3–4-h-long incubation of microtiter plates with IgG and with a conjugate in an incubator at a temperature of 37 °C. The aim of the research was to replace this type of incubation process with a technique of mechanically shaking the plates using a shaker to induce a vibrating movement. Three durations of shaking, performed at room temperature, were adopted: 30, 60 and 90 min with two incubation periods at a temperature of 37 °C: 60 and 180 min which were applied at the stage of coating the IgG plates, following addition of the conjugate. The assessment was made for three dilutions of lyophilized sap from leaf of potatoes (1:10, 1:100, 1:1,000). Replacing the stages of plates incubation with IgG and conjugate at 37 °C with mechanical shaking allowed the whole process of DAS-ELISA to be reduced below 3–4 h without any significant impact on its quality. The process turned out to be equally efficient as the 3-h-long incubation in an incubator for PVY, PVM and PLRV detection by means of DAS ELISA. Applying the 90-min-long incubation on a shaker in comparison to a 3-h-long incubation in an incubator gave comparable or even slightly improved results. The reaction background, i.e. the value of absorbance for sap from healthy plants (negative control) was very low in all the combinations irrespective of the time of reading after the substrate was placed. No significant differences for this parameter were found between the combinations and times of reading. Only in the case of PLRV was a clearly visible decrease in test sensitivity found (no positive reactions) at diluted sap over 1:10. Moreover, it was observed that an increase in dilutions impacted the length of reaction. The dilution 1:10 seemed to be the most favorable (maximum 1:100 for PVY and PVM), wherein the sensitivity and pace of staining the substrate for each of the methods did not provoke any doubts regarding the reliability of the test.  相似文献   

17.
Summary In trials with potato tubers infected with tobacco rattle virus (TRV), symptoms of spraing in cvs Bellona, King Edward, Maris Bard, Matilda, Sv 82146 and Sv 82149 increased during storage when the tubers were cut. Storage of intact tubers at a constant temperature of 9°C or at fluctuating temperatures (2 weeks at 18°C, 2 weeks at 9°C and 2 weeks at 18°C) did not increase the frequency of symptoms.  相似文献   

18.
Summary The tuber-to-tuber variability in storage behaviour of seed tubers from true potato seed was compared with that in clonal seed tubers after storage in the dark, in diffused light, or in diffused light with a single desprouting. The variability was estimated by calculating standard deviations of length, number and weight of sprouts, and tuber weight loss. After dark storage, the variability of these variables was greater in seed tubers from true potato seed than in clonal seed tubers. After storage in diffused light with a single desprouting, the variability of number, length and weight of sprouts of seed tubers from true seed was not statistically different from that observed in clonal seed tubers. All storage treatments resulted in a greater variability of tuber weight loss in seed tubers from true potato seed than in clonal tubers.  相似文献   

19.
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.  相似文献   

20.
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