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

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.     

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.     

Effect of time of inoculation with potato leafroll virus on development of net necrosis and stem-end browning in potato tubers

The Green Mountain cultivar was used in field tests to determine the effects of inoculating potato plants at various times with the potato leafroll virus (PLRV) on development of internal necrosis of tuber tissue. Viruliferous apterae of the green peach aphid,Myzus persicae (Sulz.), were placed on each stem in all hills to be inoculated in each 3.0 m single-row plot. Planting and inoculation dates were varied in all field experiments and, in one, several vine-killing dates were also included. All harvested tubers were stored for approximately four months at 10°C to enhance development of internal necrosis prior to examination. Similar but smaller greenhouse studies involving both apterous and alate green peach aphids were also conducted using Green Mountain, Irish Cobbler, and Russet Burbank cultivars. All results showed that as inoculation was delayed relative to plant development, more net necrosis (NN) occurred. Conversely, when plants were inoculated early, stem-end browning (SEB) rather than NN predominated. A high percentage of naturally occurring SEB tubers (cv. Russet Burbank) were found by ELISA to contain PLRV. Plants produced by these tubers only rarely developed leafroll symptoms. These findings suggest a previously unsuspected causal relationship between SEB and PLRV. Implications of this apparent relationship on the epidemiology of potato leafroll in Maine are discussed.     

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.     

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.     

Effect of time and date of inoculation,plant age,and temperature on translocation of potato leafroll virus into potato tubers

Movement of potato leafroll virus (PLRV) to tubers following placement of viruliferous aphids on foliage was studied on the medium-maturing cultivar, Katahdin and the late-maturing cultivar, Russet Burbank. Inoculation was begun on August 20 and continued at three-day intervals until mid-September. There was no trend of increasing or decreasing numbers of leafroll-infected tubers from early to late inoculation. Several variables were examined to determine their effects on the incidence of PLRV-infected tubers. Multiple regression analyses showed that inoculation date, maximum daily temperature, minimum daily temperature, plant age, and length of time from inoculation to harvest explained 38% of PLR incidence in tubers of cv. Katahdin. Length of time from inoculation to harvest and minimum daily temperature explained 40% of PLR incidence in tubers of the cv. Russet Burbank.     

Heat inactivation of leafroll virus in potato tuber tissues

Heat inactivation of leafroll virus in tuber tissues of three potato varieties (Russet Burbank, Katahdin, and Mohawk) was studied. Russet Burbank did not tolerate high constant temperatures and a low proportion of tubers and eye-pieces survived the treatments. On the other hand, Russet Burbank eye-pieces survived, with few exceptions, treatment at 40 C for four hours alternating with room temperaure (16–20 C) for 20 hours daily for as long as eight weeks. Inactivation of the virus was complete after six weeks of this treatment. Results obtained with the Katahdin and Mohawk varieties in similar tests were variable, and this possibly may be attributed to the higher room temperature (25–30 C) prevailing during these experiments.     

Volunteer potatoes as a source of potato leafroll virus and potato virus X

Volunteer potatoes were investigated as infection sources for potato leafroll virus (PLRV) and potato virus X (PVX) in a high elevation seed potato growing area of eastern Idaho. Population densities ofMyzus persicae were assessed. Percentage of PLRV and PVX infection of the volunteers and seed potato crops was determined, as well as density of volunteers and certain parameters of volunteer growth and reproduction. Volunteers apparently harbored no more PLRV than the potato crop from which they originated. But they were found to be an important reservoir of PVX with the infection increasing as much as 12.43% in one year. No aphids capable of transmitting PLRV were found although one species that can transmit potato virus Y was recorded. The mean density of volunteers varied from 0 to 84,880 stems/ha. The number of tubers remaining in the field after harvest and winter weather conditions appeared to be the only factors affecting volunteer density. Volunteer plants arising from seed pieces at an average depth of 6.1 cm were found to set an average of 2.1 new tubers per plant at an average depth of 4.0 cm. These results suggest that volunteer potatoes are a significant source of PVX infection in subsequent seed potato crops.     

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.     

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.     

Ultrastructure of potato tubers infected with potato virus X

The ultrastructure of Katahdin tubers infected with potato virus X (PVX) is compared with PVX-free tubers by electron microscopy. Electron-dense globules surrounding the inner periphery of the tonoplast were observed in PVX-infected tubers, while PVX-free tubers did not show such bodies. Other organelles were comparable in PVX-infected and PVX-free tubers     

Resistance of potato clones to the green peach aphid and potato leafroll virus

During 1980 and 1981 potato cultivars and breeding selections, including cultivated species and their hybrid derivatives, were evaluated for resistance to the green peach aphid (GPA),Myzus persicae (Sulzer), and to potato leafroll virus (PLRV). Criteria used were the number of aphids which colonized the clones in free choice field experiments and the number of plants derived from these experiments which showed symptoms of PLRV infection. Generally, greater resistance to GPA was found inSolarium tuberosum gp.andigena selections and hybrids than in gp.tuberosum cultivars. There were approximately fourfold differences in season-mean GPA levels among the clones tested each year. Forty-two families, representing a cross-section of the USDA breeding populations at the University of Idaho Research and Extension Center, Aberdeen, showed a similar range in colonization levels. Resistance to GPA colonization appeared to be more prevalent in gp.andigena, gp.phureja, and gp.stenotonum derivatives. There was a weak positive correlation (r² = .34, P = .01) between foliar total glycoalkaloids and season-mean GPA colonization levels for six clones representing the range of observed resistance to GPA. Resistance to GPA colonization was apparently not directly related to resistance to PLRV infection. Katahdin, for example, was relatively susceptible to GPA colonization but very resistant to PLRV infection whereas selection A69657-4 (gp.andigena) was among the most resistant to GPA colonization but among the more susceptible to PLRV infection. Breeding for resistance to GPA colonization therefore may not be as promising for PLRV control as developing PLRV resistant cultivars.     

Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

Detection of potato virus M in tubers,sprouts, and leaves of potato by the French bean test

Summary A simple French bean test was found useful in detecting potato virus M (PVM) in tubers, sprouts, and leaves of potato. Intact primary leaves of several bean cultivars were highly susceptible to the selected isolates of PVM fromKing Edward, Bintje, Fortuna, andUp-to-Date. Distinctive brown local lesions were formed 3 days after PVM inoculation. Certain factors, including leaf age, virus inactivation in sap, and relative virus infectivity in leaf and different parts of tuber, were studied by the French bean test. PVM was detectable by inoculating bean half-leaves directly with freshly cut pieces of tubers.

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Zusammenfassung Sieben aus Kanada und den Niederlanden stammende Isolate des Kartoffelvirus M (PVM) wurden in dieser Untersuchung gebraucht, um den Wert des Brechbohnen (Phaseolus vulgaris L.)-Testes für die praktische Verwendung beim Nachweis des PVM zu ermitteln. Die Inokulation erfolgte durch Abreiben jeder Blatth?lfte (nach Best?ubung mit Karborund, 600 Mesh) von voll ausgewachsenen Prim?rbl?ttern mit einem Baumwollappen, der vorher in ein Inokulum (mit 0,057M Phosphatbuffer, pH 8,5, verdünnt) getaucht worden war. Intakte Bl?tter und gepflückte Bl?tter. Die Empfindlichkeit der Prim?rbl?tter der Bohne nahm durch das Pflücken vom Stengel ab. Bohnensorten und Virusisolate. PVM-Isolate vonKing Edward, Bintje, Fortuna undUp-to-Date führten bei den folgenden Bohnensorten zu Lokall?sionen:Red Kidney, Bataaf, Prinsesco, Processor, Saxa, Walcherse Witte undWagenaars. Imuna, Noordhollandse Bruine undPinto UI 114 waren resistent gegen PVM (Tabelle 1).Alter des Blattes. Die Empf?nglichkeit der Bohnenbl?tter ?nderte mit zunehmendem Alter. Die gr?sste Empf?nglichkeit wurde 11 Tage nach der Saat beiRed Kidney, Bataaf, Processor undWalcherse Witte erreicht und 12 Tage nach des Saat beiWagenaars (Abb. 1). Verdünnungsendpunkt. PVM wurde in allen Knollenteilen bis zu einer Verdünnung von 10⁻³ entdeckt, nicht aber bei Verdünnung von 10⁻⁴, w?hrend PVM im Blatt bis zu einer Verdünnung von 10⁻⁴, aber nicht von 10⁻⁵ gefunden wurde (Tabelle 2). Langlebigkeit des Virus. Die Langlebigkeit des PVM war in den Knollenextrakten stets gr?sser (48 Std.) als in jenen von Blattextrakten (24 Std). bei 21–22°C (Abb. 2). Die Infektiosit?t von Blattmustern, tiefgefroren bei −15°C, nahm nicht stark ab, wie dies bei Saftmustern, aufbewahrt bei 22°C, der Fall war (Tabelle 3). Nachweis des PVM direkt in der Knolle. PVM wurde festgestellt in Augen, Rinde, Keim und Mark der Knollen, die w?hrend 8 Monaten bei 4°C gelagert wurden (Tabelle 4). Die Nachweisrate von 84% wurde durch direkte Inokulierung von halben Bl?ttern vonRed Kidney-Bohnen mit Knollenstücken erzielt (Tabelle 5). Daraus wurde geschlossen, der Bohnentest zur Erfassung des PVM in Knollen, Keimen und Bl?ttern der Kartoffel sei von praktischem Belang. Ferner wurde angenommen, dass der Bohnentest auch nützlich sein k?nnte bei der Auslese von Kartoffellinien bei der Untersuchung ihrer Resistenz gegen PVM und beim Auslesen von virusfreien Pflanzkartoffeln im Hinblick auf die nachfolgende Weitervermehrung.

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Résumé Dans la présente recherche, l'auteur a utilisé sept isolats du virus M de pomme de terre (PVM) provenant du Canada et de Hollande pour déterminer la valeur du test sur haricot vert (Phaseolus vulgaris L.) dans la pratique de détection du virus PVM. L'inoculation a été réalisée en frottant chaque moitié de feuilles primaires pleinement développées, poudrées avec du carborundum 600, avec un tampon de coton trempé dans l'inoculum dilué dans 0,057M, tamponné de phosphate, de pH 8,5. Feuilles de plantes intactes à feuilles détachées. Lors du sectionnement, la sensibilité des feuilles primaires de haricot diminue significativement de la tige au pétiole. Variétés de haricot et isolats de virus. Les isolats de PVM deKing Edward, Bintje, Fortuna etUp-to-Date provoquent des lésions locales chez les variétés de haricot suivantes:Red Kidney, Bataaf, Prinsesco, Processor, Saxa, Walcherse Witte etWagenaars. Imuna, Noordhollandse Bruine, etPinto UI 114 sont résistants au PVM (Tableau 1). Age de la feuille. La susceptibilité des feuilles de haricot varie avec leur age. La susceptibilité maximum au PVM est obtenue 11 jours après le semis chezRed Kidney, Bataaf, Processor etWalcherse Witte, et 12 jours chezWagenaars (Fig. 1). Point extrême de dilution du virus. PVM est détecté dans toutes les parties du tubercule jusqu'à la dilution de 10⁻³ mais non plus à la dilution 10⁻⁴, tandis que dans la feuille le PVM est détecté jusqu'à la dilution de 10⁻⁴, mais non plus à la dilution de 10⁻⁵ (Tableau 2). Longévité du virus. La longévité du PVM est toujours plus grande dans les extraits du tubercule (48 heures) que dans ceux de la feuille (24 heures) à 21–22°C (Fig. 2). L'infection d'échantillons de feuilles, gelées à −15°C, ne diminue pas fortement comme c'est le cas dans les échantillons conservés à 22°C (Tableau 3). Détection directe du PVM dans le tubercule. Le PVM se détecte dans l'oeil, la couche corticale, le germe et la mo?lle des tubercules conservés 8 mois à 4°C (Tableau 4). Le taux de détection est de 84% lorsqu'on inocule directement des demi-feuilles de haricotRed Kidney avec des fragments coupés de tubercule (Tableau 5). La conclusion est que le test sur haricot est pratique pour la détection du PVM dans les tubercules, les germes et les feuilles de pomme de terre. L'auteur suggère en outre que le test sur haricot peut être pleinement utile dans l'élimination de clones de pomme de terre lors de l'étude de la résistance au PVM, de même que dans la sélection de tubercules de pomme de terre libres de virus en vue d'une multiplication ultérieure.

     

Overwintering and monitoring of potato leafroll virus in some wild crucifers

A potato leafroll virus (PLRV) isolate has been successfully transmitted to and recovered from two wild crucifers,Sisymbrium altissimum L. (Jim Hill or tumble mustard) andCapsella. bursa-pastoris (L.) Medic. (shepherd’s purse) by the green peach aphid (GPA),Myzus persicae (Sulzer). Virus antigen in both plant species was found to be higher in root tissue than in foliar tissue, based on enzyme-linked immunosorbent assay (ELISA) determinations.C. bursa-pastoris was apparently a relatively poorer source of inoculum for the GPA thanS. altissimum. Using two geographically-separated biotypes ofC. bursa-pastoris, a Washington biotype was found to contain higher antigen titer in both leaf and root tissue than a California biotype, as determined by ELISA. Field studies demonstrated that both weed species can serve as overwintering sources of PLRV