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.     

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.     

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.     

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     

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     

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.     

Comparison of the micro-precipitin test andChenopodium quinoa for the detection of potato virus S

Summary The micro-precipitin test and a bioassay using the local-lesion hostChenopodium quinoa were compared for their efficiency in detecting potato virus S in some potato cultivars grown in the open. TheC. quinoa bioassay was more sensitive than the serological test but used more space and was time-consuming. Guest worker from April–October 1976 as a fellow of the International Agricultural Centre, Wageningen, the Netherlands     

Acquisition of potato leafroll virus byMyzus persicae from secondarily-infected potato plants of different genotypes

Summary The acquisition of potato leafroll virus (PLRV) byMyzus persicae nymphs from the top leaves of potato plants was studied throughout a growing season in relation to the antigen titre in those leaves and the feeding behaviour of the aphid. Secondarily-infected plants of eight potato genotypes with different levels of field resistance served as virus sources. Early in the growing season, plants were efficient sources for virus acquisition. The amount of viral antigen detected inM. persicae nymphs fed on the top leaves was strongly correlated with the titres of viral antigen in these leaves. Virus acquisition from the top leaves of older potato plants was markedly impaired and could not be correlated with their virus titre. With increasing age of the potato plants and the development of virus symptoms, the virus titre in the leaves declined and the initial weak correlation between the virus titre and field resistance ratings disappeared. Thus, screening secondarily-infected potato plants for field resistance to PLRV based on the concentration of viral antigen in leaves or in aphids fed on them should be avoided later in the growing season. The feeding rate ofM. persicae, measured by the number of honeydew droplets excreted, did not account for the reduced uptake of virus from older plants since it was not influenced by the age of the plant. Throughout the growing season, the feeding rate ofM. persicae nymphs on PLRV-infected plants was higher on genotypes with low levels of field resistance to PLRV than on genotypes with high ones.     

A comparison of some methods for detecting potato leaf roll virus in potato tubers in Israel

Summary The results obtained by four methods for detecting PLRV in potato tubers grown in Israel were compared with the results obtained by aphid transmission to test plants. The correlation coefficients for potatoes lifted in July (seed for autumn) and September (seed for spring) were: Igel-Lange test, 0.18 and 0.25; datura grafting with tuber sprouts, 0.71 and 0.63; aphid transmission from sprouts to test plants, 0.89 and 0.81; and visual assessment in the Florida test (for spring seed), Up-to-Date 0.58, Blanka 0.67 and Désirée 0.33. The Igel-Lange test proved unreliable; datura grafting is time consuming and PLRV symptoms were similar to reactions ofDatura stramonium to wounding and to stress. The results of the Florida test were somewhat variable and were obtained too late. The sprout test was deemed the most suitable as it is accurate and the results can be obtained within two months. Contribution No 156-E, 1979 series, Volcani Center.