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     

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.     

Transmission of potato leafroll virus to or from tuber slices by aphids feeding through a membrane

Summary Eye-bearing slices, cut from healthy potato tubers and placed between Parafilm membranes, were inoculated with potato leafroll virus (PLRV) byMyzus persicae. PLRV was detected by ELISA and by transmission tests in tuber slices and in plants grown from the slices of the susceptible cv. Désirée, but not in those of the resistant cv. Arkula. These results suggest that PLRV replication and transport within tuber phloem is controlled by specific mechanisms of resistance.M. persicae was also able to acquire and transmit PLRV toPl floridana from slices cut from tubers of infected plants. The aphids effectively transmitted PLRV from slices cut from the sprouting rose end but they failed to transmit it from slices cut from the heel end of tubers.     

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.     

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.     

Interactions betweenPotato leafroll virus and potato plants and their implications for assessment of infection resistance

The effects of Potato leafroll virus (PLRV) isolates and inoculation method on the resistance to PLRV in potato clones and cultivars were investigated. In the season of inoculation, aphid inoculation resulted in higher proportions of infected plants and higher virus concentration in infected plants, as compared to graft inoculation. In these respects, grafting showed superiority over aphid inoculation in the tuber progeny of inoculated plants, though distinct dominance of aphid inoculation was observed for some tested clones. This leads to the conclusion that both methods should be applied as complementary tests for the efficient selection of highly resistant potato clones. For inoculations, two isolates of PLRV were applied, and one of them, isolate L7, was routinely used in screening breeding materials for resistance to the virus. The second isolate originated from plants of the highly resistant clone DW84-1457, which were incidentally infected with PLRV. The virus isolated from these plants was able to infect only specific clones, which were resistant to isolate L7. At the same time, potato clones with resistance derived from a source different from that present in DW84-1457, and standard susceptible and resistant cultivars, showed generally lower infection ratings after inoculation with the new isolate. This result suggests that the isolates may be different strains of PLRV. It is possible that under the strong selection pressure of resistance genes present in clone DW84-1457, a new isolate I-1457 could evolve the ability to overcome resistance to infection controlled by these genes.     

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.     

Use of IKI leafroll test to reduce net necrosis storage losses of potatoes

An iodine-potassium-iodide (IKI) starch staining procedure was adapted for use on potato plant leaflets to estimate the frequency of potato leafroll virus (PLRV) infection in potato fields prior to harvest. Large increases in the percentage of tubers with net necrosis occurred during storage among tubers from fields with high PLRV infection rates. Such fields were reliably identified prior to harvest by the IKI test, so their tubers could be processed at harvest to avoid net necrosis storage losses. The test could be performed on hundreds of samples per hour by untrained personnel with commonly available equipment. PLRV infection frequency varied widely in Columbia Basin potato fields. Most infected plants expressed no symptoms but could be detected by the IKI test before harvest. High rates of virus dissemination apparently occur late in the growing season in the Columbia Basin     

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.     

Unusually mild symptoms of potato leafroll virus in the progeny of late-infected mother plants

Summary In a field experiment in which the date of potato leafroll virus (PLRV) inoculation was controlled, progeny plants derived from late infection of mother plants in the previous year showed much milder symptoms than progeny plants derived from mother plants that became infected earlier in the season. Plants with these milder symptoms contained as great a concentration of PLRV as progeny plants with more obvious symptoms derived from early primary infections.     

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.     

Potato virus X infection of Netted Gem potato

When Netted Gem potato plants were inoculated with potato virus X 7 to 31 days before defoliation the tubers were not uniformly infected. Testing of a single germinating eye chosen at random from each tuber detected fewer than half of the tubers from which stem-end, mid, or apical-end sections produced infected plants. The number of completely and partially infected tubers increased as the length of time between inoculation and defoliation was increased. Infection was not correlated with tuber size. Partially infected tubers produced equal numbers of infected plants from stem-end and apical-end sections.     

The effect of temperature on the development of wilting and on progeny tuber infection of potatoes inoculated with South African strains of biovar 2 and 3 ofPseudomonas solanacearum

Summary Potato plants, after inoculation with a South African strain of biovar 2 or a strain of biovar 3 ofPseudomonas solanacearum were grown in growth chambers at temperatures ranging from 14/16°C (dark/light) up to 25/30°C. When grown at 14/16°C or higher, plants inoculated with the biovar 2 strain developed wilting symptoms and internal symptoms on progeny tubers and the disease was transmitted by the progeny tubers. When the plants were inoculated with the biovar 3 strain, wilting symptoms developed from 18/20°C, internal symptoms on progeny tubers from 20/22°C and the disease was transmitted by progeny tubers from plants grown at 16/18°C or higher.     

Host genes and transgenes that confer resistance to a Scottish isolate of potato leafroll virus are also effective against a Peruvian isolate

Summary Potato genotypes with host gene-mediated resistance (host-MR) and coat protein-mediated transgenic resistance (CP-MR) to potato leafroll virus (PLRV), were inoculated with a Scottish and a Peruvian isolate of PLRV. The coat protein transgene had been cloned from the Scottish PLRV isolate which had also been used during the screening and selection of genotypes with host resistance. Significantly less PLRV accumulated in plants with either host-MR or CP-MR than in plants of susceptible genotypes or in non-transformed control plants, but the two forms of resistance were equally effective against both PLRV isolates.     

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.     

Progeny tests to identify diploid potato clones homozygous at loci controlling resistance to PLRV

Summary Using potato parental lines homozygous at a locus or loci controlling resistance to potato leafroll virus (PLRV) can give advantages in the selection of resistant forms. In order to identify homozygous diploid clones their test-cross families were evaluated. All the clones that were test-crossed expressed resistance in primarily- and secondarily-infected plants and etiolated sprouts, and were derived from mating genotypes highly resistant to PLRV. Genotypes from test-cross families varied in resistance to PLRV, and one family was found which had only resistant genotypes, suggesting that the resistant parent of this progeny was homozygous at resistance loci. Evidence was gathered that resistance in some diploid clones may result from resistance to virus multiplication as well as restricted virus transport from leaves to tubers.     

An evaluation on intensity of infection of Pseudocercospora leaf spot disease of cowpea cultivars (Vigna unguiculata L. Walp), with respect to infector rows, dates of inoculation and cultivars, grown under field conditions in Northeast Thailand

This investigation was carried out at the Experimental Farm, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand to evaluate intensity of infection of Pseudocercospora Leaf Spot disease of cowpea cultivars, grown under field conditions with the use of Yasothon soil series (Oxic Paleustults). The experimental design used was a strip split plot design with four replications. The experiment consisted of three factors, i.e., with and without infector rows (Factor A), with and without inoculation and inoculated dates, i.e. none inoculation, inoculated at days 15 and 30 after sowing of seeds in main plots (Factor B) and three cowpea cultivars of KVC7, KKU25 and IT81D-1228-14-1 (Factor C). The results showed that the use of infector rows of cowpea plants (KKU25) being sown at 15 days before sowing seeds of cowpea cultivars in main plots gave a similar intensity of disease as those infector rows being sown at 30 days before sowing seeds of cowpea cultivars in main plots but significantly higher than without infector rows. Pathogenic disease being inoculated to the cowpea plants at 15 days after the sowing of seeds in main plots gave significantly higher intensity of disease than none inoculation. The establishment of infector rows at 15 days ahead of the sowing of seeds in main plots together with an inoculation of the disease at 15 days after the sowing of seeds in main plots gave the best results in evaluating amount of the infected disease. Thus infector rows surrounded the main plots are required whenever trials on disease evaluation are taken place.     

Multiplication ofPseudomonas solanacearum in resistant potato plants and the establishment of latent infections

When 1-mo-old plants of a wilt-resistant clone ofSolanum phureja (1386.15) were stem-inoculated with three strains ofPseudomonas solanacearum (K60, S123, and S206), the bacteria multiplied rapidly at the point of inoculation and then moved in the vascular system to other parts of the stem. Resistant plants showed a remarkable ability to support relatively high populations of the bacterium in the absence of disease symptoms. Although multiplication in this resistant clone was substantially less than in susceptible Russet Burbank potato plants, large numbers of bacteria (up to 624 × 10⁴ cells of K60 per 5-cm stem segment) reached the base of the stem of plants maintained at high temperature (28°C) for 20 days after stem inoculation. From the base of the stem, the bacteria moved rapidly into the roots and tubers. Strains ofP. solanacearum differed in their ability to cause latent tuber infection in different resistant potato clones. When 11S.phureja ×S. tuberosum hybrids were stem-inoculated, maintained at 28°C for 3 wk and then grown to maturity at 20°C., most of the clones yielded tubers infected by one or more strains. The race 1 strain (K60) was the most infectious; 53.8% of all tubers harvested from all plants inoculated with this isolate carried latent infections. Because one clone (BR 53.1) never yielded infected tubers, there appear to be genetic factors which may be useful in breeding programs aimed at eliminating latent tuber infection.     

Latent infection of potato tubers byPseudomonas solanacearum

Strains ofPseudomonas solanacearum differed in their ability to infect tubers of different resistant potato clones grown in infested soil. When eight resistant clones (Solanum phureja orS.phureja ×S. tuberosum hybrids) were grown at 24–28°C in soil infested with a race 1 or a race 3 strain of the bacterium, relatively few plants had wilt symptoms at harvest, but 26.7% and 9.2% of the tubers harvested from plants infected with the race 1 and race 3 strains, respectively, carried latent infections. Some infected clones never yielded diseased tubers, however. The development of symptoms above ground was not correlated with the incidence of tuber infection in any particular clone. No tuber infection occurred in tolerant or resistant clones grown in infested soil at cool (12–22°C) temperatures. Tubers were inoculated directly in an attempt to evaluate the ability of bacteria to multiply in these tissues at different temperatures. Highly virulent strains ofP. solanacearum survived in susceptible tubers in higher numbers and for longer periods than in resistant ones. Low temperature (4°C) had a deleterious effect on survival of the bacterium in tubers, but did not completely eliminate the pathogen even after 40 days.