Detection of potato viruses X and S in tissue culture plantlets

The latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) were evaluated in separate studies for their ability to detect potato virus X (PVX) and potato virus S (PVS) in tissue culture plantlets. Healthy and infected clonal lines of several potato cultivars were used. LAT was unsatisfactory because only low levels of agglutination were obtained with infected samples, and because variable and inconsistent results were obtained with both healthy and infected clones. ELISA, however, consistently gave high spectrophotometric readings and intense visual reactions for infected but not for healthy clones. The results indicate that ELISA can be used to detect PVX and PVS in tissue culture plantlets, and in programs where tissue culture is employed, early detection and elimination of infected plantlets is possible.     

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.     

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.     

Kinetin,thermotherapy, and tissue culture to eliminate potato virus (PVX) in potato

PVX infected plantlets from two potato cultivars grownin vitro with 0.3, 3, 30 or 300 ppm kinetin were exposed to temperatures of 28 or 35 C. After 3 wk, axillary buds were isolated and grown aseptically in organogenic media, followed by PVX testing by ELISA. The serological test was also run on whole plantlets at the end of the kinetin-temperature exposure. No donor plants exposed to 28 C nor the plantlets derived from their buds gave an ELISA (-) reaction, regardless of the kinetin content of the media or that of the cultivar. At 35 C the virus was suppressed to undetectable levels in several whole plantlets. In the cultivar Alpha, 2 out of 6 resulting plantlets after isolation of buds were virus-free in the presence of 3.0 mg/1 kinetin during the treatments. From Atzimba, about 15–40% of the regenerated plants were ELISA (-), without any relationship to the cytokinin content in the media. Heat had a stronger influence on virus elimination than kinetin and the results varied with the cultivar.     

Potato leafroll virus purification and antiserum preparation for enzyme-linked immunosorbent assays

Potato leafroll virus (PLRV) was purified from potato foliage and stems with an average yield of 0.14 mg of PLRV/kg of potato. Modifications of an existing purification procedure are reported. Five low dosage (38-118 μg of PLRV) intravenous injections were used to produce a PLRV antiserum for use in enzyme-linked immunosorbent assays (ELISA) from tubers. PLRV was readily detected in ELISA testing of potato tubers and leaves and inPhysalis floridana Rybd. Non-specific reactions were low with all tissues. In parallel tests, a Canadian antiserum produced higher nonspecific reactions with tuber and leaf tissue. The results indicated that the use of low dosage-intravenous injections might be necessary methodology for producing PLRV antiserum for use in ELISA diagnostic tests with tuber tissue where high non-specific reactions have been reported.     

Maintenance,symptoms and distribution of potato viruses X,S, Y,A, M and leafroll in potato tissue culture plantlets

Maintaining potato viruses X, S, Y, A, M and leafroll in tissue culture plantlets is a convenient, cost and space effective alternative to the use of greenhouse plants. Of these six viruses, only certain strains of PVX induced symptoms in tissue culture plantlets. Nevertheless, all infected tissue culture plants were found to be more reliable than greenhouse grown plants as virus-infected controls in enzyme-linked immunosorbent assay (ELISA) testing. Another advantage of maintaining viruses in tissue culture plantlets was the elimination of contamination by other viruses or other pathogens. Leaves, stems, and roots of virus infected plantlets were tested separately for antigen levels by ELISA. In these tests, the stems and leaves of all but PVA infected tissue culture plants consistently gave positive ELISA values. In contrast, root tissue from PVY infected tissue culture plantlets was not reliable for PVY detection. In all cases, the viruses detected in the original source material were detected in the resulting tissue culture plantlets.     

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.     

A two-step ELISA for rapid,reliable detection of potato viruses

The reliability of the standard double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) was compared with a shorter, two-step DAS procedure in which sample and conjugate were mixed and incubated together in one step. The two assays were compared using beet western yellows virus and potato leafroll, M, S, X, and Y viruses. The two-step procedure was more sensitive,i.e., it detected small quantities of virus with greater statistical reliability than the standard procedure. At high virus concentrations, the standard produced stronger ELISA reactions than the two-step assay, but both assays were reliable. Since all of the viruses tested withstood high incubation temperatures, the incubation period for the two-step procedure could be reduced to 1 hr at 30 or 37 C. Therefore, assays could be completed within 2 hr using the two-step procedure compared with 2 days for the standard procedure. Reliable results were achieved with samples prepared by grinding tissues in buffer or, more simply, by adding pure, pressure extracted juice directly to conjugate in assay wells. Coating plates with gamma globulins or with F(ab′)₂ fragments of gamma globulins gave equally reliable results with all viruses except potato leafroll, where coating with gamma globulins was superior.     

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.     

Reinfection of potato seed stocks with potato virus S and potato virus X in Wisconsin

Reinfection of potato seed stocks with the potato viruses S (PVS) and X (PVX) varied with cultivar, virus, and grower. Rapid recontamination was observed for the cultivars Norgold Russet and Ontario with PVS and for the cultivars LaChipper, Norchip, and Norgold Russet with PVX. Recontamination was low for the cultivars LaChipper and Monona with PVS and for the cultivars Kennebec, Monona, Norland, and Superior with PVX. Survey results suggest that PVS and/or PVX can be eliminated from cultivars which appear to possess field resistance to infection, but that further evaluation of cultivars which are very susceptible to reinfection will be necessary.     

Association of viral,bacterial and fungal pathogens with vascular discoloration of Russet Burbank potato tubers

Russet Burbank potatoes from the 1979 and 1980 crop years, collected from Chicago, IL repack warehouses and retail markets, were sampled for vascular discoloration. The amount of discoloration varied among sampling months and states of origin and decreased from 7.7% in 1979 to 1.6% in 1980. Highest levels of vascular discoloration were detected in December and January samples. Vascular discolored and non-discolored (control) tubers were assayed for the presence of potato leafroll, potato virus X, and beet western yellows viruses by the enzyme-linked immunosorbent assay (ELISA). Tubers were also assayed for 2 subspecies ofErwinia carotovora and forVerticillium albo-atrum andV. dahliae. Potato leafroll virus was detected in 31 of 831 vascular discolored tubers.V. albo-atrum was detected in 1 of 180 discolored tubers. Beet western yellows virus was not detected in discolored or non-discolored tubers. Two subspecies ofE. carotovora and potato virus X were equally common in discolored and non-discolored tubers.     

Eradication of potato virus X from potato by ribavirin treatment of cultured potato shoot tips

Ribavirin treatment of cultured potato shoot tips was tested as a means of eradicating potato virus X (PVX) from two potato cultivars. Shoot tips were cultured on liquid medium containing 0, 1, 10 or 100μg/ml ribavirin. Cultures were evaluated periodically for viability, and scored for vigor on a relative growth scale. Developed plantlets were assayed for PVX by transmission tests toGomphrena globosa. Ribavirin treatment was phytotoxic at all tested concentrations, and lethal to all cultivars treated at 100 μg/ml. Treatment also delayed plantlet development by up to 2 months at 1 and 10 μg/ml as compared with nontreated controls. PVX assays indicated that 80 and 83% of the plantlets were free of PVX following treatment with 10 μg/ml for cultivars Russet Burbank and Red McClure, respectively. Five and 6% of the plantlets developed from the 1 μg/ml treatment were PVX-free, whereas 0 and 2% of the controls were PVX-free for the same cultivars. Six to 8 months were required to develop plants from shoot-tip cultures treated with 10 μg/ml ribavirin.     

Bison,a new red-skinned potato variety

Bison, a new red potato, was introduced by North Dakota State University. This new red variety has smooth tuber type and bright red skin color. Bison yields somewhat less than Norland and Red Pontiac but the advantage of Bison over these two varieties is its uniformity and bright red color. Bison is about medium in total solids and makes chips comparable in color to Norchip but lighter in color than Kennebec. Bison is resistant to race 0 of the late blight organismPhytophthora infestans, but susceptible to race 1–4.     

Comparison of nitrogen and phosphorus requirements between PVX free and regular Russet Burbank potato seed stocks

A Potato Virus X (PVX) free seed stock resulted in higher total yield and U.S. No. 1 potatoes than regular seed stock which carried PVX. Petiole samples from PVX free seed stock were higher in 2% acetic acid extractable PO₄-P than those from regular seed at all rates of fertilizer phosphorus. A high positive correlation was found between petiole phosphate and yield at early tuber set. Increasing rates of nitrogen increased higher total and U.S. No. 1 yields of potatoes, but also slightly decreased specific gravity. The results of this study indicate that phosphorus fertilizer recommendations for growers using Russet Burbank seed low in PVX will necessarily be different from those made for growers planting ordinary seed.     

A rapid and simple method for determination of potato chloroplast DNA type

A procedure for restriction enzyme analysis of the potato chloroplast DNA (ctDNA) is described. The advantages of this method are: 1) rapid determination of ctDNA type, 2) no ultracentrifugation, and 3) low cost of analyses. This method makes it easy to distinguish the ctDNA types of wild and cultivated potato accessions.     

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.     

Relationship of virus concentration with the field resistance to potato virus Y in potatoes

The concentration of potato virus Y (PVY) was determined, using ELISA values (A405 nm), in twenty-six potato cultivars belonging to five resistance groups, grown in the field and in the greenhouse. On the basis of virus concentration, potato cultivars of group A, B, and C did not differ significantly and constitute the most susceptible group; those of group D and E differ significantly with each other and with group A, B, C, and constitute moderate and highly resistant groups, respectively. In the second year of infection, virus concentration was higher in each group, irrespective of resistance level. Thus, the infected plants of resistant groups, in a second year of growth, could be as rich sources of virus to aphids as plants from susceptible groups.     

Eradication of potato virus Y and S from potato by chemotherapy of cultured axillary bud tips

Ribavirin (virazole) treatment of cultured axillary bud tips (3-4 mm) was tested as a method of eradicating potato virus Y (PVY) and potato virus S (PVS) from two potato cultivars, Norchip and Desiree. Ribavirin treatment was phytotoxic at all concentrations tested, but cultivars treated at 5 mg/1 were visually similar to the nontreated control cultures after 20 weeks. The buds treated with ribavirin at 20 mg/1 had a survival rate of only 30–40%. Virus assays indicated that 2,74,82, and 89% of the plants were free of PVY, and 1, 71, 83 and 90% were free of PVS, 20 weeks following treatment with 0, 5, 10, and 20 mg/1, respectively. Virus assays indicated that 2, 74, 82, 89% of the plants were free of PVY, and 1, 71, 83 and 90% were free of PVS, 20 weeks following treatment with 0, 5, 10, and 20 mg/1 ribavirin treatment respectively for cultivar Norchip. Desiree cultivar assayed 2, 69, 80 and 86% free of PVY and 2, 74, 85 and 93% free of PVS, 20 weeks following treatment with 0, 5, 10, and 20 mg/1 ribavirin treatment respectively.     

Pre and early post-emergence weed control with Paraquat in potatoes

Studies using Paraquat herbicide for early post-emergence control of broadleaved and grass weeds in Katahdin and Russet Burbank potatoes were conducted in Maine during four growing seasons. All rates and times of application of Paraquat gave good commercial control of grass and broadleaf weeds when compared to Premerge and Dowpon treatment as checks. Paraquat applied to Katahdins 2 weeks after ground crack reduced the yield of tubers but did not significantly affect specific gravity. Yield and specific gravity of Russet Burbank was reduced by Paraquat applied one and 2 weeks after ground crack. Paraquat can be used effectively for weed control in Katahdin up to one week after ground crack without crop damage. In Russet Burbank it appeared that application at ground crack was about as late as Paraquat could be applied without affecting yield or specific gravity of tubers.