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.     

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.     

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.     

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.     

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.     

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.     

Comparison of latex agglutination,enzyme-linked immunosorbent assay,and indicator plants for detection of potato viruses S and X in potatoes

Potato virus S (PVS) was consistently detected by the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination test (LAT) at dilutions (PVS sap: healthy sap) of 1:499 and 1:19, respectively. Mechanical inoculation ofNicotiana debneyi gave variable results with detection possible 38% of the time at dilutions of 1:499. Potato virus X (PVX) was consistently detected by ELISA and LAT at dilutions (PVX sap: healthy sap) of 1:199 and 1:49, respectively. Mechanical inoculation ofGomphrena globosa with PVX resulted in consistent detection of the virus at a dilution of 1:499.     

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.     

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.     

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.     

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.     

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.     

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.     

Response of selectedSolanum species to virus eradication therapy

Thirty-fourSolanum genotypes infected with one or more potato viruses were evaluated in anin vitro virus eradication system combining heat therapy and the antiviral chemical ribavirin. This system was compared within vitro heat therapy or ribavirin treatment alone. The effect of expiant size on virus eradication following therapy was also evaluated. Infected plantlets were established in ribavirin lacking and amended medium; half of the plantlets in each medium were subjected to a 30-day heat treatment (35 C/31 C, 4-hr alternating periods), while the remaining plantlets were kept at room temperature (23 C). Following therapy, plantlets were tested quantitatively (OD at A₄₀₅) for the appropriate virus by enzyme linked immunosorbent assay (ELISA) and divided into 3 categories: low(< -0.05 OD), medium (> 0.05 to < 1.0 OD) and high (> -1.0 OD). Three plantlets were selected per group and further propagated as nodal cuttings or meristemtips. Regenerants which tested negative (< -0.05 OD) were transplanted to plug trays in a greenhouse and were retested after a 30-day grow-out period. Seventy-four and 91% of the genotypes yielded virus-free (VF) plants following propagation as nodal cuttings and meristem-tips, respectively. The combined therapies generated twice as many VF plants as thermotherapy alone and 5% and 15% more VF plants for nodal cutting and meristemtip derived plantlets, respectively, than for chemotherapy alone. Eleven of the 34 genotypes exhibited susceptibility to heat, but VF plants were obtained from survivors or by shortening the thermotherapy period. The efficiency of the system was enhanced by selecting plantlets for propagation which tested < -0.05 OD after treatment with 92%, 93%, 71%, and 100% of them remaining VF from potato viruses S, X, Y and leafroll, respectively. Twice as many VF plantlets were produced when treated plantlets were then propagated from meristem-tips than from nodal cuttings, but the rapid regeneration rate of plantlets from nodal cuttings makes it an attractive propagation method. Plantlet evaluation by ELISA after therapy to select plantlets with a high probability of producing VF plantlets and after further propagation to assure virus freedom are strongly recommended.     

Net necrosis of potato tubers associated with primary,secondary, and tertiary infection of leafroll

Tuber net necrosis, caused by leafroll virus, was found to occur as a result of primary, secondary, and tertiary infection in six clones of potato (Solanum tuberosum L.). Differences were observed between clones in their reaction to the different types of infection. In most clones there was a higher percentage of net necrosis from primary than from secondary or tertiary infection. No significant difference was observed in the severity of the net necrotic symptoms due to the different types of infection.     

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.     

Anatomical evidence for the existence of roots on potato tubers and stolons

Roots on potato tubers and stolons displayed the normal root anatomy which consisted of a central vascular cylinder surrounded by endodermis with Casparian strips, the cortex and epidermis. Tuber roots appear to initiate from the parenchyma cells adjacent to the vascular tissue. Shoot tips were similar to normal apical meristems. These observations support our research demonstrating the growth of functional roots from potato tubers and stolons.     

A rapid method for determining blackspot susceptibility of potato clones

Two methods of determining susceptibility of potato clones to blackspot were compared: (1) bruising by weight dropping and (2) bruising by abrasive peeling. A highly significant positive correlation was obtained between the intensity of enzymatic discoloration following abrasive peeling and the amount of blackspot that developed by weight dropping (r=0.93). Abrasive peeling was more rapid than the weight-dropping method. Tuber samples were abraded 30 sec and the amount of enzymatic discoloration evaluated after 24 hr. The need for individually bruising and hand peeling of tubers was eliminated with this method. Because of the rapidity of the abrasive peeling method, it can be used effectively in potato breeding programs to screen large numbers of clones for blackspot susceptibility. Results indicate that tuber maturity affects enzymatic discoloration and blackspot susceptibility. Immature tubers, dug while the vines are still green, are more resistant to blackspot than mature tubers. Tuber maturity therefore must be considered when screening clones for susceptibility to blackspot.