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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

Effect of inoculum concentration ofPhytophthora infestans on potato late blight

Zoospore suspensions ofPhytophthora infestans applied to potato cultivars in the field with an exponential inoculum sprayer resulted in defoliation within a range of severity that was log-normal with respect to inoculum concentration. Regression lines for different cultivars differed in position and, depending upon moisture conditions, slope, indicating that the cultivars differed in resistance to penetration and to invasion of tissue by the pathogen.     

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.     

Objective methods for testing potato flake quality as influenced by free starch and surfactants

A method was developed which uses changes in rates of retrogradation measured by a Brabender Amylograph to assess the complexing ability of surfactants for free starch in potato flakes during cooling. The rate of retrogradation was linearly related to surfactant concentration. A Blue Value Index method, adapted from use with cereal flours for determination of starch damage, was used to monitor the amount of free starch created by the required particle size reduction for Amylograph analysis. The change in Blue Value Index was also used to monitor the ability of surfactants to complex free starch and thereby influence potato flake quality. Distilled monoglyceride appeared to be more effective than sodiumor calcium stearoyl–2-lactylate. However, the combination of 0.3% distilled monoglyceride with 0.2% sodium stearoyl–2-lactylate was nearly as effective as 0.5% distilled monoglyceride in terms of free starch complexing ability. Instron back extrusion tests for mealiness of reconstituted flakes were found to be influenced by the level of free starch; therefore, back extrusion data should be accompanied by Blue Value Index data.     

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.     

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.     

Rapid extraction and solubilization of potato starch with Dimethylsulfoxide

Dimethyl sulfoxide (DMSO) solubilization of starch from lyophilized potato tissue is equally quantitative and more convenient than solubilization in dilute alkali, especially when analysis of a large number of samples is necessary. Starch extraction with DMSO decreases the handling time of each sample by eliminating sonication, filtration, and centrifugation steps required by the NaOH solubilization technique.     

Influence of Colorado potato beetle sample counts and plant defoliation on potato tuber production

Field experiments were designed to subject Superior potato plants to various levels of defoliation by the Colorado potato beetle (CPB)Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae. Defoliation occurred during each of five consecutive plant growth periods and CPB population, plant leaf area, and tuber weight data were recorded for each period. A visual defoliation rating scheme provided an accurate estimate of actual potato plant leaf area of defoliated plants. Data generated from regression analysis demonstrated a significant dependence of leaf area on CPB numbers per plant during some plant growth periods, but numbers of CPB accounted for very little of the total variation in tuber weight. Plant leaf area was the most important independent variable in the tuber weight regression model.     

Variability in the concentration of three heat stable proteinase inhibitor proteins in potato tubers

The variability of three well characterized proteinase inhibitors, Inhibitor I, molecular weight 39,000, Inhibitor II, molecular weight 21,000, and Carboxypeptidase Inhibitor, molecular weight 4,100, were determined in apical cortical tissues of individual potato tubers of the Russet Burbank variety. The three inhibitors varied within ± 20% among sixty-five tubers and cumulatively represented about 7% of the total soluble proteins. The inhibitors were highly variable among tubers of 106 clones from randomly chosen varieties. Inhibitor I varied about twelve-fold (60 to 745 μg/ml juice), and Inhibitor II varied about seven-fold (158 to 1,025 μg/ml juice). Carboxypeptidase Inhibitor varied from as low as zero (seven varieties) to over 850 μg/ml tuber juice. With 80 tubers from fourteen varieties of potatoes, a positivecorrelation was found between the concentrations of Inhibitor I and Inhibitor II and total soluble protein. Carboxypeptidase Inhibitor did not correlate well with total soluble protein. The positive correlations of Inhibitors I and II (a correlation coefficient of 0.70) with total soluble protein indicated that the proteinase inhibitors may be excellent markers for genetic studies for selecting high protein potato tuber varieties.