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.     

Detection of potato virus X in tubers with the enzymelinked immunosorbent assay (ELISA)

Summary Tubers of field-grown plants of ten Dutch potato cultivars, secondarily infected with potato virus X (PVX) or free from this virus were submitted to ELISA after storage at 4°C. About 40 weeks after lifting PVX could be detected reliably. Extinction values with the apical parts of the tubers were slightly higher than those with the basal parts.     

Detection of potato viruses S and X in dormant potato tubers by the latex agglutination test

A simple procedure for detection of potato viruses S and X from dormant tubers by a latex agglutination test was developed. Eyes were pricked with a toothpick after having been covered with 50μ1 of buffer. Approximately 20μ1 of buffer then were drawn into a capillary pipette containing 10μ1 of antibody-sensitized latex and a latex agglutination test was performed. Both viruses were detected in all infected tubers tested. For potato virus S, the mean agglutination value was shown to vary significantly among cultivars. Tests on one eye were sufficient for detection with some cultivars, but tests on three eyes were necessary for detection with other cultivars. Eye location on tubers did not appear to influence test results, but better results were obtained when eyes were tested than when other tuber locations were tested.     

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.     

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.     

The incidence of potato viruses X,Y and S in the Chilota potato collection

The 530 potato clones of the Chilota collection of the germplasm bank from the Universidad Austral de Chile were tested for potato viruses X (PVX), Y (PVY) and S (PVS) by means of NCM-ELISA (nitrocellulose membrane enzyme-linked immunosorbent assay). Four clones (0.8%) healthy for all 3 viruses simultaneously were detected. These clones could be resistant to the above mentioned viruses.     

Comparison of penicillinase-based and alkaline phosphatase-based enzyme-linked immunosorbent assay for the detection of six potato viruses

Summary A penicillinase (PNC)-based enzyme-linked immunosorbent assay (ELISA) was compared with one based on alkaline phosphatase (ALP) to detect potato viruses A, S, V, X and Y, and potato leafroll virus. Tests were made with different ratios (w/w) of γ-globulin∶enzyme in the conjugation reaction. The γ-globulin fraction of antisera to the viruses was conjugated to ALP or to PNC by the single step glutaraldehyde bridge method using the ratio of 1∶1 (γ-globulin∶enzyme). The ALP conjugates worked well but PNC conjugates gave substantial non-specific reactions and in general were not suitable for ELISA. However, PNC conjgates made with a ratio of 1∶0.05 (γ-globulin∶enzyme) gave little or no non-specific reaction and were at least as sensitive as ALP conjugates. Both ALP and PNC conjugates were found to be useful in three variants of ELISA (double antibody sandwich; plate-trapped antigen and a simplified plate-trapped antigen form of ELISA) to detect the six potato viruses. Although PNC-based ELISA often required longer incubation with substrate to achieve the same sensitivity of detection as with ALP-based ELISA, this is only a minor disadvantage and PNC should prove to be more economical and safer to use than ALP.     

The effect of separate infections by potato viruses X and S on Netted Gem potato

Numbers and specific gravity of Netted Gem potato tubers were unaffected by mottle and latent strains of potato virus X (PVX) and by potato virus S (PVS). Neither latent PVX nor PVS affected yield but mottle PVX alone and latent PVX combined with PVS reduced tuber size and yield (P = 0.05).     

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.     

A microplate reader assay for rapid enzymatic quantification of sugars in potato tubers

Summary A sensitive, rapid and inexpensive method has been developed for quantifying the glucose, fructose and sucrose content of potato tubers. The method, based on selective enzyme-coupled reaction systems and the reduction of NAD to NADH, uses a microplate reader fitted with a 340 nm filter. In one microtiter plate 96 samples can be analysed for all three sugars in less than two hours. Several plates can be processed in parallel.     

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.     

Treatments that improve serological detection of potato viruses X,S, M and Y

Summary It was found that addition of an equal volume of 0.2% sodium sulphite or a mixture of sodium sulphite and sodium azide (both 0.2%) to expressed leaf sap improved serological detection of viruses and prevented non-specific reactions. Incubations of the preparations at 20–24°C gave better serological detectability than incubating at 10–12°C. If the sap was centrifuged, better results were subsequently obtained if centrifuging had been done at 4200 or 5900 g than at lower values.     

Detection of potato viruses A,M, S,X, Yand leafroll and potato spindle tuber viroid from tissue culture plantlets using single leaf discs

Using enzyme-linked immunosorbent assay (ELISA) and dotimmunobinding assay (DIBA) for potato viruses A (PVA), M (PVM), S (PVS), X (PVX), Y^N (PVY^N), Y^O (PVY^O) and leafroll (PLRV) and nucleic acid spot hybridization (NASH) for potato spindle tuber viroid (PSTVd), virus and viroid were detected reliably from single leaf discs (6 mm) of tissue-culture plantlets. Leaf discs taken from leaf positions (1 to 8) (bottom to top) can be used for detection of all viruses except PLRV where the lower leaves had higher concentrations of virus than the leaves from the upper part of the plantlet. Virus cultures were maintained for 1 to 4 years in several potato cultivars. The levels of virus remained reproducible except for PVM concentration, which was found to be very low in cv. Green Mountain. Using densitometry software, the DIBA spots were quantified and results were comparable to A₄₀₅ values obtained by ELISA. PSTVd concentration as measured by densitometry from spots of NASH indicated no loss of viroid over 1–4 years in tissue culture in two potato cultivars.     

Comparative levels of potato viruses S and Y infection of microplants and tuber-propagated plants in the field

The practice of transplanting microplants from tissue-culture to the field was compared to normal tuber propagation with respect to the transmission and translocation of potato virus S (PVS) and potato virus Y (PVY) to the daughter tubers of Red Pontiac, Shepody, Kennebec, and Russet Burbank cultivars in Prince Edward Island in 1984 and 1985. In general, the use of microplants to produce seed stocks appeared to increase the risk of infection with both viruses, although the results for tuber infection with PVY were only significant in one of the years.     

Resistance to potato viruses M,S, X and the spindle tuber virus in tuber-bearingSolanum species

On two separate occasions, some available accessions of tuber-bearingSolanum species were tested for resistance to potato viruses. Challenge inoculations were made mechanically with infective sap; and assessment was by sub-inoculation to differential host plants, supplemented by serological tests. In 1956–57, four accessions remained free from potato virus S (PVS), and nine remained free from PVM. In 1968–70, one or more clones of 11 accessions showed resistance to PVS—one of them,S. megistacrolobum, a diploid, apparently being hypersensitive. Resistance to PVX was found in seven accessions, and to PVM in two. There appeared to be resistance to the potato spindley tuber virus in five accessions.     

Use ofNicotiana debneyi to detect viruses S,X and Y in potato seed stocks and relative susceptibility of six common varieties to potato virus S

Summary Nicotiana debneyi Domin. proved a reliable test plant for potato viruses S, X, and Y. Test plants with several leaves 6 cm long, were dusted with carborundum and rubbed with potato leaf sap, or with raw surfaces of cut tubers. The plants were then held at 20°C under approximately 250 ft cd light intensity on a 16–18 h photoperiod. Each of the 3 viruses, or X and Y combined, could be distinguished, but symptoms of S were obscured by those of X or Y. At two elite seed farms, virus S spread into virus-free stocks of the varietyNetted Gem. but little intoKatahdin. In greenhouse tests, 3 older varieties,Green Mountain, Irish Cobbler andNetted Gem, as a group, were more susceptible to PVS, when inoculated with infective sap, than the newer varieties,Katahdin, Kennebec andSebago.