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.     

Losses due to the potato spindle tuber virus

A survey of 80 table-stock yields in New Brunswick revealed an incidence of 3.8% of potato spindle tuber virus among the three major varieties Kennebec (3.3%), Katahdin (2.5%), and Netted Gem (Russet Burbank (4.6%)). Concurrentily, in a field trial, three isolates of the mild strain of the virus (MPSTV) reduced yield in the variety Saco by 17%, 24%, and 24%, respectively, and the severe strain (SPSTV) reduced yield by 64%. An earlier preliminary survey had shown that in Eastern Canada, MPSTV prevailed over SPSTV on a ratio of 11∶1, thus a hypothetical loss of 1% could be calculated, conservative compared with Le Clerg et al. (1944) (2.6% loss by 4% incidence of PSTV), perhaps because of a lower incidence of SPSTV.     

Extreme resistance to infection by potato virus X in genotypes of wild tuber-bearingSolanum species

Summary Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X¹ (also called RX_act).     

Resistance to potato virus M in certain wild potato species

Summary Resistance to PVM has been found in some progenies ofSolanum gourlayi Haw., andS. spegazzinii Bitt. Clones from two progenies ofS. gourlayi, INTA 7330 and 7356, remained uninfected even after repeated graft inoculation.     

Seed transmission of potato spindle tuber virus

Seed transmission of potato spindle tuber virus (PSTV) has special significance for potato breeding programs. Transmission of PSTV has been reported to occur via true seed. Test families in the Frito-Lay breeding program were obtained from self- and cross-pollinations to determine the incidence of infected seedlings from healthy and diseased parents. Diseased seedlings from PSTV-infected Katahdin selfed were scored by visual and tomato index procedures as 100% and 87% infected, respectively. A similar result was obtained among seedlings from the cross of PSTV-infected Sebago x PSTV-infected Katahdin. Symptoms of PSTV infection were not detected by either method among seedlings from healthy Katahdin, selfed. The Frito-Lay, Inc., potato breeding program employs a number of phyto-sanitary procedures to exclude or reduce the possibility of PSTV contamination and spread.     

Additional indicator plants for potato spindle tuber virus

Thirteen hybrids ofLycopersicon esculentum Mill. (tomato) and threeLycopersicon species proved susceptible to sap inoculation with the potato spindle tuber virus (PSTV). These hosts showed the characteristic symptoms of epinasty and rugosity of the leaves, vein necrosis of leaves and petioles, and some stem necrosis. No local lesions were observed. These hybrids and species can possibly then be used as additional hosts showing systemic symptoms of the virus in addition to the most commonly used indicator, tomato cultivar Rutgers.     

Hosts of potato spindle tuber virus in suborder Solanineae

Additional hosts for potato spindle tuber virus (PSTV) found in the suborder Solanineae are:Nolana sp. (Nolanaceae);Antirrhinum sp.,Diascia barberae, Nemesia sp., N. foetens, Penstemon sp. (Scrophulariacceae); andBrowallia speciosa (B. major) andSolanum melongena in the Solanaceae. PSTV produced no local lesions upon inoculation of these hosts. Inoculated plants ofS. melongena (eggplant, ‘Black Beauty’) were dwarfed and epinastic. These rarely used genera in the Solanineae can be readily infected with the virus; leaf and floral parts are profuse and the tissues, upon grinding, leave very little residue.     

Detection of potato leaf roll virus and potato viruses M,S, X and Y by dot immunobinding on plain paper

Summary Potato leafroll virus and potato viruses M, S, X and Y in green leaves were detected by dot immunobinding (DIB) on plain paper and on nitrocellulose membranes. On both materials, DIB could detect the presence of very small amounts of virus, e.g. 30 pg of purified PVX. The sensitivity of the DIB test on plain paper and on nitrocellulose was compared to Double Antibody Sandwich ELISA (DAS-ELISA) by serial dilutions of infected plant sap made in healthy plant sap. Detection of potato viruses by DIB on plain paper and nitrocellulose was found to be equally sensitive whereas DAS-ELISA was 2 to 8 times more sensitive. Possible simplifications of the DIB procedure to suit the requirements of a routine method were examined. The use of the DIB method for routine testing of potato viruses is discussed.     

Resistance to virus S in the potato

On the basis of data from 18 crosses, resistance to potato virus S of the type encountered in Saco potato, segregated as an homozygous recessive. Neither Saco nor seedlings found to be resistant in greenhouse tests became infected when grown in the field and brushed with PVS-infected haulms. Meanwhile, susceptible seedlings from a cross segregating resistant and susceptible became infected in both greenhouse and field as readily as did seedlings from a cross producing only susceptibles. Resistance to PVS segregated independently of resistance to PVX.     

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.     

Experimental host range of the potato spindle tuber ‘virus’

Two hundred thirty-two plant selections (species and varieties) were tested for susceptibility to the potato spindle tuber ‘virus’ (PSTV). One hundred thirty-eight selections were found to be susceptible to PSTV but no ‘virus’ was recovered from the remaining 94. Susceptible plants were found in the families Boraginaceae, Campanulaceae, Caryophyllaceae, Compositae, Convolvulaceae, Dipsaceae, Sapindaceae, Scrophulariaceae, Solanaceae, and Valerianaceae. Most of the susceptible selections were symptomless carriers of PSTV. Visible symptoms were produced by both mild and severe strains of PSTV inLycopersicon esculentum cv. Allerfruheste-frëiland,Scopolia anomala, S. corniolica, S. lurida, S. sinensis,S. stramonifolia, S. tangutica, Solanum aviculare, andS. avicular var.albiforme; and only by the severe strain inGynura aurantica, Petunia hybrida var. Burpee Blue, andSolanum depilatum. Temperature of 21.1-22.8 C (70-73 F) with a light intensity of about 400 ft-c favored local lesion development inScopolia sinensis. S. sinensis appeared to be more susceptible than otherScopolia species.     

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.     

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.     

The elimination of potato viruses X,Y, S and M in meristem and explant cultures of potato in the presence of Virazole

Summary Incorporation of Virazole into potato explant and meristem culture media results in a higher percentage of virus-free progeny plants from virus-infected explant and meristem donor plants than are produced in tissue cultures in the absence of Virazole. Viruses eliminated, singly and as complexes, include potato viruses X, Y, S and M. The need for sensitive virus tests to distinguish between virus suppression and elimination is discussed and protein-A-linked immunoelectron microscopy is described and quantified for potato virus X.

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Zusammenfassung In der vorliegenden und einer früheren Arbeit (Cassells & Long, 1980) zeigte Kultivierung von Explantaten auf einem Medium für Gewebekultur mit Virazole (Ribavirin) einen signifikanten Anstieg virusfreier Adventivschosse gegenüber der Kontrolle. Diese Adventivschosse wie auch Kartoffelpflanzen zweiter Generation verhielten sich auch gegenüber den empfindlichsten immunoelektronen-mikroskopischen Tests virusfrei. In Gegenwart von Virazol ergab auch Meristemkultur einen h?heren Anteil virusfreier Pflanzen als bei Fehlen dieser Verbindung. Die Kartoffelviren X, Y, M und S sowie Viruskomplexe konnten aus den Sorten May Queen, King Edward, Kerr's Pink und Golden Wonder eliminiert werden (Tabellen 2 und 3). Die Ergebnisse der Anwendung und der Quantifizierung der Protein A-gebundenen Immuno-Elektronenmikroskopie (PALIEM) werden ebenfalls dargestellt (Abb. 1).

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Résumé Cette étude a montré, comme d'autres travaux préliminaires (Cassels & Long, 1980), que le fait de cultiver des boutures sur un milieu de culture de tissus contenant du virazole (ribavirine) permettait d'augmenter de fa?on significative le nombre de tiges aventices indemmes de virus par rapport au témoin. Ces tiges adventices, dans le cas de plants de deuxième génération, sont apparues indemmes de virus par l'analyse des tests les plus sensibles de microscopie immuno-électronique. En présence du virazole, la culture de méristèmes montre également un taux plus élevé de plants indemmes de virus que sans l'application de virazole. Les virus concernés sont les virus X, Y, M, S ainsi, que les complexes viraux, provenant des variétés May Queen, King Edward, Kerr's Pink et Golden Wonder (tableau 2 et 3). Sont également indiqués les résultats qualificatifs et quantitatifs de la microcopie immunoélectronique avec utilisation de la protéine A (PALIEM) (figure 1).

     

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.