马铃薯纺锤块茎类病毒不同株系及不同接种方法对产量的影响

马铃薯纺锤块径类病毒(Potato spindle tuber viroid)是危害黑龙江省马铃薯生产的一种重要病害。为了查明不同类病毒变株对主栽品种克新1号和4号所引起的产量损失和症状反应,应用往复聚丙烯胺凝胶电泳法(Peturn-Polyacmdegel electophoresis)鉴定类病毒。试验结果表明,接种当年,接种类病毒的处理平均减产37%,按商品产量计算减产达47%。当年表现症状的植株平均为59.6%,块茎产生症状的植株平均为57.4%。用于接种的3个马铃薯纺锤块茎类病毒变株的致病力,以当地的类病毒弱系为最强,其次为北美的类病毒强系,北美的类病毒弱系的致病力较弱。类病毒对产量的影响与马铃薯品种和类病毒变株的组合有极为密切的关系。克新1号接种当地的类病毒弱系时薯块严重畸变,商品产量只为不接种对照的21%。用往复电泳法检测各个试验处理被类病毒侵柴的情况,芽接种处理,于出苗后1周即可检测到类病毒,叶接种处理,于接种后2周即能检测到类病毒。出苗后7周,植株中类病毒浓度达到高峰,随即急剧下降,到出苗后10周,大部分接种植株已检测不到类病毒。块茎直径达1厘米时,即能检测到类病毒,随着块茎膨大,类病毒浓度也相应增高,到收获前,块茎中的类病毒含量只略低于植株含类病毒高峰期的浓度。用于本试验的克新1号、4号无类病毒和主要马铃薯病毒的核心材料,在1988年已交付给省内外的良种场繁殖使用。于当年秋季抽样检测克山第二良种场连续种植二年的种薯,当年种植试管苗生产的种薯,以及试管苗等,均未检测到类病毒。     

Detection of potato spindle tuber viroid by nucleic acid spot hybridization: Evaluation with tuber sprouts and true potato seed

A diagnostic test for the potato spindle tuber viroid (PSTV) that is based on hybridization of highly radioactive, recombinant DNA complementary to PSTV with PSTV bound to a nitrocellulose membrane and autoradiographic detection of the resulting DNA-RNA hybrids has been evaluated with tubers from 20 potato clones maintained at the International Potato Center (CIP) and with true seed obtained from healthy or PSTV-infected potato plants. The nucleic acid spot hybridization test detected PSTV not only in tuber sprouts from 10 clones that had previously tested positive in polyacrylamide gel electrophoretic (PAGE) analysis, but also in tuber sprouts from 9 clones that had previously tested negative in PAGE analysis. Further tests confirmed the presence of PSTV in these clones. The spot hybridization test detected PSTV in mixtures of seed extracts equivalent to as few as one seed from a PSTV-infected plant and 80 seeds from healthy plants. The spot hybridization test was shown to be more sensitive and reliable than PAGE analysis; it is suitable for the testing of large numbers of samples with a minimum expenditure of labor and materials.     

Detection,distribution and long-term persistence of potato spindle tuber viroid in true potato seed from heilongjiang,China

Return-polyacrylamide gel electrophoresis (R-PAGE) and tomato bioassay followed by R-PAGE were compared for the detection of potato spindle tuber viroid (PSTVd) from individual true potato seed (TPS). Both methods detected PSTVd from single TPS. TPS extract formulated as sap or nucleic acids in two different buffers did not affect the percentage of viroid detection on tomato plants. There was some evidence of viroid inhibitor in TPS extracts but not in nucleic acid extracts of TPS. Because R-PAGE is more rapid than the tomato bioassay followed by R-PAGE, the former was used to determine the extent of PSTVd in TPS of China’s Keshan Potato Research Institute breeding material. Over 1700 individual TPS were tested. Twenty-four of the 46 seedlots tested (inbred and outcrossed) contained PSTVd. The viroid was detected in 70% of lots from inbred lines compared to 38% of lots from outcrosses. TPS (20 lots) stored in paper bags at room temperature as far back as 1965 were also tested, and PSTVd was detected in TPS stored for 21 years.     

Detection of potato spindle tuber viroid in true potato seed by bioassay on Rutgers tomato

Various methods of preparing inoculum from single and composite samples of true potato (TP) seeds and TP seedlings were evaluated by bioassay on Rutgers tomato. An inhibitor of infection was found in concentrated plant sap prepared from composite TP seedlings that could be sequestered with RNasin or purified bentonite. Sap extracts from TP seedlings prepared in buffer containing bentonite were more infectious than nucleic acid extracts from an equivalent fresh weight of tissue. PSTV was detected in single seeds when sap preparations were inoculated on tomato and amplified to levels detectable by polyacrylamide gel electrophoresis.     

Spindle tuber virus in seeds and pollen of infected potato plants

The presence of potato spindle tuber virus (PSTV) in seed and pollen of diseased plants was demonstrated. Transmission through the seed from open-pollinated female parents to the seedlings occurred frequently (average 31%) but varied in individual collections from zero to 100%. The amount of transmission did not appear to be correlated with variety or with age of the seed. All PSTV infections encountered appeared to be caused by mild strains. It is suggested that only healthy parents should be used for breeding purposes.     

Detection of strains of potato virus S by nucleic acid spot hybridization (NASH)

Summary Two complementary DNA (cDNA) clones (0.9 kb and 1.6 kb) reacting to the ordinary strain of potato virus S (PVS) were compared with single-stranded randomly primed cDNA (prepared to total genomic RNA) as probes for various strains of PVS, using the technique of nucleic acid spot hybridization (NASH). The cDNA clones detected 11 PVS isolates well, including both Andean and ordinary strains, while one of each strain was detected weakly. As little as 5 femtograms of PVS RNA could be detected. A low cross hybridization was observed with cDNA probes to PVS with potato virus M and carnation latent virus both members of the carlavirus group. No cross homology was detected with seven other potato viruses. NASH was as sensitive and specific as enzyme-linked immunosorbent assay (ELISA), with no pretreatments of crude sap being required before spotting on nitrocellulose filters. Cloned cDNA probes would be suitable for mass screening of PVS.     

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.     

Evaluation of six insect pests for transmission of potato spindle tuber viroid

Six insect pests of potato, green peach aphid,Myzus persicae (Sulzer); potato leafhopper,Empoasca fabae (Harris); Colorado potato beetle,Leptinotarsa decemlineata (Say); tarnished plant bug,Lygus lineolaris (Palisot de Beauvois); red-legged grasshopper,Melanoplus femurrubrum (DeGeer); and southern army worm,Prodenia eridania (Cramer), were tested for their ability to transmit potato spindle tuber viroid (PSTV). PSTV infection was determined by a gel electrophoretic assay made 6 weeks after transfer of insects from infected to non-infected plants of the cultivar, ‘Katahdin.’ Plants grown from tubers of the original non-infected hosts were also assayed. PSTV was detected in only 2 of 183 test plants; these 2 plants had been infested with the tarnished plant bug. These results suggest that the insect species studied are relatively insignificant as vectors of PSTV.     

New disease symptoms observed on field-grown potato plants with potato spindle tuber viroid and potato virus Y infections

Summary Potato spindle tuber viroid (PSTV) and potato virus Y (PVY) were isolated from plants of cultivar Kennebec with severe necrotic symptoms in the field. In the greenhouse, severe necrotic symptoms were reproduced only when potato plants were infected either simultaneously with PSTV+PVY, or with PSTV prior to PVY infection. Thirteen additional potato cultivars were tested in the greenhouse for this synergistic reaction and eight developed necrotic responses similar to cv. Kennebec. PVY concentration was significantly higher in doubly infected plants, compared with those infected with PVY alone.     

Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labelled DNA probe

Summary The potential use of a simple, sensitive and non-radioactive method for detecting potato spindle tuber viroid (PSTVd) in germinated true potato seeds, based on nucleic acid hybridization with a PSTVd-specific DNA probe labelled with digoxigenin, was investigated. Two simple procedures for the clarification of plant extracts suitable for a non-radioactive detection system were also investigated. The nucleic acid hybridization technique could detect one PSTVd-infected seed in more than 150 healthy seeds. The benefits of this non-radioactive detection system are discussed.     

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.     

Ineractions between a mild and a severe strain of potato spindle tuber viroid in doubly infected potato plants

Potato (Solarium tuberosum) plants co-infected with a mild and a severe strain of potato spindle tuber viroid (PSTVd) were analyzed by return-polyacrylamide gel electrophoresis for the presence of both strains in vegetative and reproductive plant parts. Both strains were detected in the anthers, flowers, inflorescences, leaves, ovaries, ovules, petals, pistils, roots, sepals, stolons and tubers. Only mild strain was detected from pollen in the cultivars tested. True potato seed (TPS) were not doubly infected when they were obtained from co-infected maternal parent plants pollinated with pollen from healthy plants. Also, when maternal plants infected with severe strain were pollinated with pollen from healthy plants or from those infected with the mild strain, TPS were not doubly infected. A small number of TPS with double infection was obtained when co-infected or mild-strain-infected plants were pollinated with pollen containing the severe strain (singly or doubly infected). The number of TPS containing mild strain predominated in a ratio of 7:1 over TPS containing the severe strain. This study indicates that segregation of strains from doubly-infected plants probably takes place during pollen formation and persists through seed development.     

Survey of potato spindle tuber viroid in seed potato growing areas of the United States

Fourteen United States (U.S.) seed potato certification agencies surveyed all U.S. seed potato growing areas for presence of the potato spindle tuber viroid (PSTVd). The survey included general surveillance, which involved searching for the occurrence of PSTVd in state seed potato certification records from 1990 through 2000, and a field survey, which involved testing selected crops for PSTVd infection by nucleic acid dot blot hybridization during 1999 through 2001. No PSTVd incident was documented in any of the state certification records, nor was PSTVd detected in the field surveys. All U.S. seed-growing areas were determined to be free of PSTVd. It is concluded that PSTVd has been eradicated and freedom from potato spindle tuber viroid has been successfully maintained in all of the seed potato growing areas in the United States.     

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.     

Albinism of potato spindle tuber viroid-infected Rutgers tomato in continuous light

Potato spindle tuber viroid (PSTV) caused albinism in the new growth of Rutgers tomato under continuous light. The albinism response was obtained with 4 isolates causing severe symptoms in tomato and with 1 mild isolate which is normally symptomless. Rutgers tomato inoculated with the 4 severe strains of PSTV developed typical ‘bunchy top’ symptoms in 12 to 16-hr day lengths. Symptoms developed slower and much less distinctly on plants incubated in 6-hr days. New growth in PSTV-infected plants was green in short days (12-hr illumination) and chlorotic to white in continuous light. Infectivities of extracts from white portions of plants were from 3 to 10 times greater than those from green portions of infected plants. Albino symptoms also developed in other tomato varieties when inoculated with PSTV and kept under continuous light. Albinism was most pronounced under continuous light at 30°C, somewhat less intense at 24°, and at 16°, new growth of infected leaves and stems remained green. Albinism did not develop in otherSolanum species which are symptomless hosts of PSTV nor in seedling potato plants grown in continuous light.     

Detection on polyacrylamide gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid

A diagnostic procedure for potato spindle tuber disease is described which allows detection of the causal viroid in small amounts of potato and tomato tissue. The method involves extraction of cellular nucleic acids, their separation by polyacrylamide gel electrophoresis and staining with toluidine blue O. The procedure has been used to index greenhouse and field potatoes for mild and severe strains of the pathogen     

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).     

Loss of potato spindle tuber viroid from tuber tissues after repeated freezing

Potato spindle tuber viroid (PSTV) infected foliage and tuber tissues when subjected to repeated freezing and thawing under controlled conditions (?18 to ? 20 C and 5 C), lost PSTV more rapidly in tuber tissues than in foliage. Tubers of 17 potato cultivars showed similar tendencies, but with some differences. In 7 of the 17 cultivars, PSTV was significantly reduced after one freeze-thaw treatment, and in 6 of the 17 cultivars, infectious PSTV was still recoverable after exposure to 4 cycles of freeze-thaw. Exposure of PSTV-infected tubers to outdoor temperature fluctuations (13.1 C to ?15.1 C) resulted in complete loss of PSTV from the tubers.     

A rapid micro-starch quantitation method for potato callus and its application with potato tubers

The procedure described was developed as a micro-starch method for potato callus tissue (0.1-0.2% starch on a fresh weight basis) and then evaluated with macro-starch samples of potato tubers harvested during three physiological stages of development. This method consisted of the following steps: (a) a mild alkaline-sonic extraction of starch from lyophilized 80% aqueous ethanol-washed samples, (b) starch hydrolysis of the neutralized, filtered extracts by 1 N HC1, and (c) the determination of liberated glucose by the specific glucose oxidase system. Results with this procedure were as sensitive and accurate (SD ± 0.86%) as with other starch-specific methods with more extensive starch isolation and purification steps.     

Solanum × berthaultii,a sensitive host for indexing potato spindle tuber viroid from dormant tubers

Summary When infected with mild and severe strains of potato spindle tuber viroid (PSTV), some clones ofSolanum × berthaultii developed severe symptoms, consisting of necrotic spotting of petioles and main stems and of leaf collapse. The necrotic, rolled leaves eventually dried and remained attached to the stem, any new leaves were small and the plant was severely stunted. Symptoms developed after inoculation with sap from potato foliage, sprouts, or tubers. Tubers could be indexed immediately after harvest or after storage. Single tubers could be tested by rubbing onto leaves ofS. × berthaultii either the cut surfaces of the suspect tuber directly or an extract of nucleic acid. Crude tuber extract proved unsatisfactory for inoculation. PSTV infection could be detected from as little as 31 to 7 mg of infected tuber tissue mixed with 1969 to 1993 mg of healthy tuber tissue (at a dilution factor of 64 to 256).

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Zusammenfassung Nach Infektion mit einem schwach oder stark aggressiven Stamm des Kartoffel-Spindle-Tuber-Viroids (PSTV), zeigten einige Klone vonSolanum x berthaultii die Entwicklung starker Symptome. Dabei ergaben sich nekrotische Flecken an Petiolen und Haupttrieben, begleitet von Kollabieren der Bl?tter. Die nekrotischen, eingerollten Bl?tter trocknen schliesslich aus, brechen jedoch nicht ab oder fallen herunter (Abb. 1). All neu entstehenden Bl?tter sind im Wachstum reduziert, die ganze Pflanze ist stark gestaucht. Symptome erscheinen nach Inokulation mit Saft aus Bl?ttern, Trieben oder Knollen (Tab. 1). Knollen konnten sowohl durch Abreiben der Schnittfl?chen der zu untersuchenden Knollen direkt auf Bl?tter oder durch Abreiben mit einem Extrakt von Nukleins?ure getestet werden (Tab. 1, 2). Rohe Knollen-Extrakt war nicht geeignet für Inokulation, aber rohe Sprossextrakt war ebenso brauchbar wie Knollen-Nukleins?ure (Tab. 1). PSTV-Infektion konnte noch bei Mischung von 31 bis 7 mg infizierten Gewebes mit 1963 bis 1993 mg gesunden Knollengewebes ermittelt werden (Tab. 2). Das Verh?ltnis von infiziertem zu gesundem Gewebe ist ?quivalent mit einer infizierten Knollen zu 63 bis 255 gesunden Knollen oder einem Verdünnungsfaktor von 64 zu 256. Bei Verwendung vonS. x berthaultii konnte PSTV von Knollen ermittelt werden, die von Kartoffelpflanzen abstammten, welche mit stark verdünntem PSTV-Inokulat behandelt worden waren (Tab. 3); es ist also m?glich, PSTV an im laufenden Jahr infizierten Kartoffelpflanzen zu entdecken. In einer Untersuchung von im Feld aufgewachsenen PSTV-verd?chtiger Knollen, konnte PSTV sowohl bei Knolleninfektion mit mild als auch stark aggressiven St?mmen ermittelt werden (Tab. 4). S. x berthaultii kann leicht bei Herstellung von Stengelabschnitten verbreitet werden. Ein Klon (Klon 26), der im Rahmen dieser Arbeit entwickelt wurde und einheitlich in Wachstum und seiner Reaktion gegen PSTV ist, sollte für den PSTV-Test verwendet werden. Diese Arbeit vermittelt ausserdem eine Methode zur Entwicklung weiterer PSTV-sensitiver Klone mitS. x berthaultii-Anteil, wenn Pflanzenquarant?ne-Regulierungen den Import fremderSolanum-Arten verbieten.

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Résumé Certains cl?nes deS. x berthaultii ont donné des sympt?mes graves après contamination par souches peu ou très agressives du viro?de en fuseau de la pomme de terre. Les sympt?mes se manifestent par des taches nécrotiques sur les pétioles et les tiges principales, avec un affaissement du feuillage. Les feuilles nécrosées et enroulées peuvent éventuellement se dessècher, mais elle restent attachées à la plante (fig. 1). La croissance de quelques jeunes feuilles est ralentie et la plante entière reste très chétive. Les sympt?mes appara?ssent après inoculation de jus obtenu à partir des feuilles, des germes ou des tubercules de pomme de terre (tableau 1). Les tubercules douteux pourraient être testés soit par frottis des surfaces coupées directement sur les feuilles soit par frottis avec un extrait d'acides nucléiques (tableaux 1, 2). L'extrait brut de tubercules ne donne pas entière satisfaction, alors que l'extrait de germes permet d'obtenir d'aussi bons résultats que l'extrait d'acides nucléiques provenant de tubercule (tableau 1). L'infection du viro?de en fuseau dans les tubercules peut être décelée pour des quantités faibles allant de 31 à 7 mg de tissu infecté et mélangé à une quantité allant de 1969 à 1993 mg de tissu de tubercules sains (tableau 2). Le rapport tissue infecté sur tissue sain équivaut à 1 tubercule infecté dans un lot de 63 à 255 tubercules sains, soit un facteur de dilution de 64 à 256. Le viro?de en fuseau a été détecté en utilisantS. x berthaultii à partir de tubercules provenant de plants infectés par un inoculum du viro?de très dilué (tableau 3). Mais il est préférable de le déceler dans la même année sur plantes contaminées. Au cours d'un contr?le d'essai de plein champ réalisé sur tubercules douteux, le viro?de en fuseau a été décelé sur des tubercules contaminés par des souches peu ou très virulentes (tableau 4). S. x berthaultii se multiplie facilement par boutures et un cl?ne (cl?ne 26) expérimenté dans cette étude a montré une croissance régulière et une réaction uniforme au viro?de en fuseau; il peut être ainsi utilisé pour tester le viro?de en fuseau de la pomme de terre. Cette étude peut servir également à rechercher d'autres cl?nes sensibles au viro?de et issus deS. x berthaultii, supprimant ainsi l'importation d'espèces étrangères deSolanum soumises à une mise en quarantaine.