马铃薯纺锤块茎类病毒株系鉴定

 以加拿大的马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTV)强毒、中毒和弱毒株系样品作对照,用改进的往返-聚丙烯酰胺凝胶电泳方法,鉴定了发生在我国北方种植的马铃薯上的PSTV株系类型。从60个马铃薯品种488个样品中,所有检测到的PSTV的电泳迁移率与加拿大的弱毒株系迁移率相同。表明所有检测到的PSTV都属弱毒株系。首次明确发生在我国北方种植的马铃薯上的PSTV以弱毒株系为主要类型。没有分离到强毒株系或其它株系。     

Exclusion of viroids from potato resources and the modified use of a cDNA probe1

Threats from potato spindle tuber viroid (PSTV) to potato breeding and centralized elite seed-tuber production have been identified in world potato genetic resources. In the UK effective diagnostic testing has proved essential in preventing acquisition. Inoculation of potato nucleic acids to tomato and subsequent viroid detection by polyacrylamide gel electrophoresis (PAGE) has proved a sensitive, but cumbersome, test over 8 years. Additionally, over 2 years, ³²P-labelled PSTV cDNA was used to probe denatured sap and nucleic acid extracts: 10^-4 of peak viroid concentrations in tissue could be detected. Spurious positives were seen in particular circumstances, but could be avoided. Probing of non-denatured samples was not as sensitive. Tubers became infected and PSTV was readily detected by PAGE in leaves of potato experimentally inoculated and maintained below 20°C, but the cDNA probe could not detect infection in tuber sprouts growing at 8–10°C in darkness. Otherwise similar green-leaved sprouts were faintly positive. Detection for all sprouts was unproblematic after movement to 25°C and light for 10 days.     

Simplified application of return gel electrophoresis for the routine detection of potato spindle tuber viroid

The return gel electrophoresis method for the detection of potato spindle tuber viroid has been modified to simplify its use for large-scale testing. The quantities of dissolvents for the extraction of nucleic acids were reduced and adapted to the use of disposable plastic tubes. The vertical electrophoresis system was replaced by a horizontal one, which was easier to handle. Migration distance for the separation of PSTV bands was shortened, saving time for the electrophoretic run. A single-buffer system was used. The detection limit was estimated as 0.3 ng per slot. It was possible to detect both mild and severe strains of PSTV.     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.     

Detection of chrysanthemum stunt and potato spindle tuber viroids by polyacrylamide gelelectrophoresis

Stunt viroid can be detected in chrysanthemums with the polyacrylamide gelectrophoresis (PAGE) method developed by Morris and Smith (1977) for potato spindle tuber viroid. The time of sample preparation can even be shortened considerably. The reliability of the short and the complete PAGE method proved to be similar to that of the biological Mistletoe test in a parallel experiment. Combined samples can be tested in the complete PAGE method easily permitting the detection of one diseased chrysanthemum top in a total of ten.Although potato spindle tuber viroid is not known to occur in the Netherlands we searched for methods to detect possible infections. Artificial infections of tomato and potato plants and of sprouts of potato tubers could readily be detected by Morris and Smith's method. Using this method it was possible to demonstrate infections by severe and weak isolates even when not yet producing symptoms. In tomato plants the viroid could be detected four to eight days before symptoms appeared.Samenvatting Het dwergziekteviroïde (CSV) kon in chrysanten worden aangetoond met een door Morris en Smith (1977) voor het aardappelspindelknolviroïde (PSTV) ontwikkelde polyacrylamide-gelelektroforesemethode (PAGE). Het bereiden van de monsters voor elektroforese kon evenwel aanzienlijk worden vereenvoudigd. De volledige, evenals de korte PAGE-methode bleek even betrouwbaar als de biologische Mistletoe-toets. De PAGE-methode was zo gevoelig dat toepassing ervan op mengmonsters verantwoord is: één besmette top van een chrysantheplant in een totaal van tien kon nog betrouwbaar worden aangetoond.Howewel het PSTV niet in Nederland voorkomt, werden de mogelijkheden onderzocht om infecties met dit viroïde te kunnen vaststellen. Kunstmatige infecties met het viroïde in tomate- en aardappelplanten en in aardappelspruiten konden met de door Morris en Smith beschreven PAGE-methode worden aangetoond. Dit gold zowel voor sterke als voor zwakke isolaten, ook als ze geen symptomen veroorzaken. In tomaat kon met de PAGE-methode het PSTV al vier tot acht dagen vóór de symptomen verschenen worden aangetoond.     

Occurrence of a viroid in chrysanthemum in Brazil

A viroid was detected in Chrysanthemum plants showing symptotns of stunting in a commercial field in Brazil. Analysis by return polyacrylamide gel electrophoresis (R-PAGE) of the nucleic acid preparations of leaves and flowers revealed the presence of a nucleic acid of low molecular weight with mobility within the range of viroids. The viroid-like band was completely eliminated by ribonuclease treatment or alkaline hydrolysis. The Chrysanthemum viroid was readily transmissible to Chrysathemum , tomnato and Gynura , which suggests that it may be an isolate of chrysanthemum stunt viroid.     

Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*

The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro‐ and glasshouse‐grown potato plants for the purposes of post‐entry quarantine and the production of pathogen‐tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10–20 mg of PSTVd‐infective tissue for R‐PAGE; 0.25–0.5 mg for DIG‐probe; 0.062 mg for RT‐PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG‐probe and RT‐PCR. The DIG‐probe and R‐PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant.     

Potato germplasm poses the highest risk of introducing potato spindle tuber viroid in potatoes in the Netherlands: analysis and evaluation of an outbreak in potato breeding

In 2014, potato spindle tuber viroid (PSTVd) was identified in a potato clone originating from a breeding company in the Netherlands. This clone was submitted for micro propagation and therefore tested for PSTVd and a number of other pathogens. This finding of PSTVd initiated actions to track and eradicate the infections. In addition to the finding at the breeding company, PSTVd was also found at a research institute. At both locations the viroid was eradicated following extended testing and discarding of infected plants. Additional surveys including testing of each individual plant in all crossing glasshouses and random samples of pre-basic and basic seed potatoes, revealed no further infections in the Netherlands. This result concurred with the fact that mechanical spread of PSTVd in the field is not likely under climatic conditions in the Netherlands. Therefore, vegetative propagation seems the most important pathway for maintaining and spreading of PSTVd. Based on the evaluation of this outbreak, it was concluded that potato germplasm poses the highest risk of introducing this viroid in potatoes in the Netherlands.     

Transmission of the coconut cadang-cadang viroid to six species of palm by inoculation with nucleic acid extracts

Seedlings of Areca catechu (betel nut palm), Corypha elata (buri palm), Adonidia merrillii (manila palm), Elaeis guineensis (oil palm), Chrysalidocarpus lutescens (palmera) and Oreodoxa regia (royal palm) were inoculated with nucleic acid extracts from coconut palms with cadang-cadang disease. Within 2 years of inoculation, analysis using a ³²P-labelled DNA probe complementary to the coconut cadang-cadang viroid (CCCV) showed that RNA sequences identical to CCCV were present in the inoculated seedlings. Electrophoresis in polyacrylamide gels showed that these palms also contained an RNA with mobility identical to CCCV. Four to five years after inoculation, the infected palms of four species were usually stunted compared with uninoculated palms, while betel nut and palmera were not stunted. Yellowing of leaflets was observed with defined spots or mottling of the older fronds in all except betel nut palms. All infected palms showed mild or severe yellow-leaf spotting. These results widen the known host range and. hence, the potential number of viroid reservoir species in the field.     

A new disease of greenhouse tomatoes in Israel caused by a distinct strain ofTomato apical stunt viroid (TASVd)

Using the sequential PAGE method for detection of small circular RNA molecules we isolated a viroid from greenhouse-grown tomato plants exhibiting severe stunting in Israel. The viroid was transmitted to tomato and to several other solanaceous plants by graft and mechanical inoculation, but only tomato plants showed symptoms of disease. Cloning and sequencing revealed that the viroid RNA is composed of 363 nucleotides, has 92% identity with the type strain (Ivory Coast strain) ofTomato apical stunt viroid (TASVd) and 99% identity with the Indonesian strain of this viroid. The experimental host range of TASVd-Is differs significantly from that of the type strain of TASVd. The possible epidemiological consequences leading to TASVd spread in geographically distant areas are discussed. http://www.phytoparasitica.org posting Sept. 18, 2002. Corresponding author     

Problems associated with potyviruses in potato certification-field inspection and serological testing1

In UK, the tobacco veinal necrosis strain of potato virus Y (PVY^N), potato virus A (PVA) and potato virus V (PVV) each occur in the field only in limited ranges of potato cultivars in which they mostly cause mild symptoms or even symptomless infection; little is known about incidence of strain C of PVY (PVY^C). The ordinary strain of PVY (PVY°), however, is widespread causing symptoms ranging in severity from very severe through to very mild, depending on cultivar sensitivity/tolerance. During field inspections, very mild potyvirus symptoms may be missed, so inspectors are trained to be particularly vigilant when examining problem cultivars which react in this way. PVA is almost invariably treated, along with PVX, as a mild kind of virus infection, but infections with PVY°, PVY^N and PVV are treated as severe with stricter tolerances being applied for them (especially for PVY^N) regardless of symptom severity. Wide variation within the same cultivar in the behaviour of variants within the PVY° strain group also sometimes causes difficulties in interpretation at inspection. To detect PVY, PVA and PVV in routine serological testing on potato certification samples, it is necessary to employ specific antisera to each of them. PVY^N-specific monoclonal antibodies can be used in ELISA to distinguish PVY^N from PVY°.     

Development of a standard method for detection of potato spindle tuber viroid in potato plants

A standard test method for detecting viroids was designed, to be applied on imported plant material, for which a zero-tolerance exists in the Netherlands towards potato spindle tuber viroid (PSTV).Partial purification of nucleic acids after homogenizing leaf material with a Polytron homogenizer, followed by increasing the viroid concentration by inoculation of an intermediate tomato host, and complete purification of the small nucleic acids from the tops of these plants, followed by polyacrylamide gelectrophoretic analysis, proved successful. With this procedure, now used as a standard method, more samples could be handled than with other methods tested.Desalting by Sephadex filtration proved to be superior to dialysis. An attempt to develop a serological test for PSTV failed. Albinism, induced in PSTV-infected tomato plants by certain environmental conditions, was not of diagnostic value.

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Samenvatting Voor het viroïde, dat de aardappelspindelknolziekte veroorzaakt (ASKV) geldt in Nederland een nultolerantie. Al het geïmporteerde aardappelmateriaal wordt daarom getoetst op het voorkomen van het viroïde. Voor dat doel is een betrouwbare en, zo mogelijk, ook snelle toets noodzakelijk, die niet alleen secundaire infecties maar ook jonge, primaire infecties kan aantonen.Een standaardmethode, die werd ontwikkeld, bleek zeer betrouwbaar, hoewel niet snel. Zij toont meer infecties aan dan snellere methoden die in het buitenland beschreven zijn. De toets omvat de volgende stappen: 2–5 g bladmateriaal wordt vermalen en op het homogenaat wordt een eenvoudige nucleïnezuurextractie en-concentratie toegepast. Dit preparaat wordt gebruikt om vier jonge tomatezaailingen te inoculeren. Door deze zaailingen 4 weken onder optimale omstandigheden te houden wordt het eventueel aanwezige viroïde vermeerderd. Geeft tenminste één van de tomateplanten symptomen, dan wordt het oorspronkelijke monster ziek verklaard. Vertoont geen van de vier tomateplanten symptomen dan wordt een nucleïnezuurextractie uitgevoerd van de topjes van deze planten. Kleine nucleïnezuurmoleculen worden geïsoleerd, geconcentreerd en tenslotte geanaliseerd met behulp van polyacrylamide gelelektroforese.Om overdracht van het ene naar het andere monster te voorkomen werd voor het vermalen gebruik gemaakt van verwisselbare schachten bij de Polytron homogenisator.Ontzouten van de nucleïnezuurextracten met Sephadexfiltratie gaf betere resultaten en was sneller uitvoerbaar dan dialyse.Pogingen om een specifiek antiserum tegen ASKV te maken zijn niet gelukt. Onder onze omstandigheden was het ook niet mogelijk om op een betrouwbare manier albinisme in geïnfecteerde planten te induceren als middel om infecties met ASKV op te sporen.

     

Potato latent virus: a proposed new species in the genus Carlavirus

A previously undescribed carlavirus, potato latent virus (PotLV), was found infecting the potato cultivar Red La Soda imported from the USA. The particles were filamentous and slightly curved, with modal lengths of 530 and 670 nm. The 11 kDa protein encoded downstream from the coat protein contained a 'zinc-finger' motif characteristic of carlaviruses, and RT–PCR using a carlavirus-specific primer gave a PCR product of 857 bp. Antibodies produced to PotLV did not detect other carlaviruses when used in ELISA and the coat-protein nucleic acid sequence of PotLV showed < 67% similarity with the other carlaviruses tested. The closest similarity was with the Andean strain of potato virus S. Unusually for a carlavirus, PotLV systemically infected Nicotiana bigelovii , N. glutinosa , N. rustica , N. tabacum and Physalis floridana .     

Comparison of bi-directional electrophoresis and molecular hybridization methods to detect potato spindle tuber viroid and chrysanthemum stunt viroid1

A comparison was made of methods for viroid detection. Molecular hybridization using cDNA is a very sensitive method that can handle large quantities of samples at the same time but it has the disadvantage that only small amounts of the sample can be applied to the nitrocellulose filter. The method therefore can only detect viroid in plants when its concentration is 10–20 ng g^-1 of leaves, using ³²P as a marker system. Bi-directional electrophoresis can detect viroid in plants when its concentration is 10 ng g^-1 of leaves, because it uses larger samples. It does not need hazardous chemicals like ³²P and formamide, and the reading of the results of the test is less liable to failures because it is based on two criteria (position and intensity of RNA band). The Dutch Plant Protection Service and the Dutch General Inspection Service for Ornamentals therefore use a modified bi-directional electrophoresis method to detect potato spindle tuber viroid and chrysanthemum stunt viroid, respectively.     

Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

European Journal of Plant Pathology - To investigate the current status of viroid infection in potato fields in Russia, potato spindle tuber viroid (PSTVd) and chrysanthemum stunt viroid (CSVd)...     

Application of real‐time RT‐PCR for large‐scale testing of potato for Potato spindle tuber pospiviroid*

The real‐time RT‐PCR protocol of Boonham and coworkers performed extremely well in a recent ring test, comparing different methods for detection of Potato spindle tuber pospiviroid (PSTVd) in several laboratories. Since, in addition, real‐time PCR technology has proved suitable for high‐throughput testing, this method was chosen as the starting point for the development of a protocol for the large‐scale testing of potato. The initial experiments focused on the specificity of the primers and probes with regard to different isolates of PSTVd and other (pospi‐) viroids. Further experiments were performed with leaf material from both primarily and secondarily infected plants. The parameters studied were sampling position, growing‐on temperature and bulking rate. In addition, different grinding, nucleic‐acid extraction and disinfection methods were compared. To monitor false negatives and positives, different controls were included and tested in duplex and triplex formats. The final protocol was tested using a hundred samples from the Dutch potato‐monitoring programme. The results of this pilot experiment were promising. Future plans include the development of a protocol for direct tuber testing and inter‐laboratory ring testing of the protocols.     

柑桔裂皮病类病毒分子生物学研究进展

本文就柑桔裂皮病类病毒(citrus exocortis viroid,CEVd)的分类地位,分子结构及其生物学鉴定、聚丙烯酰胺凝胶电泳分析和分子杂交、分子生物学检测等方法进行了综述。