不同探针大小、标记物及反应底物对马铃薯类病毒杂交检测的影响

 灵敏可靠地检测马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)对脱毒种薯的生产具有重要意义。本研究探索了影响核酸杂交检测技术的关键因素。通过基因克隆技术构建了插有PSTVd全长单体、双体及片段的载体。分别以地高辛和同位素为标记物,利用PCR和转录标记技术制备cDNA和RNA探针。比较探针大小、标记物、标记方法、反应底物等对检测灵敏度的影响。结果显示,以地高辛为标记物,利用PCR标记制备的PSTVd双体cDNA探针,在以CDP-Star为底物,通过在柯达X-OMAT BT胶片进行化学发光反应来分析结果的检测灵敏度最高,可以检测到0.05 pg总RNA中的PSTVd,是国外报道检测灵敏度的500倍。利用核酸斑点杂交技术检测PSTVd具有灵敏度高,一次可检测样品数量多等特点,对于大规模PSTVd检测更加方便可行。     

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.     

Composting to sanitize plant‐based waste infected with organisms of plant health importance

The potential for using the composting process to sanitize plant waste infected with one of three plant pathogens was investigated using bench‐scale composting equipment. Two of these pathogens, the potato wart disease fungus Synchytrium endobioticum and Potato spindle tuber viroid (PSTVd) are currently subject to European quarantine regulations. The third, Polymyxa betae, a parasite of sugar beet, is regulated in some European countries when in association with Beet necrotic yellow vein virus (BNYVV), the causal organism of rhizomania disease of sugar beet. Survival of test organisms following various combinations of compost temperature, exposure time and moisture was determined using RNA‐based detection methodology and/or plant‐based bioassays. Mathematically definable relationships between compost treatment (temperature/time) and organism viability were identified for P. betae and S. endobioticum; these give some indication of the practicality of using composting for dealing with infected wastes. However, for PSTVd, the considerable variability in measured susceptibility of the viroid to the composting process meant that no such definable relationship could be determined and further work would be needed to extrapolate to practical situations.     

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.     

Comparison of direct-RT-PCR and dot-blot hybridization for the detection of Potato spindle tuber viroid in natural host plant species

Potato spindle tuber viroid (PSTVd) is an EPPO A2-listed quarantine pathogen and its detection in large scale surveys requires complex decision schemes. In this study, a simple and rapid application of direct-RT-PCR was evaluated together with dot blot hybridization for the detection of PSTVd in dormant potato tubers harvested from primary infected plants, as well as in tomato and solanaceous ornamental plants. In all infected dormant potato tubers tested, both direct-RT-PCR and dot blot hybridization detected two different PSTVd isolates, with direct-RT-PCR being ten times more sensitive than dot blot. Similarly, in infected tomato and Brugmansia spp., PSTVd was detected by direct-RT-PCR with higher sensitivity compared to that of dot blot hybridization. However, in Brugmansia spp., a ten-fold decrease of the typical working concentration of the sap was required for an unequivocal detection of the viroid by direct-RT-PCR. The potential to use direct-RT-PCR for routine PSTVd examination is discussed.     

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.     

马铃薯纺锤块茎类病毒的检测和防治

马铃薯纺锤块茎类病毒病(potato spindle tuber viroid,PSTVd)是一种严重为害马铃薯生产的病害,降低产量20%—30%。防治的主要措施是应用无类病毒的种薯。由于目前还没有脱掉类病毒的有效措施,只能从未被饱和侵染的群体中鉴定筛选出未被侵染的个体,再脱掉其它病毒,作为核心繁殖材料。1987年以来,利用自制的电泳设备,以往复聚丙烯酰胺凝胶电泳法(return-polyacrylamide gel electrophoresis,R-PAGE)检测类病毒,筛选出未感病的个体,再用茎尖组织培养法脱掉其它病毒。经用马铃薯卷叶病毒等8种病毒酶标抗体鉴定筛选,获得既无类病毒也无主要马铃薯病毒的克新1、2、3和4号等主栽马铃薯品种的核心种。并已提供给省内外的良种场繁殖推广。1989和1990年抽样检测克山良种场繁殖的原种、一级和二级良种,未检测到类病毒。     

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)...     

Reliability of nucleic acid amplification techniques: modified target RNA as exogenous internal standard for a real‐time RT‐PCR for Potato spindle tuber pospiviroid*

Techniques based on nucleic acid amplification techniques, like PCR, are quick, sensitive and specific, and therefore very suitable for the development of diagnostic tests. These techniques enable the detection of very small amounts of target organisms by specific amplification of part of its genome. This exquisite sensitivity puts a high demand on measures to prevent false‐positive reactions due to contamination of the laboratory with nucleic acid, hence the need for inclusion of negative controls. In addition, to exclude false negatives, the performance of the reactions must be measured by the inclusion of several positive controls, e.g. cytochrome oxidase (COX) primers (and probes) to monitor efficiency of the nucleic acid extraction and an internal control to monitor inhibition of the target PCR. For the RT‐PCR assay for Potato spindle tuber pospiviroid (PSTVd) recently described by Boonham and coworkers, we have developed an exogenous internal standard, i.e. in vitro RNA transcribed from a plasmid containing a modified PSTVd sequence (a 17‐bp sequence of cloned PSTVd (isolate Howell) was substituted for a 118‐bp sequence of Escherichia coli). To this exogenous sequence, a specific probe was designed with a fluorescent label different from that of the PSTVd‐specific probe. By making use of the same primers as the target organism PSTVd, this internal standard provides a tool to measure the performance of the specific reaction. Moreover, by using another fluorescent probe, this standard can easily be discriminated from the target organism. The approach used for the construction of the internal control for PSTVd offers a tool for the construction of internal standards for other pathogens.     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.     

Epidemiological evidence that vegetatively propagated, solanaceous plant species act as sources of Potato spindle tuber viroid inoculum for tomato

In autumn 2006 in the Netherlands, Potato spindle tuber viroid (PSTVd) infections were detected in 42·3 and 71·9% of professionally grown lots of Brugmansia spp. and Solanum jasminoides respectively. The infected lots contained 73 985 and 431 374 plants, respectively, demonstrating the presence of many potential viroid sources for tomato ( Solanum lycopersicum ). PSTVd was identified in cultivars of Brugmansia × candida , B. × flava , B. sanguinea , B. suaveolens and unspecified Brugmansia species/cultivars. Most infected lots of Brugmansia spp. originated from a single Dutch nursery; most infected lots of S. jasminoides originated abroad. Sequence analysis revealed that the PSTVd genomes from Brugmansia spp. contained an average of 360 nt, whereas all genomes from S. jasminoides except one consisted of 357 nt. Furthermore, the collective PSTVd genotypes showed polymorphism at four or more positions, except for two cases in which genotypes from Brugmansia spp. and S. jasminoides were identical. Phylogenetic studies showed that PSTVd genotypes from Brugmansia spp. and S. jasminoides grouped apart from each other and from PSTVd isolates from potato ( Solanum tuberosum ) and Physalis peruviana . The PSTVd genotypes from tomato did not form a separate cluster, but were dispersed over clusters of vegetatively or partly vegetatively propagated plant species, i.e. potato, P. peruviana and S. jasminoides . Moreover, mechanical inoculation of the predominant PSTVd genotypes from S. jasminoides to tomato was successful. These results provide evidence that vegetatively propagated, solanaceous plant species have been sources of infection for tomato crops in the past.     

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.     

Direct tuber testing for Potato Y potyvirus by real‐time RT‐PCR and ELISA: reliable options for post‐harvest testing?*

The method currently used for testing potato tubers for viruses following harvest involves a growing‐on test. This takes up to 6 weeks to complete, and there is therefore a demand for more rapid test results. The sensitivity and reliability of direct tuber testing by DAS‐ELISA and real‐time RT‐PCR (TaqMan) were compared with the growing‐on test. In addition, the reliability of all three methods for the detection of Potato Y potyvirus (PVY) in tubers was compared over post‐harvest intervals of 6, 10, 14 and 18 weeks. The test material came from plots of tubers (cv. ‘Maris Piper’) containing a primary infection of strains PVY^N and PVY^O, following aphid transmission from marked infector plants grown during the 2003 season. Sample material was homogenized and divided, to provide comparative test material for detection of PVY by ELISA and real‐time RT‐PCR. Tuber eye‐plugs were then taken and subjected to the growing‐on test. The remainder of the tuber was also grown on and tested, to ensure infection was not missed as a consequence of an uneven distribution of virus throughout the tuber material. The results obtained using the two methods for direct testing of the tubers, and those results obtained from the traditional growing‐on test, are compared. The advantages and disadvantages of each method are discussed.     

Testing of Imported Potato Genotypes for Potato Spindle Tuber Viroid with a Tomato-intermediate /Electrophoresis Combined Method

The polyacrylamide gel electrophoresis (PAGE) test of Morris & Smith (1977) was evaluated for detection of potato spindle tuber viroid (PSTV) in breeding material. Number, density and mobility of nucleic acid bands in the electropherograms were influenced by genotype and growing temperature. So direct testing of genotypes was not reliable. After an intermediate viroid multiplication in tomato host plants at about 30oC and high irradiance, PSTV was reliably detectable with PAGE in inocula of potato samples of diverse origin. A 4-week incubation period proved to be suitable for inocula with low and high concentrations of a mild strain of PSTV (m-PSTV) as well as a severe strain of PSTV (s-PSTV). If incidence of PSTV is expected to be low, testing can be speeded up by bulking samples. With the combined tomato-intermediate/ PAGE assay, one m-PSTV or one s-PSTV infected leaf disk in 200 healthy ones was consistently detectable. Occasionally gels with a nucleic acid band of about the same relative mobility as the viroid band were found. Evidence that these bands were not caused by viroid is presented. A procedure to resolve such questionable test results is described. Infectivity of s-PSTV was higher than that of m-PSTV. Concentration of viroids in the inoculum influenced appearance of mild or severe symptoms and the rate of symptom production.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

Test performance study for detection of Ralstonia solanacearum and Clavibacter sepedonicus in potato tubers with TaqMan PCR

A test performance study (TPS) was organized in 2018 with ten official testing laboratories to evaluate the performance of different real-time PCR tests for the detection of Clavibacter sepedonicus and/or Ralstonia solanacearum in potato tubers. Participants were sent spiked potato extracts with low (0.8–1.2 × 10⁴ cfu mL^-1), medium (1.6–2.4 × 10⁵ cfu mL^-1) and high (1.6–2.4 × 10⁷ cfu mL^-1) bacterial loads, DNA extracts thereof and heel-end cores from symptomatic potato tubers. The four real-time PCR tests in this TPS for detection of C. sepedonicus were considered fit for purpose as principal screening methods. Two real-time PCRs in this TPS were considered fit for purpose as principal screening methods for detection of R. solanacearum. A third real-time PCR missed 23% of the DNA samples from low-level R. solanacearum spikes and is considered not fit for purpose as a principal screening method. Correct identification of spiked samples was lower when DNA extraction from the spiked samples was performed by the participating laboratories, highlighting the importance of appropriate DNA extraction protocols.     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).