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.     

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)...     

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.     

An outbreak of Potato spindle tuber viroid in tomato is linked to imported seed

In 2011, an outbreak of the quarantine-regulated pathogen Potato spindle tuber viroid (PSTVd) occurred in a commercial glasshouse-grown tomato crop in Queensland, Australia. Phylogenetic studies showed that the genotype of this isolate grouped in a cluster of PSTVd genotypes from tomato and Physalis peruviana, and exhibited an interesting mutation (U₂₅₇→A) that has previously been linked to lethal symptom expression in tomato. Transmission studies showed that the viroid could be mechanically transmitted from crushed fruit sap, but not from undamaged fruits. A low rate of asymptomatic infection was determined for plants in the affected glasshouse, demonstrating the efficacy of using symptoms to detect PSTVd infections in tomato. No PSTVd infections were detected in solanaceous weeds located outside of the infected glasshouse, excluding them from playing a role in the viroid epidemiology. Monitoring and subsequent testing of new tomato crops grown in the facility demonstrated successful eradication of the pathogen. A trace-back analysis linked the outbreak of PSTVd to an infected imported tomato seed-lot, indicating that PSTVd is transmitted internationally through contaminated seed.     

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.     

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.     

Mechanism underlying potato spindle tuber viroid affecting tomato (Solanum lycopersicum): loss of control over reactive oxygen species production

Journal of General Plant Pathology - Severe infection of the tomato cultivar ‘Rutgers’ with potato spindle tuber viroid (PSTVd) variants resulted in systemic stunting, leaf...     

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。     

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.     

Occurrence and molecular variability of Potato spindle tuber viroid and Tomato apical stunt viroid in ornamental plants in Croatia

Viroids of the genus Pospiviroid are able to induce diseases in a wide range of host plants including important crop species. Although occasional disease outbreaks of Potato spindle tuber viroid (PSTVd) and closely related pospiviroids have been reported in potato and tomato, recent studies found an increase in number of latent infections in ornamental solanaceous species. In order to verify the presence of PSTVd and other pospiviroids in Croatia, a survey was conducted between 2009 and 2012. A total of 182 samples belonging to five ornamental species and two solanaceous crops were analyzed. Eight plants belonging to two different species (Solanum jasminodes and Lycianthes rantonnetii) were found infected by PSTVd and, in addition, one S. jasminoides plant infected by Tomato apical stunt viroid (TASVd). Viroid infection was confirmed by mechanical inoculation on tomato plants to observe symptom expression. Molecular characterization of the isolates was done and complete viroid sequences were submitted to the GenBank. This is the first evidence of the presence of PSTVd and TASVd and their variability in Croatia.     

马铃薯受马铃薯甲虫危害后冠层光谱特征的初步研究

使用美国ASD野外便携式光谱仪测量马铃薯受马铃薯甲虫危害后冠层光谱反射率,进行光谱微分、相关分析,得出结论：在750～975 nm范围内,受害后的马铃薯与健康的马铃薯相比冠层光谱反射率下降,红边斜率降低。选择了马铃薯受害后的敏感波段为736～920 nm。     

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.     

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。     

Mechanical transmission of <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> between plants of <Emphasis Type="Italic">Brugmansia suaveoles,Solanum jasminoides</Emphasis> and potatoes and tomatoes

Potato spindle tuber viroid (PSTVd) has been recently found in many solanaceous ornamental plant species. This study reports on the effectiveness of mechanical transmission between Brugmansia suaveolens, Solanum jasminoides, potato and tomato. Inoculation with ‘infected’ plant sap diluted in water, rubbing with contaminated finger tips and cutting with contaminated razor blades all resulted in transmission of PSTVd. Temperature, plant species and source of inoculum were found to be critical factors. An average temperature of 15°C only resulted in a few infections, whereas transmission at 20 and 25°C was more successful. Tomatoes were more susceptible to PSTVd than B. suaveolens, S. jasminoides and potatoes. Furthermore, S. jasminoides was a better source of inoculum than B. suaveolens. No transmission was obtained after repeated addition of inocula to tomato roots. These results indicate that PSTVd can be transmitted between plant species in practice by crop handling.     

Identification of Pathotypes of the Potato Cyst Nematode1

Since the discovery of pathotypes of the potato cyst nematode (Heterodera rostochiensis Wollenweber, 1923), surveys on the distribution of these pathotypes have been carried out in different European countries. Test varieties of potato were selected according to findings resulting in the present situation of varying distinctive host-plants and of diverging programs for breeding potatoes resistant to the potato cyst nematode. With respect to rational selection and use of resistant potato varieties, there is a great need for standardization in pathotype identification (nomenclature), maintenance and supply of appropriate genotypes of Solanum species and methods for testing resistance in new potato varieties. Anticipating a cross-experiment with pathotypes and test varieties from Great Britain, the Federal Republic of Germany and the Netherlands in 1974, a preliminary test with all recognized or presumed pathotypes from these countries, but with only part of the potential differential host-plants was carried out by the Plant Protection Service at Wageningen. The results of this experiment are discussed.     

An Integrated Approach in the Control of Potato Root Eelworm

During the last decade, new control possibilities for the potato root eelworm have come to the fore, namely resistant potato varieties and soil disinfection. These have led to an integrated approach to the control of the potato root eelworm in the Netherlands. For this purpose, a number of potato rotations, aiming at the prevention or elimination of non-detectable populations of the potato root eelworm, are prescribed for non-infested fields. These regulations allow the farmer to grow potatoes more frequently on non-infested soils if he uses control measures (i.e. resistant potato varieties, soil disinfection). More stringent restrictions are in force for infested fields on which the growing of susceptible crops and propagation material is, of course, prohibited.     

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.     

Resistance of potato varieties to field populations of the potato cyst nematode Globodera pallida in the northeastern part of the Netherlands

In the northeastern part of the Netherlands starch potatoes are grown intensively. From 392 fields in that area, soil samples that contained the potato cyst nematodeGlobodera pallida were used to analyze the resistance of 7 varieties of starch potatoes.Differences and similarities in resistance of the varieties to the field populations ofG. pallida could well be explained by their descent from distinct sources of resistance.TheG. pallida-populations that showed a relatively high virulence to the varieties Producent, Elles, and Darwina were not equally distributed over the area, but were confined to a small number of regions.     

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.