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.     

Production of anti-virus,viroid plants by genetic manipulations

Many pathogenic plant viruses are RNA viruses, which initiate production of double-stranded RNA intermediates when they replicate in host plant cells. Introduction of double-stranded RNA-specific ribonucleases such as the Schizosaccharomyces pombe derived pac I protein and animal cell derived interferon-induced 2',5'-oligoadenylate synthetase (2-5 Aase)/ribonuclease L (RNase L) system into various plants may make plants resistant to various pathogenic viruses and viroids. We have demonstrated that pac I and 2-5 Aase/RNase L transgenic tobacco plants are resistant to various viruses including tobacco mosaic virus, cucumber mosaic virus and potato virus Y. In addition, pac I transgenic potato plants are resistant to potato spindle tuber viroid. Using Agrobacterium-mediated transformation, we have established a transformation system for chrysanthemum plants and have recently developed pac I transgenic chrysanthemum (Dendranthema grandiflora cv Reagan) resistant to chrysanthemum stunt viroid and have grown them in isolated fields for an evaluation of their effects.     

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.     

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.     

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...     

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.     

Characterization of a new viroid strain from hops: evidence for viroid speciation by isolation in different host species

A new viroid was detected in hops cultivated in Akita Prefecture, Japan where it is prevalent in many hops fields. In a survey of hop samples collected during the 1986–2002 growing seasons, the new viroid was present in the major Japanese hop-cultivating areas as early as the 1980s. A single-stranded circular RNA of 368–372 nucleotides that assume a highly basepaired, stable, rod-like secondary structure, shares 93%–98% sequence homology with Apple fruit crinkle viroid (AFCVd) isolated from apple and 85%–87% with Australian grapevine viroid (AGVd) isolated from grapevine. Taking into account the present concept of viroid species, we conclude that the viroid is AFCVd. Circumstantial evidence suggests that AFCVd from apples and hops were endemic in Japan only where cultivation of the two host plants overlapped, thereby strongly supporting the possibility that AFCVd (or an ancestral viroid) was transmitted across the species barrier from apples to hops or hops to apples somewhere in the region. Phylogenetic analysis of AFCVd from hops, AFCVd from apples, and AGVd together with the other members of the genus Apscaviroid revealed that the Akita isolates of AFCVd from hops (AFCVd-hop) formed a cluster that is distinct from AFCVd-apple and AGVd. Accumulation of host-specific sequence variation following their isolation in different host species may be leading to the formation of two viroid species from a common ancestor.     

植物类病毒脱除技术进展

类病毒是已知最小的植物病原物,可侵染蔬菜、果树以及花卉等多种农作物,并对农业生产造成严重影响。目前,对感染类病毒的植株进行脱毒处理是一种有效的防控措施,本文针对果树、蔬菜和花卉上几种常见的类病毒,综述了茎尖培养、热处理结合茎尖培养、低温处理、超低温处理、化学处理及不含叶原基的顶端分生组织再生等植物类病毒的脱除技术,分析了不同方法的应用效果及所适合脱除的类病毒种类,同时展望了类病毒病害在未来植物病害防控中的重要地位,并提出综合考虑各种因素、制定合理方案、选用适宜方法的类病毒脱除建议。     

利用两种方法检测苹果锈果类病毒

以克隆ASSVd的部分序列,通过RT-PCR成功合成了地高辛标记的cDNA探针,提取苹果和梨树枝条的总RNA,用斑点杂交技术对其进行了检测试验,结果表明,探针具有很高的灵敏度和特异性。地高辛标记的cDNA探针不与阴性对照枝条RNA以及感染PBCVd、AFCVd、ADFVd枝条总RNA发生杂交,仅与感染ASSVd样品的总RNA杂交。     

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.     

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。     

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.     

Symptoms and molecular characterization of apple dimple fruit viroid isolates from apples in Japan

Apple dimple fruit viroid was detected from an apple tree (‘Jonagold’) bearing apples with mild dapple apple symptom. The isolates in Japan were distinct from those in apples in Italy and China and in fig in Italy. Graft-inoculation experiments showed that the symptoms were variable depending on the cultivar, and the symptom on ‘Starking Delicious’ was virtually similar to those reported in Italy. Symptoms induced by apple dimple fruit viroid were similar in part to those by apple fruit crinkle viroid or apple scar skin viroid, indicating that they cannot be discriminated by symptoms on any specific variety.     

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.     

First report of tomato chlorotic dwarf viroid isolated from symptomless petunia plants (Petunia spp.) in Japan

A viroid was detected for the first time in symptomless petunia plants (Petunia spp.) and identified as Tomato chlorotic dwarf viroid (TCDVd) based on an analysis of the complete genomic sequence. These petunia plants are a likely source of inoculum for tomato or potato plants because TCDVd induces severe symptoms on these plants. The genomic sequence of this petunia isolate from Japan shared 100 % identity with petunia isolates from the Netherlands and United Kingdom and a tomato isolate from Japan. Phylogenetic analysis showed that all petunia isolates and the tomato isolate from Japan formed a monophyletic clade.     

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.