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.     

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.     

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.     

Evaluation of biochemical methods for the diagnosis of the avocado sunblotch viroid in Israel

The reliability of biochemical diagnostic methods for avocado sunblotch viroid (ASBV) was evaluated for the Israeli avocado propagation program. Polyacry-lamide gel electrophoresis (PAGE) was compared with hybridization toin vitro ³²P-labeled cDNA and ASBV-RNA probes. Although hybridization to a cDNA probe was the most sensitive method, not all known infected plants were detected. In the light of these results, the problem of diagnosing ASBV in the Israeli propagation program is discussed.     

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.     

A novel technique using the Hendrickx centrifuge for extracting winter sporangia of <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> from soil

A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia. The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down to 0.02 sporangia g⁻¹ soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service. It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated pests is recommended as an alternative method for detection of sporangia in soil.     

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.     

Effects of resistance of Eremocitrus glauca and Microcitrus australis to viroid infection: replication,accumulation and long‐distance movement of six citrus viroids

In studies to identify genotypes resistant to infection with citrus viroids, Eremocitrus glauca and Microcitrus australis were selected because their evolution in their habitat in Australia and New Guinea may have led to the selection of unusual traits. The movement and accumulation of Citrus exocortis viroid (CEVd), Hop stunt viroid, Citrus bent leaf viroid, Citrus dwarfing viroid, Citrus bark cracking viroid and Citrus viroid V (CVd‐V) in self‐rooted as well as in graft‐ propagated E. glauca and M. australis plants was assessed by northern hybridization, RT‐PCR and by topworking to the sensitive selection 861‐S1 of Etrog citron. In both plant species the inoculated viroids were undetectable unless these plants were grafted to a susceptible Citrus partner, the rough lemon rootstock and/or the topworked Etrog citron, which acted as viroid sources. The results obtained indicate that M. australis and in particular E. glauca are poor viroid hosts in which viroid replication/accumulation does not occur or is extremely inefficient. However, viroid downward and upward movement to grafted Citrus partners in which viroid replication and accumulation occurs efficiently was not impaired. Eremocitrus glauca and M. australis showed differences regarding their properties as viroid hosts, but for both species CEVd seemed to have the lowest affinity among the viroid species tested and CVd‐V the highest. Even though E. glauca and M. australis do not appear to be truly resistant to viroid infection, they are interesting genotypes for further characterization of the mechanisms involved in viroid infection.     

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.     

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.     

苹果锈果类病毒在八棱海棠种子中的分布及氢氧化钠脱毒效果分析

为明确苹果锈果类病毒在八棱海棠果实和种子中的分布特征、种传率以及药剂脱除效果,以带毒母株上的八棱海棠果实和种子为试材,运用RT-PCR技术分析了八棱海棠果实不同部位锈果类病毒的带毒率、实生后代的带毒情况以及氢氧化钠脱除病毒的效果。结果表明,八棱海棠果皮、果肉、种子以及种胚均不同程度携带苹果锈果类病毒,其带毒率分别为96.0%、96.0%、52.0%和4.0%;该病毒可经种子传递给后代,种传率为12.1%;经2%氢氧化钠溶液浸种10、15、20 min,种子的病毒检出率均为0,但后代实生苗的带毒率分别为2.5%、1.3%和0。表明苹果锈果类病毒可侵染种子不同部位并经种子传递给后代,氢氧化钠浸种是脱除该病毒的有效方法。     

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.     

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     

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.     

Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.