[Show abstract][Hide abstract]ABSTRACT:
Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing-a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21-24-nucleotides by cleaving (or "dicing") entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant.The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid-host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species.

[Show abstract][Hide abstract]ABSTRACT:
The dahlia isolate of Potato spindle tuber viroid (PSTVd-D) shares 97 % sequence homology with PSTVd-intermediate (PSTVd-I), but differs at eight positions in the nucleotide sequence from PSTVd-I: five substitutions at positions 42, 43, 127, 202, and 311, two insertions at 63/64 and 312/313, and one deletion at 119. PSTVd-D accumulates slowly and induces very mild symptoms in tomato (cv. Rutgers) plants. In contrast, PSTVd-I propagates faster and induces severe symptoms. Here we used deep-sequencing analysis of PSTVd-specific small RNAs (PSTVd-sRNA) that accumulate in PSTVd-I- and PSTVd-D-infected tomato plants to reveal that the number of PSTVd-sRNA reads extensively decreased in PSTVd-D-infected leaf and stem tissues, especially those derived from the regions containing the nucleotides 119, 127, and 202, in which the nucleotide sequence differed between the severe and mild symptom-inducing isolates. In comparison with healthy controls, relative expression levels (i.e., number of reads by deep sequencing) of various host microRNAs changed after infection with PSTVd-I and PSTVd-D. The relative abundance of miR159 and miR162 in PSTVd-I- and PSTVd-D-infected leaf and stem tissues decreased to nearly 50 % of that in healthy tissues. In PSTVd-I- and PSTVd-D-infected stem tissues, miR319, which is approximately five times more abundant in stem tissues than in leaves, also decreased to 33–63 % of that in healthy controls.

[Show abstract][Hide abstract]ABSTRACT:
Australian grapevine viroid (AGVd) is a viroid specific to grapevine with the least records in the world till date. Here, we report for the first time the presence of AGVd in grapevines in Indian sub-continent. The overall infection rate of AGVd in major grapevine producing areas in India was 9.3 %, which is conspicuously higher than the other regions of the world except for Tunisia and Iran. To understand the AGVd diversity in India, the genetic divergence was examined based on the disparity in the cultivars and the locations. Nucleotide sequence analysis revealed the existence of five major AGVd variants in India besides other 44 minor variants implying the "quasi-species" nature. Further, sequence alignment of all the Indian AGVd variants along with Australian type species underscored the presence of eleven mutation points which are archetypal for Indian AGVd, irrespective of the region, and cultivar of grapevines. Plotting of Indian AGVd sequence variants against Australian type species unveiled that all these eleven mutations are distributed on upper and lower left terminal and pathogenicity regions of the molecule. Phylogenetic analysis divulged all the major Indian AGVd variants formed two distinct clusters, suggesting the two separate evolutionary lineages of AGVd in Indian viticulture.

[Show abstract][Hide abstract]ABSTRACT:
Overwintering of the brown leaf spot fungus, Mycochaetophora gentianae, in infected gentian leaves was studied in Iwate, northern Japan. Sporophores were produced on overwintered, infected leaves when they were sampled from January to July, but not in August after incubation in high humidity at 15 °C. Symptoms developed on gentian plants grown in soil artificially infested with overwintered, infected leaves that were either left throughout the experiments or removed before planting. Few lesions developed when plants were grown in soil infested with conidia. These results indicate that M. gentianae can overwinter in infected leaves, which act as the primary inoculum source.

[Show abstract][Hide abstract]ABSTRACT:
Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco () expressing a hairpin RNA (hpRNA) of (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

[Show abstract][Hide abstract]ABSTRACT:
A simple, low-cost hybridization assay using a universal DIG-labeled riboprobe for the rapid detection and identification of coleus viroids is presented. An octamer of 32-nucleotide sequence derived from the central conserved region (CCR) of viroids in the genus Coleviroid was used to develop a universal cRNA probe (8-central-conserved-region probe, 8CCR probe) for coleus viroids. Dot-blot hybridization assays demonstrated that the sensitivity of this probe was similar to specific probes for each CbVd, and Northern hybridization results revealed that at least four coleus viroids could be distinguished readily and simultaneously using the 8CCR probe. Batch detection assay showed that hybridization using the 8CCR probe can identify coleus viroids rapidly and effectively. This rapid and low-cost molecular hybridization technique is an effective way to survey the occurrence of coleus viroids, and has reference for the detection of other viroids and possibly viruses.

[Show abstract][Hide abstract]ABSTRACT:
To date, several viroid species have been shown to infect grapevine, including Hop stunt viroid (HpSVd), Citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and a tentative new species, Grapevine yellow speckle viroid-3 (GYSVd-3). Here, we identified and analyzed the distribution, genetic diversity, and molecular properties of viroids infecting grapevine cultivated in China and Japan, including old grapevines. The analysis showed that all the five known viroids and a tentative species GYSVd-3 infecting grapevine exist in China, and three of them (HpSVd, GYSVd-1 and GYSVd-3) exist in Japan. The contrast in diversity of viroid species in old grapevines from China and Japan may reflect different history of viticulture between the two countries. In general, the species of viroids infecting grapevine in China, as well as those in Iran and Australia, were more diverse than in the other countries. The population structure of viroids infecting grapevine in China and Japan showed species-dependency; i.e., HpSVd shared similar population structures in both countries, but GYSVd-1, GYSVd-2, and AGVd showed regional disparity even within the same country, although the role of sequence diversity in the biology of viroids infecting grapevine, such as the pathogenicity and evolution, still needs further study.

[Show abstract][Hide abstract]ABSTRACT:
In July 2003, a new disease occurred on leaves of highbush blueberry (Vaccinium corymbossum L.) in Iwate, Japan. Leaves initially had brownish spots, which subsequently developed into large lesions with concentric rings, resulting in premature defoliation. Teardrop-shaped conidia infecting leaves were visible by the naked eye as small protuberances in the center of lesions. Star-shaped conidia were sporadically produced on large lesions. The causal fungus was identified as Valdensinia heterodoxa, based on cultural, morphological and genetic studies. Inoculation tests showed that the fungus reproduced lesions on detached young leaves of highbush blueberry. A field survey in 2009 indicated that symptoms initially appeared on the lower leaves of basal shoots in late May, and the disease rapidly progressed on leaves of basal shoots, eventually spreading to lateral shoots from late June to late July. Removal of all basal shoots in late June significantly reduced disease incidence on lateral shoots in late July.

[Show abstract][Hide abstract]ABSTRACT:
To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees.
First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 10(3) CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants.
The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions.

[Show abstract][Hide abstract]ABSTRACT:
Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease-small, highly structured, circular RNA molecules that lack detectable messenger RNA activity, yet are able to replicate autonomously in susceptible plant species. To better understand the possible role of RNA silencing in disease induction, a combination of microarray analysis and large-scale RNA sequence analysis was used to compare changes in tomato gene expression and microRNA levels associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd small interfering RNAs in the absence of viroid replication. Changes in messenger (m)RNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the approximately 10,000 genes present on the array. Chloroplast biogenesis was down-regulated in both sensitive and tolerant cultivars, and effects on mRNAs encoding enzymes involved in the biosynthesis of gibberellin and other hormones were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.

[Show abstract][Hide abstract]ABSTRACT:
When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411-428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection and able to support high levels of viroid replication played a critical role in viroid detection and characterization. Polyacrylamide gel electrophoresis (PAGE) was the first molecular technique to be used for the rapid (2-3 days) identification of viroid-infected plants. Because it is the only diagnostic method that is sequence-independent, PAGE under denaturing conditions continues to play a key role in the identification of new viroids. Starting in the early 1980s, dot blot hybridization began to replace PAGE for routine viroid diagnosis. The first diagnostic protocols based on reverse transcription-polymerase chain reaction (RT-PCR) appeared approximately 10 years later, and much effort has subsequently been devoted to simplifying the sample preparation procedure and identifying group-specific primer pairs. This chapter describes four simple, easy-to-follow protocols-two involving PAGE and two others based on enzymatic amplification of viroid cDNAs-that currently play key roles in viroid discovery and characterization.

[Show abstract][Hide abstract]ABSTRACT:
A new variant of Potato spindle tuber viroid (PSTVd) was detected for the first time from dahlia grown in Japan. The dahlia isolate of PSTVd formed a quasi-species and
a major sequence variant consisting of 361 nucleotides in length, including five substitutions, three insertions, and one
deletion, when compared to the intermediate strain from potato. In bioassays with the new isolate, Rutgers tomato developed
mild stunting and leaf curling.
Keywords
Potato spindle tuber viroid
–Dahlia

[Show abstract][Hide abstract]ABSTRACT:
A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds.

[Show abstract][Hide abstract]ABSTRACT:
A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance.

[Show abstract][Hide abstract]ABSTRACT:
To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in potato spindle tuber viroid (PSTVd)-infected tomato plants. Large-scale sequence analysis of viroid-specific small RNAs revealed active production from the upper portion of the pathogenicity and central domains, two regions previously thought to be underrepresented. Profiles of small RNA populations derived from PSTVd antigenomic RNA were more variable, with differences between infected Rutgers (severe symptoms) and Moneymaker (mild symptoms) plants pointing to possible cultivar-specific differences in small RNA synthesis and/or stability. Using microarray analysis, we monitored the effects of PSTVd infection on the expression levels of >100 tomato genes containing potential binding sites for PSTVd small RNAs. Of 18 such genes down-regulated early in infection, two genes involved in gibberellin or jasmonic acid biosynthesis contain binding sites for PSTVd small RNAs in their respective ORFs.

[Show abstract][Hide abstract]ABSTRACT:
Viroids are autonomously replicating, small single-stranded circular RNA pathogens that cause diseases in infected, susceptible plants. They are non-coding RNA replicon which replicate depending on host transcriptional machinery and develop disease symptoms through interactions with cellular components of the host. The small size and unique molecular structure of viroid RNA makes them an attractive system to analyze molecular features responsible for pathogenesis, RNA transport, or molecular evolution and adaptation to specific host species. Here we show the latest progress in viroid research on new disease epidemics, molecular evolution and host adaptation, and pathogenesis in relation to viroid-induced RNA silencing.

[Show abstract][Hide abstract]ABSTRACT:
When the influence of host species, inoculum density, temperature, leaf wetness duration, and leaf position on the incidence
of gentian brown leaf spot caused by Mycochaetophora gentianae, was examined, the fungus severely infected all seven Gentiana triflora cultivars, but failed to infect two cultivars of G. scabra and an interspecific hybrid cultivar. Inoculum density correlated closely with disease incidence, and a minimum of 102conidia/mL was enough to cause infection. In an analysis of variance, temperature and leaf wetness duration had a significant
effect upon disease incidence, which increased with higher temperature (15–25°C) and longer duration of leaf wetness (36–72h).
No disease developed at temperatures lower than 10°C or when leaf wetness lasted <24h. At 48-h leaf wetness, disease incidence
was 0, 28, 77, and 85% at 10, 15, 20, and 25°C, respectively. Middle and lower leaves on the plant were more susceptible than
upper leaves. In microscopic observations of inoculated leaves, >50% of conidia germinated at temperatures >15°C after 24-h
leaf wetness. More appressoria formed at higher temperatures (15–25°C) with extended duration of leaf wetness (24–72h). At
48-h leaf wetness, appressorium formation was 0, 8, 26, and 73% at 10, 15, 20, and 25°C, respectively. These results suggest
that temperature and leaf wetness duration were important factors for infection of gentian leaves.
KeywordsAppressorium-Epidemiology-
Gentiana scabra
-
Gentiana triflora
-Infection

[Show abstract][Hide abstract]ABSTRACT:
A 303-nucleotide viroid was isolated from an apple tree (Malus×domestica, ‘Fuji’) cultivated in Japan. The viroid had 84.9% overall nucleotide sequence homology to Apple dimple fruit viroid (ADFVd), a member of Pospiviroidae, reported from Italy. This viroid differed from the Italian variant by 47 mutations (38 substitutions, six deletions and
three insertions), and most of these mutations occurred on either side of the central conserved region. The leaves and branches
of the infected trees did not have any disease symptoms, but the fruits were dimpled and yellow. The infected scions were
top-grafted onto a healthy ‘Fuji’ apple tree, which tested positive for this viroid in a northern hybridization analysis,
and yellow dimple fruits were produced in the second growing season. We propose that this viroid is a new variant of ADFVd
and causes yellow dimple fruit formation in ‘Fuji’ apple trees.
Keywords
Apple dimple fruit viroid
-Apple-Yellow dimple fruit

[Show abstract][Hide abstract]ABSTRACT:
Mycochaetophora gentianae, the causal agent of brown leaf spot on gentian (Gentiana scabra), is characterized by its hyaline besom-like sporophore, although its conidiogenesis and phylogenetic position have so far
remained unknown. We isolated the causal fungus from a new host, G. triflora, in Iwate, Japan. Both the G. triflora isolate and the ex-type M. gentianae isolate produced symptoms on G. triflora but not on G. scabra. Microscopic observations of the diseased leaves indicated that conidiogenesis was blastic from short conidiophores, and
schizolytic secession of conidia left unthickened and inconspicuous conidial scars on the conidiogenous cells. Conidia were
catenate, in branched acropetalous chains; secondary conidia were blastically produced from the first or second cell at the
base of primary conidium. The G. triflora isolate was identified as M. gentianae because of its identity to the ex-type in characteristics of culture, pathogenicity, and conidia. Phylogenetic analyses using
three ribosomal DNA (rDNA) sequences combined [small subunit (SSU)+large subunit (LSU)+5.8S rDNA] indicated that both
isolates clustered with Rhexocercosporidium carotae, and the cluster was placed within Helotiales–Rhytismatales. Additional analyses using internal transcribed spacers including
5.8S rDNA sequences revealed that both isolates were monophyletic and that they were closely related to three helotialean
Pseudocercosporella-like hyphomycetous genera: Helgardia, Rhexocercosporidium and Rhynchosporium.
KeywordsConidiogenesis-
Helgardia
-rDNA sequence-
Rhexocercosporidium
-
Rhynchosporium

[Show abstract][Hide abstract]ABSTRACT:
Viroids are autonomously replicating, small single-stranded circular RNA pathogens that do not code for proteins and may cause diseases in infected, susceptible plants. They have the ability to induce both RNA-mediated transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS), or RNA silencing, in infected plants. Their induced RNA silencing has also been demonstrated in a wheat germ extract system. A possible role of RNA silencing in viroid pathogenicity and evolution has been discussed. It is suggested that RNA silencing can be employed to engineer plants for viroid resistance and attempts to produce these plants have been also discussed.