Hop Stunt Viroid Isolates Research Articles

Detection and genetic diversity of Hop stunt viroid infecting Pistachio (Pistacia vera L.) trees in Turkey

Hop stunt viroid (HSVd) infects a large number of woody plants such as grapevine, citrus, plum, peach, fig, mulberry, pear, pistachio, and almond. Pistachio (Pistacia vera L.) is an important crop in Turkey and very few reports described the presence of viruses or viroids affecting that crop. The viroid was recently detected in one P. vera tree out of a limited number of samples in Kahramanmaras which is one of the most important pistachio-growing areas. Therefore, an extensive research was carried out to investigate the prevalence and genetic diversity of HSVd in Kahramanmaras and two additional major pistachio growing areas. During 2016–2017, a total of 70 leaf samples from pistachio trees were collected and assayed for HSVd by reverse transcription polymerase chain reaction (RT-PCR). The prevalence incidence of HSVd infection was relatively low in the tested plant materials. Comparative analysis of the three Turkish HSVd-pistachio variants was performed with viroidal genome sequences from the NCBI database. Phylogenetic analysis showed that one HSVd-pistachio variant was classified with the reference HSVd isolate from Japan and formed a sub-cluster within the HSVd-hop isolates group, whereas the remaining two variants were clustered into the plum-hop/cit3 group with four Tunisian HSVd-pistachio variants. Further studies are required to verify its infectivity in healthy pistachio trees.

Characterization of viroids infecting grapevine in India

Several viroids infect grapevine worldwide. They are smallest infectious pathogens. Present study was undertaken to detect viroids in commercial grapevine genotypes in India. Four viroids viz., Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2), Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in reverse transcriptase-polymerase chain reaction (RT-PCR) based assay. These viroids were found to present either alone or in different combinations. None of the samples showed the presence of citrus exocortis viroid. Few genotypes like Beauty Seedless and Black Mustard did not showed the presence of any of the five viroids. Thompson Seedless, Sharad Seedless and Black Prince were found to be associated with all the four viroids. Blast analysis of Indian GYSVd-1 isolate revealed a close identity of 99% with Q4-III isolate from Pakistan, and Indian GYSVd-2 isolate was found to have close identity of 100% with VB-108 isolate from Croatia. While Indian HSVd isolate showed close identity of 99% with SDLY-20 isolate from China and AGVd Indian isolate was found to have close identity of 99% with AGVd-Iran isolate. GYSVd-2 association with grapevine was found for the very first time from the Indian conditions. Nucleotide substitutions, additions and deletions were observed in the sequenced isolates of these viroids. Variation was observed in the secondary structure of Indian Hop stunt viriod (HSVd) isolate. Characterization of all the detected viroids indicated that they might have been introduced in India from Asian or European countries.

First report of Hop stunt viroid infecting Vitis gigas, V. flexuosa and Ampelopsis heterophylla

Abstract Hop stunt viroid (HSVd) is one of the most common viroids that infect grapevine ( Vitis spp.) worldwide. Sixteen sequences of the HSVd genome were obtained from infected grapevines in Brazil by next generation sequencing (NGS). Multiple alignments of the sequences showed nucleotide identities ranging from 94.6% to 100%. This is the first report of HSVd infecting two wild grape species and Ampelopsis heterophylla . These HSVd isolates along with others from V. vinifera and V. labrusca were phylogenetically analyzed. Keywords Next generation sequencing . HSVd . Incidence . Genetic variability . Vitis

First Report of Hop stunt viroid in Japanese Apricot (Prunus mume) in Korea

HomePlant DiseaseVol. 101, No. 7First Report of Hop stunt viroid in Japanese Apricot (Prunus mume) in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Hop stunt viroid in Japanese Apricot (Prunus mume) in KoreaY. Jo, S. Lian, J. K. Cho, H. Chu, and W. K. ChoY. JoSearch for more papers by this author, S. LianSearch for more papers by this author, J. K. ChoSearch for more papers by this author, H. ChuSearch for more papers by this author, and W. K. ChoSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. Jo , Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea S. Lian , College of Crop Protection and Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109, China J. K. Cho , The Taejin Genome Institute, Gadam-gil 61, Hoengseong, 25239, Republic of Korea H. Chu , Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea W. K. Cho , Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea; and The Taejin Genome Institute, Gadam-gil 61, Hoengseong, 25239, Republic of Korea. Published Online:9 May 2017https://doi.org/10.1094/PDIS-01-17-0059-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Viroids are the smallest pathogens, and they consist of a circular single-stranded RNA genome that does not encode any protein (Hammann et al. 2012). To date, 46 viroid species have been identified. One of them, Hop stunt viroid (HSVd) in the genus Hostuviroid has been identified in at least 17 plant species, such as citrus, grapevine, apricot, plum, and peach species, from 38 countries (Jo et al. 2017). The Prunus mume (mei), known as Japanese apricot, belongs to the genus Prunus in the family Rosaceae, and is mostly grown in China, Japan, and Korea. It is used for juices, alcohol flavoring, pickles, and traditional medicine. In a survey of viruses and viroids in Japanese apricot trees during September 2015, leaf samples from two plants representing two cultivars, Takada and Wallyoung, were collected from an orchard in Korea. The leaves of the two plants did not display any visible disease symptoms. The leaf samples were subjected to total RNA extraction using Fruit-mate for RNA Purification (Takara, Shiga, Japan) and the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturers’ instructions. Two libraries from the two cultivars were generated and paired-end (2 × 100 bp) sequenced by Illumina’s HiSeq 2000 system (Macrogen, Seoul, Korea), as described previously (Jo et al. 2016). We obtained 37,546,342 and 37,171,434 reads for Takada and Wallyoung, respectively. Raw sequence reads were assembled by the Trinity program (Haas et al. 2013), and the assembled contigs were subjected to a MEGABLAST search against a viral reference database. We identified two and four contigs associated with HSVd from Takada and Wallyoung, respectively. Reverse transcription (RT)-PCR was performed to confirm the presence of HSVd using HSVd-specific primers (5′-GCCCCGGGGCTCCTTTCTCAGGTAAG-3′ [position 85-60] and 5′-CCCGGGGCAACTCTTCTCAGAATCC-3′ [position 78-102]) based on the HSVd reference sequence (GenBank NC_001351.1) (Astruc et al. 1996). The amplified PCR products were cloned in pGEM-T-Easy Vector (Promega, Madison, WI) and sequenced. Based on the sequencing results from using the PCR amplicon, HSVd isolate Takada (KY445746) was 99% identical to the clone chi100Y-02-4-5 from grapevine from China (AB219944.1), while HSVd isolate Wallyoung (KY445747) was 98% identical to the clone 2hsvdh2-4 from hop from China (EU365352.1). To our knowledge, this is the first report of HSVd associated with Japanese apricot.

First detection of Grapevine rupestris stem pitting-associated virus and Grapevine rupestris vein feathering virus, and new phylogenetic groups for Grapevine fleck virus and Hop stunt viroid isolates, revealed from grapevine field surveys in Spain

This study was supported by Projects Consejo Superior de Investigaciones Cientificas CSIC (2010CL0021) and BIO2011-25018 from the Spanish MINECO / UNIVERSIDAD DE CHILE 04/11-2 and 2010CL0021

First Report of Hop stunt viroid Infecting Japanese Plum, Cherry Plum, and Peach in Greece.

Dapple plum and peach fruit is a widely distributed disorder of plum and peach resulting in significant economic losses (4). During a survey for the presence of Hop stunt viroid (HSVd) on stone fruit trees in Greece, samples from 30 European plums (Prunus domestica L., cvs. President, Tuleu Grass), 45 Japanese plums (Prunus salicina Lindl., cvs. Angeleno, Diamond, Santa Rosa), 12 cherry plums (Prunus domestica L. var. insititia (L.) Fiori & Paoletti of unknown cultivar), and 107 peaches (Prunus persica (L.) Batsch, cvs. Red Haven, Elberta, June Gold, Spring Crest, Lemonato) were collected in several orchards around Greece. Their fruit skin symptomatology indicated viroid infection (reddish dappling blotches and cracks in European and Japanese plum, green dappling in cherry plum, and light colored blotches and lines in peach). Samples were screened with tissue-print hybridization (TPH) for HSVd using a full length DIG-labelled riboprobe deriving from in vitro transcription of the positive control, a citrus isolate of HSVd (G. Vidalakis, CCPP, University of California, Riverside). In total, 44 out of the 194 trees surveyed were HSVd-positive with TPH. For a small number (40) of TPH-positive field samples, TNA phenol extraction from fruit skin, leaves, and bark and one-tube two-step reverse transcription (RT)-PCR assays followed, using a standardized protocol (3) with two different primer pairs, one new primer pair (this study) and a previously reported primer pair (2). RT-PCR analysis showed the presence of HSVd in peach and Japanese plum in prefectures Pella (Central Macedonia), Achaia, and Korinthia (Peloponnesus) and in cherry plum in Achaia (Peloponnesus). Six of 11 Japanese plums (cvs. Angeleno, Santa Rosa), 2 of 12 cherry plums, and 8 of 12 peaches (cvs. Spring Crest, Red Haven) examined were found HSVd-infected, but none of the five European plums were. Nucleotide sequence analyses of purified and cloned amplicons from peaches and Japanese and cherry plums revealed sizes of 297 to 308 nt and similarity to sequence variants of other HSVd isolates previously characterized: 95 to 97% identity with the Moroccan isolates apr.9, apr.10, apr.11, and apr.12 and the Spanish isolate apr.4 from apricot (1) (GenBank Accession Nos. AJ297825 to AJ297828 and Y09346, respectively). For confirmation of HSVd presence in field trees, 10 Japanese plums cv. Angeleno, 10 peaches cv. June Gold, and 10 peaches cv. Spring Crest, HSVd-negative (TPH), were bud- or chip-grafted from two of the aforementioned Japanese plums cv. Angeleno and two of the aforementioned peaches cv. Red Haven. Two years later, five Japanese plum trees (cv. Angeleno) and five peach trees (three cv. Spring Crest and two cv. June Gold) were found HSVd-positive with TPH; no fruits were observed to produce fruit symptoms as the grafted trees were kept in an insect-proof greenhouse (no bees for cross-pollination). To our knowledge, our investigation reports for the first time the occurrence of HSVd infecting Japanese plum, cherry plum, and peach in Greece, emphasizing the need for a certification program for the prevention of spreading stone fruit tree viroids in this country.

Development of a quantitative real-time RT-PCR (qRT-PCR) for the detection of hop stunt viroid

Hop stunt viroid (HSVd) is a worldwide plant pathogen that infects a wide range of hosts. In some cases different molecular variants are associated with particular disease symptoms, e.g. the cachexia isolate on citrus. A new method for HSVd detection has been developed by designing a new primer pair and a probe for quantitative real-time RT-PCR (qRT-PCR) assay that is based on TaqMan™ chemistry. The primers and the probe were manually designed in order to detect different HSVd isolates from different hosts, including citrus cachexia variant. The protocol was then tested for its sensitivity and specificity. Specifically, sensitivity was evaluated with dilution series of in vitro transcripts from cloned whole HSVd genome. Dilutions were used as a standard for quantification of viroidal template in infected samples. Sensitivity was also tested by comparing the amplification results in quantitative and qualitative (end point) RT-PCR, obtained with serially diluted total RNA, extracted with two different methods. The qRT-PCR was evaluated for its specificity using healthy and infected samples from peach, apricot, plum, pear, citrus (infected with cachexia and non-cachexia variants), almond and grapevine and non-target controls (Apple scar skin viroid, Apple dimple fruit viroid, Peach latent mosaic viroid, Pear blister canker viroid and Potato spindle tuber viroid). The assay showed very high sensitivity and specificity. When the developed assay was applied to detect HSVd variants in different hosts that were previously tested, it was in complete agreement with previous test which confirmed its efficiency and accuracy. Moreover, the developed assay has the advantages of increased sensitivity of viroid detection as well as its quantification.

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.

FIRST REPORT OF HOP STUNT VIROID IN LEBANESE FIG TREES

In the course of a survey carried out in 2010 in the main fig-growing areas of Lebanon (Bekaa and Mount Lebanon), a total of 100 samples representative of 15 dif- ferent cultivars were collected and tested by RT-PCR for the presence of Hop stunt viroid (HSVd) using viroid-spe- cific primers (Sano et al., 2001). Distinct PCR products of the expected size (303 bp) were amplified from 12 fig trees of cvs Houmairi (6), Bayadi (5) and Sweidi (1), all originat- ing from the Bekaa valley. This result was further con- firmed by dot blot molecular hybridization using a HSVd- specific riboprobe. Only seven infected plants showed symptoms resembling those typical of Fig mosaic disease, the remaining being symptomless. Five of these HSVd iso- lates were sequenced (accession Nos HE662802- HE662806) and compared with the other HSVd isolates from GenBank. In Blast analysis, all the sequenced HSVd isolates showed 99-100% nucleotide identity among them and 94-95% identity with the HSVd type member (X0009). The only exception was the isolate D1, which confirmed the 94% identity with HSVd type but showed a lower identity level (94-95%) with the other HSVd Lebanese fig isolates. In a phylogenetic tree constructed with whole viroidal sequences the Lebanese fig isolates, ex- cept for D1, clustered in a separate group together with fig isolates from Syria and mulberry isolates from Lebanon (Elbeaino et al ., 2012a, 2012b). To our knowledge this is the first report of HSVd in Ficus carica in Lebanon.

Imbalance in expression of hop (Humulus lupulus) chalcone synthase H1 and its regulators during hop stunt viroid pathogenesis

Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar “Admiral”, including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

Molecular characterization of Chinese Hop stunt viroid isolates reveals a new phylogenetic group and possible cross transmission between grapevine and stone fruits

A survey for the incidence of Hop stunt viroid (HSVd) infection in China was conducted using dot-blot hybridization. Out of 553 tested samples, 127 samples of stone fruits (apricot, peach, plum and almond), grapevine and hop were positive for HSVd, giving a mean infection rate of 23 %. Phylogenetic analysis revealed that most of the HSVd variants isolated from stone fruits, grapevine and hop were clustered into known hop and plum groups. However, two grapevine variants, HSVd.g50 and HSVd.g57, could not be clustered into any known groups, indicating a previously unknown phylogenetic group of HSVd isolates. HSVd.g38 was the single grapevine variant that clustered with the plum group isolates, supporting cross transmission between grapevine and stone fruits and the heterogeneity of grapevine isolates.

Occurrence of Hop Stunt Viroid in Mulberry (Morus alba) in Lebanon and Italy

AbstractThe presence of Hop stunt viroid (HSVd) was detected using RT‐PCR and Northern blot hybridization in five of 60 samples from symptomless mulberry trees (Morus alba) collected in Italian and Lebanese orchards in July 2010. Infection levels were c. 10% in Lebanese and 8% in Italian samples. Nucleotide alignments showed that sequences of the mulberry HSVd isolates shared 95–96% identity with those of the same viroid occurring elsewhere. In a phylogenetic tree, mulberry HSVd isolates clustered together with those of HSVd‐citrus, regardless of their geographical origin. This is the first report of infection in mulberry trees by HSVd.

Insights on genetic diversity and phylogenetic analysis of Hop stunt viroid (HSVd) population from symptomatic citrus tree in Tunisia

Hop stunt viroid (HSVd) variants from cachexia symptomatic citrus tree were subjected to retrotranscription and DNA amplification (RT-PCR), cloning and sequencing. Here we report genetic diversity and phylogenetic analysis of HSVd Tunisian isolate. Our study revealed obvious polymorphism within Tunisian isolates and high similarity with Japanese variants. Neighbor-joining analysis was carried out on the new HSVd-citrus sequences together with 44 previously described HSVd isolates from citrus and one from grapevine. The phylogenetic analysis showed that Tunisian isolates were clustered into 4 different groups (CVd-IIa, b, and c and grapevine group). Furthermore, the predicted secondary structure was scrutinized to be more understanding on how the nucleotide change affects variable (V) and pathogenicity domain (P).

First Report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in Grapevine (Vitis vinifera) in New Zealand.

In February 2009, grapevines (Vitis vinifera) in a commercial vineyard in Auckland were showing shortened, spindly canes with tiny leaves. Approximately 10% of the vines were affected. An RNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used to isolate total RNA from leaves collected from six symptomatic (cvs. BAC0022A and Syrah) and eight symptomless vines (cvs. BAC0022A, Syrah, and Chardonnay). RNA was tested by reverse transcription-PCR for the presence of Australian grapevine viroid, Citrus exocortis viroid, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2, and Hop stunt viroid (HSVd). Four of the six symptomatic and all the symptomless vines tested positive for GYSVd-1 using primers 5'-TGTGGTTCCTGTGGTTTCAC-3' and 5'-ACCACAAGCAAGAAGATCCG-3', which amplify the complete genome (368 bp), and published primers PBCVd100C/194H (3), which amplify a 220-bp region of the genome. Amplicons from each PCR were transformed into a pCR 4-TOPO vector (Invitrogen, Carlsbad, CA), cloned, and sequenced. Sequence from both PCRs aligned identically to generate a consensus sequence (GenBank Accession No. HQ447056), which showed 99% nt identity to GYSVd-1 (GenBank No. X87906) by BLASTN analysis. All symptomatic and symptomless vines also tested positive for HSVd using primers C/H-HSVd (4) and HSVd-C60/H79 (1), which amplify the complete genome (298 bp). Amplicons from each isolate were cloned and sequenced. Sequence from both PCRs were aligned. Clones from all isolates, with the exception of one, aligned identically to create a consensus sequence (GenBank No. HQ447057) that showed 99% nt identity to Chinese HSVd isolates from grapevine (GenBank Nos. DQ371436-59) by BLASTN analysis. Sequence from the remaining isolate (GenBank No. HQ447056) was identical to a German Riesling grape isolate of HSVd (GenBank No. X06873). The presence of each viroid was further confirmed in PCR-positive plants by dot-blot hybridization with digoxigenin-labeled synthetic ssRNA probes specific to the full-length genomes of GYSVd-1 and HSVd (S. Harper and L. Ward, unpublished data). To our knowledge, this is the first report of GYSVd-1 and HSVd in V. vinifera in New Zealand. Since both viroids were detected in symptomatic and symptomless plants, the symptoms observed in the vineyard cannot be attributed to viroid infection. Symptoms described for GYSVd-1 include leaf spots and flecks, but no disease symptoms have been reported in grapes as a result of HSVd (2). Viruses found in the vines include Grapevine leaf roll virus-3, Grapevine viruses A and B, and Rupestris stem pitting associated virus, but these are not thought to be the cause of the symptoms. Two sequence types of HSVd were found, suggesting at least two separate introductions of HSVd into the vineyard. The vineyard is more than 40 years old so both viroids may have been present for some years. Export of wine from New Zealand was worth 1 billion dollars in 2009, so there is potential for these viroids to have an economic impact if symptoms are expressed. HSVd has been reported from China, Europe, Japan, Middle East, Pakistan, and the United States. GYSVd-1 has been reported from Australia, China, East Mediterranean, Europe, Japan, and the United States.

Molecular detection and characterization of Hop stunt viroid sequence variants from naturally infected pomegranate (Punica granatum L.) in Tunisia.

Tunisian pomegranate Hop stunt viroid (HSVd) variants are described. Dot-blot hybridization, S-Page, and reverse transcription polymerase chain reaction (RT-PCR) of RNA extracts from infected tissues were carried out. Results obtained by these techniques were confirmed by cDNA sequencing. The genetic diversity among the Tunisian variants was investigated, which also involved analysis of sequences of previously described HSVd variants from Tunisian citrus var. clementine and fig, and from fruit trees from other Mediterranean countries. Phylogenetic analysis showed that Tunisian pomegranate HSVd variants were clustered into two groups: a cachexia strain within the citrus type group and a recombinant citrus-plum type group. Results also showed a high haplotype diversity which was not related either to the host or to the geographical origin. Selective neutrality and genetic network tests suggest that the HSVd isolates have spread rapidly.

Molecular, Biological and Phylogenetic Analysis of Chinese Isolates ofHop stunt viroidAssociated with Citrus Cachexia Disease

Citrus cachexia is an economically important disease of citrus hosts caused by specific variants of Hop stunt viroid (HSVd) that are usually referred to as Citrus cachexia viroid (CCaVd). Eight cachexia-associated HSVd isolates were collected from six citrus growing areas of China, where citrus cachexia had not been reported previously. Forty-seven independent cDNA clones were used for genetic diversity and phylogenetic analysis. There were no sequence variant-cultivar correlation and no distinct regional specificity among or within the cachexia-associated HSVd populations analysed. Three clusters consisting of three major HSVd variants were identified by phylogenetic analysis, suggesting that most Chinese isolates contain a mixture of cachexia and non-cachexia variants. Biological properties of eight CCaVd isolates were determined by inoculating Parsons Special mandarin (Citrus reticulata). Our results suggest that the interactions between CCaVd and non-CCaVd variants might play an important role in suppressing cachexia symptom expression.

Characterisation of Hop stunt viroid (HSVd) isolates from jujube trees (Ziziphus jujuba)

Hop stunt viroid (HSVd) has been detected in many woody species including grapevine, citrus, peach, plum, apricot, and almond. Here, we report the first detection of HSVd in jujube trees (Ziziphus jujuba). Ninety-five samples were collected from jujube trees in Cangzhou, Baoding, and Handan of the Hebei province of China in June 2006. An additional 70 samples were collected from Taigu, the Shanxi province of China in August 2008. From these samples, low molecular weight RNAs were extracted for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), hybridisation, and reverse transcription polymerase chain reaction (RT-PCR) assays. HSVd was detected in three of the 165 samples, giving an infection rate of 1.8%. The sequences of the HSVd isolates from jujube trees shared 92.6–92.8% homology with the first reported HSVd sequence (GenBank: X00009). Using alignment and phylogenetic analyses, the HSVd sequence variants from jujube were classified in the plum subgroup and were found to be more conserved than other HSVd subgroups.

Occurrence and identification of citrus viroids from Sudan.

The Sudanese citrus industry is mainly based on oldline cultivars of grapefruit, sweet orange and willow leaf mandarin, grafted almost exclusively on sour orange. Infectionwith graft-transmissible agents, viroids in particular, may have deleterious effects on the productivity of these citrus species and, more importantly, limits the choice of rootstocks should sour orange be replaced by alternative rootstocks because of tristeza disease. Three cv. Foster pink grapefruit sources and one local selection of Nuri-16, a local sweet orange selection, proved to be infected by several viroids, including Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the causal agents of exocortis and cachexia, respectively. Cloning and sequencing of four HSVd isolates showed that in addition to the characteristic variants containing the “cachexia expression motif” or the “non-cachexia expression motif”, other variants were also present. To secure additional information, samples from 24 citrus species and cultivars were collected in 2008 from several citrus-growing regions and analyzed for the presence of viroids. The results confirmed that CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd), but not Citrus viroid V (CVd-V), are widely spread throughout the surveyed areas. None of the collected samples was found to be viroid-free. The relationships between viroid infection and “gummy bark” and “Kassala disease”, two affections of unknown etiology, but with cachexia-like gum impregnations in the bark, are discussed.

Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.

SEQUENCE VARIABILITY OF HOP STUNT VIROID ISOLATES FROM STONE FRUITS IN TURKEY

SUMMARY Hop stunt viroid (HSVd), genus Hostuviroid, family Pospiviroidae is a circular single-stranded RNA 294-303 nt in size, infecting a large number of woody hosts, such as grapevine, Citrus spp. and Prunus spp. In this study, we report the identification and molecular characterization of HSVd variants isolated in Turkey from different naturally infected Prunus sources, including apricot, plum and peach. We determined the nucleotide sequences of eleven isolates and found five new sequence variants of 296 (3 variants) or 297 (2 variants) nt, comparable in length to previously known HSVd isolates. Multiple alignments and phylogenetic analyses showed that one apricot isolate (HSVd.AP1) clustered with the recombinant P-H/cit3 group, whereas all the others (one apricot; HSVd.AP23, two plum; HSVd.PL49 and HSVd.PL278 and one peach, HSVd.PE73) clustered with the Hop group, confirming the molecular variability of HSVd isolates. The sequence variability seems to be more related to the geographical origin of the isolates than to their hosts.