Presence Of Hop Stunt Viroid Research Articles

Occurrence and Pathogenicity of Hop Stunt Viroid Infecting Mulberry (Morus alba) Plants in China.

To investigate the presence of hop stunt viroid (HSVd) in mulberry (Morus alba) plants in China, HSVd was detected by reverse transcription (RT)-PCR using dsRNAs extracted from symptomatic or asymptomatic mulberry leaf samples collected from a mulberry field located in Zhenjiang, China, as a template and the primer pairs for HSVd detection. The primer pairs were designed based on the conserved sequence of 25 HSVd variants deposited in the GenBank database. Four out of a total of 53 samples were HSVd-positive, confirming that HSVd is present in mulberry plants in China. The consensus full-length nucleotide (nt) sequence of two HSVd variants determined by sequencing the HSVd variants in these four HSVd-positive samples consisted of 296 nt and shared the highest nt identity of 96.8% with that from plum in Turkey but relatively low identity with those from mulberry in Iran (87.3 to 90.8%). Phylogenetic analysis showed that these HSVd variants clustered together with those of the HSVd-hop group. Analysis of the infectivity and pathogenicity to hosts by the constructed Agrobacterium-mediated dimeric head-to-tail HSVd cDNA infectious clones demonstrated that one of the HSVd variants identified in this study infects the natural host, mulberry plants, and also infects experimental plants, cucumber, and tomato. It probably induces stunting symptoms in HSVd-infected tomatoes but does not induce symptoms on mulberry leaves or in cucumbers. Although HSVd infecting mulberry has been found in Iran, Italy, and Lebanon, this is the first study to report this viroid in naturally infected mulberry plants in China.

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.

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.

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.

Viruses and viroids of stone fruits in Algeria.

Field surveys were carried out in the major stone fruit growing areas of eastern Algeria to assess the sanitary status of stone fruits. A total of 454 samples from peach, apricot, almond, sweet and sour cherry, plum and myrobalan were tested by ELISA or RT-PCR for the presence of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), Apple chlorotic leaf spot virus (ACLSV) Apricot latent virus (ApLV), Cherry virus A (CVA), Cherry green ring mottle virus (CGRMV), and Plum bark necrosis stem pitting-associated virus (PBNSPaV). The overall average infection level was 10.4%. The most frequent virus was PNRSV (56.8%), followed by PDV (27.2%) and ApMV (22.7%). The most infected species was cherry (21.9%) and the less almond (4.4%). ACLSV, ApLV, CVA, CGRMV and PBNSPaV were not detected. Of 531 samples tested for the presence of viroids by tissueprint hybridization, 28 (5.2%) were infected. Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) were detected in 15 and 13 samples, respectively. This is the first large-scale study on viruses and viroids of stone fruits in Algeria and reports for the first time the presence of HSVd in the country.

Analysis of symptoms developed in Nicotiana benthamiana plants expressing dimeric forms of Hop stunt viroid.

Viroids are sub-viral plant pathogens consisting of covalently closed circular RNAs that infect herbaceous and woody hosts. They do not code for any peptide or protein and therefore are fully dependent on the host machinery for most biological processes, including pathogenesis. Certain viroid-host combinations show severe symptoms but the biochemical bases of this process are currently unknown. In this work, we have characterized the symptoms induced by Hop stunt viroid (HSVd) when provided as a transgene in Nicotiana benthamiana plants, a natural non-host that is able to process and translocate HSVd. The presence of HSVd in this transgenic plant causes severe stunting and decrease in the leaf size, followed by strong alteration of flower development and reduced fertility. These symptoms are similar to those induced by HSVd in natural infections, indicating that in N. benthamiana HSVd expression mimics the metabolic disorders induced in natural hosts. Therefore, these transgenic plants may be useful to study the viroid-host interactions leading to pathogenesis.

First Report of Hop stunt viroid in Apricot in China.

Hop stunt viroid (HSVd), a member of the family Pospiviroidae, was first described as the causal agent of hop stunt disease in Japan. It has since been found in a wide range of hosts including herbaceous and woody hosts (e.g., hop, cucumber, grapevine, citrus, plum, peach, pear, apricot, almond, and pomegranate). It was also detected and characterized in apricot where infection appears to be latent (1). The viroid occurs frequently in apricot. In southeastern Spain, the presence of HSVd was found to infect 81% of apricot trees (2). Apricots originated in China and are extensively cultivated, but HSVd infection in this host has not been reported. In September 2005, a single symptomatic apricot tree, 'Yin Bai', one of the most popular and widely grown cultivars in China, was discovered at the Institute of Fruit Science in Changping District in Beijing, Peoples Republic of China. Observed symptoms included a number of yellow spots with an irregular border that scattered in an irregular manner over the leaf surface. Total RNA was extracted and used for return-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) (4). Results of both assays were positive for HSVd. A 297-bp full-length DNA fragment was amplified by RT-PCR using primers R1 (5'-GCTGGATTCTGAGAAGAGTT-3') complementary to HSVd residues 87-106 for the RT reaction, followed by R2 (5'-AACCCGGGGCTCCTTTCTCA-3') complementary to HSVd residues 67-84 and forward primer F3 (5'-AACCCGGGGCAACTCTTCTC-3') residues 79-96 for PCR. The primers are located in the strictly conserved central region of the conserved HSVd group and contain the unique endonuclease restriction site SmaI. The amplified products were cloned into pGEM-T (Promega, Madison, WI) and selected for further analysis on the basis of the results of restriction digests. Six individual clones were sequenced and three different sequences were obtained. Nucleic acid sequence (GenBank Accession No. DQ362901) obtained from one clone was 99.3% (nucleotide changes T206→C, C233→T) identical to HSVd.apr8 (GenBank Accession No. Y09349) (3). Sequence (GenBank Accession No. DQ362904) obtained from three clones was 99.7% (nucleotide change C233→T) and a third sequence (GenBank Accession No. DQ362905) obtained from two clones was 99.3% (nucleotide changes G107→A, C233→T) identical to HSVd.apr8. Further investigation is necessary to determine whether the symptoms observed are associated with the viroid infection. To our knowledge, this is the first report of HSVd isolated from apricot in China.

First Report on "Hop Stunt Viroid" (HSVd) from Some Mediterranean Countries.

Hop stunt viroid (HSVd) has a very wide host range including most stone fruit trees. Among them, apricot is one of the most important host crops in the Mediterranean basin. In this study non-isotopic molecular hybridisation revealed, for the first time, the presence of HSVd on apricot in four Mediterranean countries (Cyprus, Greece, Morocco and Turkey). The results obtained by this technique were confirmed by northern-blot and RT-PCR analyses. The data presented in this work indicate a wider geographical distribution of this viroid than hitherto known and emphasise the need for this kind of study as part of the control effort.

Hop Stunt Viroid in Australian Grapevine Cultivars: Potential for Hop Infection.

A nucleic acid probe was made for the detection of hop stunt viroid (HSV). The HSV probe was used in the screening of grapevine cultivars and rootstocks from Victoria and South Australia for the presence of HSV by northern blot analysis. HSV was detected in all samples tested except seedlings and vines derived from fragmented shoot apex culture. Cultivars containing HSV are grown in some of the hop-growing regions of Australia raising the possibility of HSV transmission from grapevines to hops.