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

The 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.     

Molecular,Biological and Phylogenetic Analysis of Chinese Isolates of Hop stunt viroid Associated 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 Parson’s 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.     

Behaviour of Apricot Cultivars Against Hop Stunt Viroid

The apricot species is susceptible to infection by different viruses and viroids including the Hop stunt viroid (HSVd), widely distributed around the world and described as the causal agent associated with fruit degeneration disease in apricot. However, to date, there are no ‘ad hoc’ studies about the resistance or susceptibility of apricot cultivars to this viroid. In this study, we tested the resistance/susceptibility to HSVd of 26 Mediterranean and North American apricot cultivars in controlled greenhouse conditions. All apricot cultivars assayed were infected by HSVd, showing a different range of susceptibility. As no sources of resistance among the apricot cultivars evaluated have been detected yet, it is necessary to continue the search for such sources to be included in apricot breeding programmes. In addition, the high level of susceptibility found highlights the importance of identifying this viroid in routine tests performed by nurseries and plant protection services.     

High‐throughput sequencing of Hop stunt viroid‐derived small RNAs from cucumber leaves and phloem

Small RNA (sRNA)‐guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self‐replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid‐derived sRNAs (vd‐sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd‐sRNAs are largely unknown. Here, we analyse, by high‐throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd‐sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd‐sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd‐sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd‐sRNAs. Our sequence data suggest that viroid‐derived double‐stranded RNA functions as one of the main precursors of vd‐sRNAs. Remarkably, phloem vd‐sRNAs accumulated preferentially as 22‐nucleotide species with a consensus sequence over‐represented. This bias in size and sequence in the HSVd vd‐sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd‐sRNAs to the phloem tissue of infected cucumber plants.     

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

We have developed a multiplex RT‐PCR protocol for the simultaneous detection of three viroids in three different genera that infect hops: Hop latent viroid (HLVd; Cocadviroid), Hop stunt viroid (HSVd; Hostuviroid) and Apple fruit crinkle viroid (AFCVd; Apscaviroid). The method was validated by testing 175 hop samples collected from the Xinjiang autonomous region of China. All samples were found to be positive for HLVd but negative for AFCVd, confirming the widespread or even ubiquitous infection of HLVd and the low incidence of AFCVd in hops in China. In addition, HSVd was detected in 22.86% of the samples tested. This rapid and reliable multiplex RT‐PCR assay provides an effective method for detection of three important viroid species in large‐scale surveys for disease management in hops.     

Genetic diversity of hop stunt viroid from symptomatic and asymptomatic citrus trees in Iran

Hop stunt viroid as the causal agent of cachexia disease has detected from citrus trees in different areas in Iran. Although cachexia has not been reported as a decline disease for citrus trees, it can impair crop quality and reduce plant yields. This study was undertaken to molecularly detect HSVd among different commercial citrus cultivars and determine genetic diversity of this viroid in Mazandaran province of Iran. Sampling was performed from symptomatic and symptomless citrus cultivars in Mazandaran province. HSVd specific primers were used for molecular detection. SSCP and sequencing were applied to assay HSVd genetic diversity. Results showed the detection of HSVd in all symptomatic Satsuma (25 out of 25), Clementine (25 out of 25), sweet lime (20 out of 20) and sweet orange cv. Valencia (7 out of 7), as well as, 31% (14 out of 22), 100% (12 out of 12) and 33% (5 out of 15) of page mandarin, lemon and grapefruit trees, respectively. 10 different HSVd genomes were identified by sequencing the SSCP profiles among which HSVd‐IR1 had the most frequency.     

Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.     

Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions

Taxonomy Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling‐circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid.Physical properties Hop stunt viroid consists of a single‐stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod‐like or quasi‐rod‐like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region.Hosts and symptomsHSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases (citrus cachexia, cucumber pale fruit, peach and plum apple apricot distortion, and hop stunt). It is distributed worldwide.TransmissionHSVd is transmitted mechanically and by seed.     

Viroid-induced symptoms in Nicotiana benthamiana plants are dependent on RDR6 activity

Viroids are small self-replicating RNAs that infect plants. How these noncoding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data have led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidence of a direct relation between key components of the RNA silencing pathway and symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (RDR6) is constitutively silenced. Here, we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd, indicating the requirement of RDR6 for viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild-type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptom expression associated with viroid pathogenesis.     

Assessment of UK hops for the occurrence of hop latent and hop stunt viroids

The occurrences and distributions of hop stunt (HSVd) and hop latent (HLVd) viroids were assessed by a nucleic acid hybridisation assay, using samples from 476 commercial hop plantings in the UK. These samples represented about half of the UK production.
HLVd was detected in c. 17% of the samples, with infection in different cultivars ranging from 0% to 89%. This viroid was found in all cultivars sensitive to Verticillium wilt except cv. Sunshine, an old cultivar grown on only one farm in the UK. Two minor wilt-tolerant cultivars were also found to be infected at low frequencies, but the main commercially-important wilt-tolerant cultivars were all uninfected. A high proportion of the nuclear stock mother plants in the "A +" house at the Institute of Horticultural Research Dept of Hops Research, Wye College were infected. Circumstantial evidence, based on the planting dates of infected gardens, suggests that infection became established in the hop propagation system during the late 1970s and that there was a major increase in the prevalence of HLVd as a result. Whether this contamination of propagating material arose because of spread from long-standing infections or because the viroid was newly introduced into the UK, is not known.
All samples were also tested for HSVd but this viroid was not detected in any UK hop material.     

Identification of an in vitro ribonucleoprotein complex between a viroid RNA and a phloem protein from cucumber plants.

We used the interaction of Hop stunt viroid (HSVd) and cucumber plants to investigate the involvement of phloem proteins in the systemic transport of RNA molecules. A ribonucleoprotein complex, stable even at high salt and temperature conditions, was detected in vitro between HSVd-RNA and the phloem exudate obtained from sectioned internodes from cucumber plants. The phloem protein 2 was recovered from this ribonucleoprotein complex and its RNA-binding properties as demonstrated by gel retardation analysis. The involvement of this protein in the movement of RNAs in cucumber is discussed.     

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.