E.M. Torchetti

Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids.

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each targeted viroid were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, GYSVd-1, AGVd, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.

Identification and characterization of a viroid resembling apple dimple fruit viroid in fig (Ficus carica L.) by next generation sequencing of small RNAs.

Viroids are small (246-401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21-24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (-) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs.

A single polyprobe for detecting simultaneously eight pospiviroids infecting ornamentals and vegetables.

The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.

FIRST REPORT OF CHRYSANTHEMUM STUNT VIROID IN ARGYRANTHEMUM FRUTESCENS IN ITALY

Chrysanthemum stunt viroid (CSVd) is a quarantine pathogen for chrysanthemum (Dendranthema spp.) in the European countries (Plant Health Directive 2000/29/EC), because this host is severely affected, thus comes down with a disease characterized by stunting, leaf chlorosis and floral disorders. CSVd spread in Europe has efficiently been restrained so far, although several outbreaks were recorded in the past. Here we report the first occurrence of CSVd in Italy, as detected in several symptomless cultivars of Argyranthemum frutescens (marguerite daisy) by RT-PCR with specific primers and by Northern blot hybridization with a specific digoxigenin-labeled riboprobe. Viroid identity was ultimately ascertained by cloning and sequencing cDNA amplicons. Molecular characterization of CSVd isolates from six different A. frutescens cultivars disclosed viroid RNA populations with a prevalent size of 354 nt and sequences 98-100% identical to those of CSVd variants reported previously from D. grandiflora and A. frutescens. These results call for a prompt extension of surveys for assessing the presence of CSVd in symptomless A. frutescens and other ornamentals, which could constitute hidden reservoirs of this pathogen. In view of this, a tissue-printing hybridization method for detecting CSVd in A. frutescens was tested and validated.

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.

GRAPEVINE VIROIDS AND GRAPEVINE FANLEAF VIRUS IN NORTH- WEST IRAN

Grapevine-infecting viroids do not induce symptoms, except for Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2), the agents of yellow speckle (YS), a disease characterized by yellow spots or flecks scattered on the leaf blade. The association of these viroids with Grapevine fanleaf virus (GFLV) is thought to elicit vein banding (VB), a syndrome characterized by chrome yellow flecks localized along the main veins and progressing into the interveinal areas of affected vines. The occurrence of these diseases and their causal agents was investigated in north-west Iran with a survey in which 137 vines were tested by multiplex RT-PCR for the presence of the five known grapevines viroids. GYSVd-1, GYSVd-2, Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in 91%, 64%, 95%, and 100% of the tested samples, respectively, whereas Citrus exocortis viroid (CEVd) was not found. Combinations of three and four different viroids were present in most plants (88%) whereas GFLV was found in 50 samples (37%). The Iranian isolates of GYSVd-1, GYSVd-2, HSVd and AGVd showed minor molecular changes compared with the respective reference strains from grapevine. VB occurred in 22 vines infected by GYSVd-1, GYSVd-2 and GFLV, whereas YS symptoms, which occurred in 10% of the tested plants, were always shown by vines infected by GYSVd-1 and/or GYSVd-2. These findings are in line with the notion that assigns to GYSVd-1 and GYSVd-2 a role in the induction of YS and to both viroids and GFLV the genesis of VB.

The role of plant viroids in diseases - new developments

Work in B. Navarro’s and F. Di Serio’s laboratory has been supported by a grant from the Italian Ministry of the Agriculture (OIGA-‘PSTVd-free’ 2009–2011) and a dedicated grant from the Italian Ministry of Economy and Finance to the CNR (Legge no. 191/2009), and in R. Flores’s laboratory by the Ministerio de Ciencia e Innovacio´n of Spain (grant nos. BFU2008-03154 and BFU2011-28443).

Other Apscaviroids Infecting Pome Fruit Trees

Abstract Apple dimple fruit viroid, first reported in Italy, was later identified in Lebanon, China, and Japan, always in apple trees affected by dimple fruit disease. It has been found recently in fig in Italy, but not associated with a specific symptom. Apple fruit crinkle viroid was first reported in apples showing fruit crinkle disease in Japan. Other isolates were reported in hops with symptoms of stunting and leaf curling, and in asymptomatic Japanese persimmons, both cultivated in Japan. Pear blister canker viroid naturally infects pear and quince. It has been reported worldwide, particularly in the Mediterranean basin, although the economic impact is limited because most pear cultivars are tolerant. Apscaviroids infecting pome fruit trees can be detected readily by molecular hybridization and RT-PCR, and controlled by using viroid-free propagation material.

A nuclear-replicating viroid antagonizes infectivity and accumulation of a geminivirus by upregulating methylation-related genes and inducing hypermethylation of viral DNA.

DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection with TYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd infection promotes a strong hypermethylation of TYLCSV DNA, thus supporting a mechanistic link with the antagonism of the viroid on the virus in co-infected tomato plants. These results describe the interaction between two nuclear-replicating pathogens and show that they differentially interfere with DNA methylation pathways.