F. Di Serio

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.

Close structural relationship between two hammerhead viroid-like RNAs associated with cherry chlorotic rusty spot disease.

Summary.Analysis of the population of cherry small circular RNAs (cscRNAs) from trees affected by cherry chlorotic rusty spot (CCRS) showed two groups of variants with similar sequence but differing in size (394–415 and 372–377 nt for cscRNA1 and cscRNA2, respectively) because of the presence or absence of a 27-nt fragment folding into a hairpin in their predicted quasi-rod-like secondary structures. These structures were preserved by co-variations and compensatory mutations, as well as by additional complex rearrangements. The variability also preserved the central conserved core and the stability of the helices of the plus and minus hammerhead ribozymes, supporting their role in replication of cscRNAs. The smaller variants most likely derive from the larger through recombination events. Possible functional relationships between cscRNAs and certain mycoviral-like double-stranded RNAs, also associated with CCRS, are discussed.

Sequences of the smallest double-stranded RNAs associated with cherry chlorotic rusty spot and Amasya cherry diseases

Cherry chlorotic rusty spot (CCRS) and Amasya cherry disease (ACD) are two disorders that affect cherry in Italy [1] and Turkey [2]. Both diseases have similar symptoms and are associated with a complex pattern of doublestranded RNAs (dsRNAs), absent from healthy-looking material [1]. The dsRNAs were presumed to have a viral origin, and their characterization showed that: (1) four comprise the genomic components of a member of a new tentative species of the genus Chrysovirus [3], (2) two comprise the genomic components of a member of a new tentative species of the genus Partitivirus [4], and (3) another four display their highest similarity with members of the family Totiviridae [5]. Because members of these three taxa typically infect fungi, and because microscopy observations have identified a fungus-like organism in the affected leaf tissues [6], these data support a fungal etiology for CCRS and ACD diseases. In addition to the ten large dsRNAs, PAGE analysis of CCRS-affected material revealed at least another two bands in the lower part of the gels [1]. We presumed that they were generated by small dsRNAs, given that the bands were detected in preparations enriched in dsRNAs. Here we report their sequences.

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.

Viroids Infecting the Grapevine

Viroids are nonprotein-coding, small, circular RNAs infecting plants in which they may induce specific symptoms. Five different viroids have been identified in the grapevine in the period elapsed from 1985 to 1990. Since then, no new viroid has been reported from grapevines until the application of next-generation sequencing allowed the discovery of an additional viroid and a new viroid-like RNA. Possibly, new small, circular RNAs will be identified in the future by metagenomic approaches, but bioassays, which are time intensive and require phytopathological expertise, will always be needed for establishing conclusively their true identity as viroids. Although viroids generally do not elicit severe symptoms in grapevines, some of them are the agent of diseases in certain environmental conditions or in combination with certain viruses. Some of grapevine-infecting viroids may cause severe diseases in other crops. This chapter reviews the molecular, biological, and epidemiological features of viroids and viroid-like RNAs infecting grapevines and the methods for their detection and control and discusses the future perspectives of research.