Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.

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A rapid and sensitive real time reverse transcription-PCR (RT-PCR) assay based on SYBR Green I chemistry was developed for the quantitative detection of Citrus viroid III (CVd-III) in citrus samples. CVd-III titre was determined at different times in green bark of sour orange, Troyer citrange, trifoliate orange and alemow seedlings inoculated with a CVd-IIIb source. Ten weeks after inoculation the viroid was detected in the four species, without substantial differences in viroid titre among them. Nine weeks later an overall increase of viroid titre was observed. The copy number of CVd-III in sour orange and Troyer citrange was monitored up to 52 weeks after inoculation and a further increase of viroid titre was observed at 35 weeks. For validation purposes, field samples were tested from 58 citrus trees with mixed infections of CVd-III, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), as well as from healthy controls. Based on the sensitivity (100%), specificity (96·7%), accuracy (99·2%) and repeatability (Cohen's kappa index 0·98) of the assay, it is suggested that its employment in breeding programmes would be helpful in the evaluation of host resistance and viroid accumulation in plants.

As with viruses, viroids infect their hosts as polymorphic populations of variants. Identifying possible sources of genetic variability is significant in the case of the species Citrus dwarfing viroid (CDVd) which has been proposed as a dwarfing agent for high-density citrus plantings. Here, a natural CDVd isolate (CMC) was used as an inoculum source for long-term (25 years) and short-term (1 year) bioassays in different citrus host species. Characterization of progenies indicated that the genetic stability of CDVd populations was high in certain hosts (trifoliate orange, Troyer citrange, Etrog citron, Navelina sweet orange), which preserve viroid populations similar to the original CMC isolate even after 25 years. By contrast, CDVd variant populations in Interdonato lemon and Volkamer lemon were completely different to those in the inoculated sources, highlighting how influential the host is on the genetic variability of CDVd populations. Implications for risk assessment of CDVd as a dwarfing agent are discussed. The GenBank/EMBL/DDBJ accession numbers for the complete sequences of the Citrus dwarfing viroid variants are JF970266.1 forH2-2, JF970267.1 for H2-7, EU938647.1 for H6-2, EU938651.1 forH6-10, JF970268.1 for H10-7, EU938652.1 for H14-13, EU938653.1for H14-14, JF970269.1 for H14-16, EU938648.1 for H15-9,EU938649.1 for H16-2, JF970265.1 for H16-9, EU938654.1 forH16-13, EU938650.1 for H20-3, JF970270.1 for H20-7, EU938641.1for PR-1, EU938642.1 for PR-3, EU938643.1 for PR-7, EU938644.1for CR-1, EU938639.1 for VR-4, JF12070.1 for VR-15, JF812069.1LS-4, EU938640.1 for LS-10 and JF970264.1 for LS-11.

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An outbreak of Spartium witches’ broom (SpaWB) in Sicily prompted us to identify and characterize associated phytoplasmas. Over 80 samples of Spanish broom (Spartium junceum) and around 270 individuals of the potential vector Livilla spectabilis were collected and analysed. Single and mixed infections of 16SrV and ‘Candidatus Phytoplasma spartii’ were detected in Spanish broom samples and for the first time in L. spectabilis. The 16SrV isolates were further characterized by multilocus sequence typing (MLST) to determine their phylogenetic relationship with flavescence dorée phytoplasma (FDp) and to evaluate the risk of host‐jumping to grapevine. Phylogenetic analysis of most of the analysed genes using the MLST approach grouped S. junceum 16SrV‐C isolates with FDp isolates infecting grapevine and Scaphoideus titanus. Notably, phylogenetic analysis of the vmpA gene clustered the S. junceum isolates with FDp genotypes transmitted by S. titanus. This study extends the knowledge of SpaWB epidemiology, focusing on the possible risk of a 16SrV host jump from Spanish broom to grapevine. Spanish broom was identified as a reservoir and potential inoculum source of phytoplasmas that cause severe disease in cultivated crops. Furthermore, the L. spectabilis psyllid may be involved in the epidemiology of this 16SrV‐C phytoplasma, although in the absence of in vivo transmission trials. The study further confirms the strong ability of phytoplasmas to adapt to new hosts and vectors, thus leading to potential phytosanitary emergencies.

Citrus cultivation in China has increased since the late 1970s, with China now having the largest area of citrus in culture in the world that is spread in 22 provinces and municipalities. Hunan Province has undergone a program to become one of the major citrus producers in China. Poncirus trifoliata is the main rootstock, so citrus viroids are a limiting factor for further citriculture development. In mainland China, only the presence of Citrus exocortis viroid (CEVd) has been reported from Etrog citron indexing, sPAGE (sequential polyacrylamide gel electrophoresis) analysis (2), and reverse transcription (RT)-PCR (3). Three viroid-like RNAs, a1, b1, and d, based on sPAGE patterns were detected years ago in our laboratory in budsticks received from Sichuan Province. To identify different viroids and determine their distribution, a survey has been undertaken. Field trees showing stunting, bark scaling and cracking of the rootstock, and poor yield were tested using biological indexing and PCR for the most frequent citrus viroids. Samples from six trees of a local sweet orange variety and three of a Clementine variety introduced from abroad, both grafted on P. trifoliata and showing a variable degree of bark scaling and cracking, were collected near Changsha and in the County of Xin Ning at the end of summer 2006. Small pieces of bark were inserted in stems of young E. citron budwood grafted on rough lemon and maintained in a warm greenhouse (24 to 32°C). Indexing on E. citron showed mild epinasty and leaf roll typical of citrus viroid infections. To identify specific viroids, bark was ground to a fine powder with liquid nitrogen and total RNA was extracted with TRIZOL Reagent (Invitrogen, San Diego, CA) and tested by RT-PCR to detect CEVd, Hop Stunt viroid (HSVd), and Citrus viroid III (CVd-III), as well as to identify the cachexia variants of HSVd. Four primer pairs were used to test the RNA extracts by RT-PCR (1). All samples were infected by HSVd, eight with CVd-III, and six with CEVd. The cachexia variants of HSVd were detected in four of nine samples. Mixed infections were as follows: one sample had CEVd and HSVd, eight had HSVd and CVd-III, and five were infected by the three viroids. A second sampling 3 months after inoculation gave the same amplification patterns. The results show that at least three viroids are present in citrus orchards in Hunan Province. To our knowledge, this is the first report of cachexia variants of HSVd and CVd-III in China. The common occurrence of these viroids supports the need for proper indexing of mother trees and a specific shoot tip grafting program to create healthy budwood sources to provide healthy plants. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes, 20:105, 2006. (2) L. Han et al. Viroids. CSIRO Publishing, Melbourne, 283, 2003. (3). Q. Hu et al. Acta Bot. Sin. 39:613, 1997.

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