Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

An assay to identify interactions between Citrus dwarfing viroid (CDVd) and Citrus tristeza virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd. V iroids are infectious, unencapsidated, single-stranded, and highly structured noncoding circular RNAs of 250 to 400 nucleotides (nt). Despite their simplicity, viroids interact with cell factors, which redirect them to replicate and traffic throughout the plant while avoiding the RNA silencing machinery they trigger in their hosts (1).Indexing tests have shown that viroids are widespread in commercial citrus orchards, wherein trees may be coinfected with other graft-transmissible pathogens. Data regarding virus-viroid interactions are very scarce, but the information available indicates that such interactions may alter symptom expression (2, 3). Since coinfection of citrus species with Citrus tristeza virus (CTV) and viroids is not rare, assays were conducted to establish whether interactions between these two types of pathogens actually exist.The assays were performed by comparing single and double infections of the two pathogens in etrog citron (Citrus medica L.) (viroid bioindicator) and Mexican lime (Citrus aurantifolia [Christm.] Swingle) (CTV bioindicator). Citrus dwarfing viroid (CDVd) (GenBank accession number EU934004), inducing moderate but specific symptoms in etrog citron (4), and CTV (T-305 strain), inducing symptoms in Mexican lime (5), were chosen for this study. To synchronize infection, Volkamer lemon (Citrus volkameriana Ten. & Pasq.) seedlings were graft inoculated with CDVd and/or CTV before being used as rootstocks. Etrog citron and Mexican lime were graft propagated to produce groups of five plants for each of the following treatments: (i) plants singly infected with CDVd, (ii) plants singly infected with CTV, (iii) plants coinfected with CTV and CDVd, and (iv) noninfected controls. CTV and CDVd infection was assessed by immunoprinting enzyme-linked immunosorbent assay (ELISA) (6) and dot blot hybridization (7), respectively.After an incubation period of 4 months at 25°C, plant height was measured (Fig. 1) and data were subjected to multifactor analysis of variance (ANOVA). The results indicated that etrog citron singly infected with CTV (P value, 0.00001) or CDVd (P value, 0.05) was stunted, as was Mexican lime singly infected with CTV (P value, 0.0013) but not that with CDVd (P value, 0.98). Height reduction in coinfected plants resulted from the added effect of each pathogen, indicating no interaction between CDVd and CTV in either host (interaction P value for etrog citron, 0.29; interaction P value for Mexican lime, 0.44). Similarly, symptom expression in stems and leaves resulted from the added effect of each pathogen, revealing agai...

Citrus dwarfing viroid has been proposed as an agent to control tree size in high-density plantations. Thirty-three field isolates have been characterized, and the most frequent sequence/s have been identified. Five distinct variants were selected for biological characterization. Symptom expression analysis demonstrated a good correlation between leaf/stem symptoms and plant growth. The discriminating nucleotide sequence differences included two deletions and an insertion resulting in a reorganization of the base pairing of the terminal left loop, two (G42 --> A and C52 --> U) changes found in one of the variants, and as many as thirteen changes located in the right and left regions flanking the CCR.

A field-source mixture of citrus viroids was characterized and shown to contain Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). Sequencing results showed that: (i) CEVd contained the PL and PR characteristic of class A variants; (ii) HSVd was a noncachexia variant; (iii) CBLVd was related to CVd-Ia variants; (iv) CDVd was a mixture of two types (CVd-IIIa and CVd-IIIb) of variants. The presence of the same type of variants in inoculated clementine (Citrus clementina ‘Nules’) and sweet orange (C. sinensis ‘Navelina’) trees on Carrizo citrange (Poncirus trifoliata × C. sinensis) rootstocks was confirmed. The effect of infection was determined by assessing the performance of infected and noninfected trees growing in the field. Infection resulted in small trees with reduced canopy, yielding a reduced crop. Fruit characteristics were also affected: (i) clementine and sweet orange fruits from infected trees were larger than those from noninfected trees; (ii) clementine fruits from infected trees differed in shape from those of noninfected trees; (iii) sweet orange fruits from infected trees had maturity indexes and juice contents higher than those from noninfected trees; (iv) in both species, the density of the juice, the amount of soluble solids, and the acidity of the fruits from infected trees were lower than those of fruits from noninfected trees. Infected trees had a poorly developed root system with fibrous roots containing fewer amyloplasts than noninfected trees. The results of an in vitro assay on the induction and development of roots in cultured explants are discussed.

In studies to identify genotypes resistant to infection with citrus viroids, Eremocitrus glauca and Microcitrus australis were selected because their evolution in their habitat in Australia and New Guinea may have led to the selection of unusual traits. The movement and accumulation of Citrus exocortis viroid (CEVd), Hop stunt viroid, Citrus bent leaf viroid, Citrus dwarfing viroid, Citrus bark cracking viroid and Citrus viroid V (CVd-V) in self-rooted as well as in graft-propagated E. glauca and M. australis plants was assessed by northern hybridization, RT-PCR and by topworking to the sensitive selection 861-S1 of Etrog citron. In both plant species the inoculated viroids were undetectable unless these plants were grafted to a susceptible Citrus partner, the rough lemon rootstock and ⁄ or the topworked Etrog citron, which acted as viroid sources. The results obtained indicate that M. australis and in particular E. glauca are poor viroid hosts in which viroid replication ⁄ accumulation does not occur or is extremely inefficient. However, viroid downward and upward movement to grafted Citrus partners in which viroid replication and accumulation occurs efficiently was not impaired. Eremocitrus glauca and M. australis showed differences regarding their properties as viroid hosts, but for both species CEVd seemed to have the lowest affinity among the viroid species tested and CVd-V the highest. Even though E. glauca and M. australis do not appear to be truly resistant to viroid infection, they are interesting genotypes for further characterization of the mechanisms involved in viroid infection.