Studies on Atalantia citroides, a citrus relative, revealed the existence of a viroid not described previously. The new viroid has a GC-rich genome of 293-294 nucleotides and contains the central conserved region characteristic of members of the genus Apscaviroid, and the terminal conserved region present in this and other genera of the family Pospiviroidae. The secondary structure of minimum free energy predicted for the new viroid is a rod-like conformation with 68.7% paired nucleotides and showing sequence identities with other viroids always lower than 90%, the conventional limit that separates different species within a given genus. Infectivity assays showed that the new viroid induces mild but characteristic symptoms on the indicator Etrog citron. Co-inoculation of CVd-V with either Citrus bent leaf viroid or Citrus viroid III, two other members of the genus Apscaviroid infecting citrus, disclosed synergistic interactions manifested in enhanced leaf symptoms and very pronounced dwarfing. Viroid titers, however, remained unaltered in co-infected plants. Possible mechanisms underlying the observed synergistic effects are discussed. According to its molecular and biological properties and its unusual ability to replicate in A. citroides, the new viroid, tentatively named Citrus viroid V (CVd-V), should be considered a new species of the genus Apscaviroid.

Preliminary transmission assays conducted under greenhouse conditions demonstrated that Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) can be mechanically transmitted from citron to citron (Citrus medica) by a single slash with a knife blade. The impact of mechanical transmission of viroids by pruning and harvesting operations was also demonstrated in experimental and commercial field plots. Transmission efficiency under field conditions ranged from 4% in ‘Nules’ clementine to 10% in ‘Navelina’ sweet orange and 21% in ‘Verna’ lemon. Transmission efficiency varied only slightly with viroid and donor hosts. The impact of viroid transmission on tree height, canopy volume, and crop harvest was minimal. When the donor host was coinfected with several viroids, the viroids were not necessarily cotransmitted. Considerations regarding viroid transmission in other climates are discussed. Measures to control viroid spread in nurseries should be mandatory in certification programs.

Progress, spread and natural transmission of Bahia bark scaling of citrus were evaluated in a trial where 240 screenhouse-nursed nucellar grapefruit plants -'Clason', 'Little River Seedless', 'Red Blush', 'Reed' and 'Howell Seedless' cvswere planted alongside and 5 m apart from a 10-year-old symptomatic 'Marsh Seedless' grapefruit orchard. Plants were distributed in 16 rows of 15 trees, with three plants of each cultivar per row. Eight trial plants were kept in screen cages. Incidence of symptomatic plants was assessed at 3-months intervals, for 5 years, and for further 2 years at irregular intervals. Cumulative maps of disease incidence were produced for each assessment date and used in all analyses. Temporal progress was analysed by nonlinear fitting of three disease progress models. Spread was characterised in three levels of spatial hierarchy by the following analyses: ordinary runs, binomial dispersion index, binary power law fitting, isopath mapping and nonlinear fitting of disease gradient models. The first symptomatic plant was detected 2 years after planting. In the last disease assessment, 5 years after the first, 98% of the unprotected plants were symptomatic. None of the screen-caged trees showed any symptoms. Bahia bark scaling progress was polyetic and best described by the logistic model. Ordinary runs analysis showed little if any evidence of transmission between adjacent trees. Diseased plants showed a very aggregated pattern inside quadrats (D > 5 and b > 1.53). Isopath mapping showed that main spread was only because of the primary inoculum source. Secondary foci were also observed, but they were never dissociated from main initial disease focus. Disease gradient followed wind direction, starting near the original inoculum source and was best described by exponential model. These results support a hypothesis of Bahia bark scaling transmission by air-borne vectors with limited dispersion ability.

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.

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