Abstract:

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 dwarfing viroid, 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. 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), has been proposed as a new species of the genus Apscaviroid.
Analysis of 64 samples from different citrus-growing areas has shown that CVd-V is present in the United States, Spain, Nepal, and the Sultanate of Oman. These results suggest that this viroid has not emerged recently and that it is relatively widespread. Transmission assays to sweet orange, mandarin, and mandarin hybrids, clementine, satsuma, lemon, sour orange, Tahiti lime, Palestine sweet lime, calamondin, bergamot, and kumquat have shown that all these citrus species and citrus relatives are hosts for CVd-V. Several indexing approaches, including slot blot, northern blot hybridization, and reverse transcription-polymerase chain reaction, have been evaluated for detecting CVd-V, either using Etrog citron as an amplification host or directly from commercial species and cultivars.
To evaluate the effect of exchanging discrete segments of the CVd-V molecule for the corresponding segments of CDVd, on symptom expression, seven chimeric viroids were synthesized and were inoculated mechanically to three citron seedling plants, each. Northern-blot hybridization and RT-PCR showed that a single plant from the three that had been inoculated with the chimeric variant Ch5 (CVd-V containing TL domain of CDVd) was actually infected. Sequencing the DNA amplicon confirmed that the chimeric variant Ch5 was stable, except for a single 42C®U substitution. Plants infected with Ch5 were symptomless, and plants co-infected with Ch5 and CVd-V or CDVd developed the same symptoms as plants singly infected with CVd-V or with CDVd, respectively. These results suggest that the terminal left domain is involved in the pathogenicity of  Citrus viroid V and that there were no interactions between Ch5 and these two related viroids. Analysis of the co-infected plants showed that CVd-V and CDVd displaced Ch5 that was found to be present in extremely low titers. 
Cachexia disease of citrus is caused by Hop stunt viroid (HSVd). In citrus, pathogenic and non-pathogenic strains differ by a "cachexia expression motif" of five to six nucleotides located in the variable domain of the proposed rod-like secondary structure. Site-directed mutants were generated to investigate if all these nucleotides were required for infectivity and/or symptom expression. Our results confirm that the "cachexia expression motif" plays a major role in inciting cachexia symptoms, and that subtle changes within this motif affect symptom severity and may even suppress symptom expression.