DETECTION AND AGRONOMIC CHARACTERIZATION OF CITRUS VIROIDS. IDENTIFICATION AND MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF VARIANTS OF CITRUS DWARFING VIROID (CDVd).

SUMMARY
      Citrus are natural hosts of several viroid species, all of them belonging to the family Pospiviroidae. Preliminary approaches to detect viroids in commercial species and cultivars yielded erratic results unless the indicator species Etrog citron was used as a bioamplification host. To avoid the use of this host in routine detection assays, a northern hybridization protocol was developed. This protocol consisted of: (i) Nucleic acid extraction of bark samples collected in different growing seasons and/or stored at different conditions; (ii) separation of the RNAs in 5% PAGE or 1% agarose and transfer to membranes; (iii) hybridization with viroid specific DNA probes labelled with digoxigenin (DIG), detection with an anti-DIG antiobody conjugated with alkaline phosphatase and developed with a chemiluminiscence substrate (CSPD). With this approach, viroids were successfully detected in all the citrus species and cultivars tested. The technique is extremely sensitive, and all the viroids described in citrus can be detected in a short period of time. 
      This technique was used to perform viroid surveys in different citrus growing areas of Colombia, Peru and Brazil. Multiple viroid infections of HSVd and CDVd or CEVd, HSVd and CDVd were found in Tahiti lime trees from Colombia. With the exception of a single Etrog citron tree that was found to be infected with CEVd and HSVd, the samples collected in the Germplasm Bank located in Palmira were viroid-free. The molecular characterization of the field isolates of HSVd, CDVd and CEVd recovered from the surveyed samples showed that: (i) With a single exception, all the HSVd isolates were devoid of the cachexia expression motif and therefore can be considered as non-pathogenic variants; (ii) the CDVd isolates contained variants with high sequence identity with the reference sequence CVd-IIIb; (iii) one of the CEVd isolates (CEVdCO) recovered from a symptomless Etrog citron presented 96.5% nucleotide identity with the reference sequence of Class A (which contains the severe variants of this viroid) and 98.9% identity with the reference sequence V1 that induces severe symptoms in Etrog citron. The nucleotide differences between CEVdCO and variant V1 are: U186A (TR domain), A235U (V domain) and AU313-314AG (P domain). Since the P domain is associated with the pathogenesis of CEVd, synthetic mutants in the nucleotides 313-314 of V1 and CEVdCO were obtained with the aim to demonstrated if the changes identified in CEVdCO are indeed responsible of the lack of symptom expression in the Etrog citron trees infected with this variant. 
      The analysis of several Tahiti lime clones from Peru and Brazil showing the “quebra-galho” syndrome (portuguesse expression meaning “breaking branche”) and presenting the characteristic bark cracking, dwarfing and open canopy associated with other desirable traits, showed that they were infected with CEVd, HSVd and CDVd (clones from de Peru) or with CEVd and CDVd (clones from Brazil). The results available from a field assay designed to find out if these viroids were responsible form the “quebra-galho” syndrome showed that CEVd caused bark cracking in Tahití lime trees two after inoculation. Additional observations in fully developed trees are required to confirm that CEVd is the causal agent of the overall symptoms characteristic of the “quebra-galho” syndrome.
      Citrus dwarfing viroid (CDVd) is very widespread in different citrus growing areas but since it does not induce specific symptoms, its effect on tree size is poorly recorded. A study was addressed to identify and characterize variants of this viroid in 33 field isolates. SSCP analysis showed that each isolate contained populations of sequence variants and the most frequent variant of each isolate was identified and sequenced. A phylogenetic analysis demonstrated their relationship with the reference variants (CVd-IIIa, CVd-IIIb y CVd-IIIc). Five sequences representing the five major groups identified in the phylogenetic tree were selected for their further biological characterization in Etrog citron. Statistical analysis of growth parameters and symptom expression data showed that two variants acted as severe strains, two as moderate strains and single variant was a very mild strain. The nucleotide differences found in these variants were associated with the modulation of symptom expression. 
      In order to determine the effect of viroid infection on the performance of field grown trees, a field plot established with viroid-infected Washington navel sweet orange trees grafted in Carrizo citrange was monitored over four growing seasons. Statistical analysis of growth parameters (tree height, stem width, canopy volume and quantity and quality of fruit crop) showed only minor slight statistical differences among treatments.