Occurrence and Characterization of Graft-Transmissible Pathogens of Citrus in Orchards and Urban Citrus in Chile with a Focus on the Apscaviroid nanocitri.
Apscaviroid nanocitri (citrus dwarfing viroid, CDVd) is a graft-transmissible pathogen that frequently coexists with other citrus viroids and affects tree vigor. This study evaluated the occurrence of citrus viroids in small growers and commercial orchards in Chile and characterized CDVd strains obtained from urban Citrus spp. trees, with unknown propagation history following the discontinuation of the national citrus certification program. A total of 301 samples from five citrus-producing regions were analyzed, revealing that CDVd is widely distributed and commonly associated with Hostuviroid impedihumuli (hop stunt viroid, HSVd). Single infections were the most frequent (41%), followed by double (7.3%) and triple infections (3.0%), indicating that mixed infections are common. CDVd strains from urban trees were characterized using biological indexing on 'Etrog' citron Arizona 861-S-1 indicator plants combined with RT-PCR detection and phylogenetic analysis. Symptom development and pathogen detection were evaluated at 6 and 18 months post-inoculation. All graft-inoculated plants developed symptoms consistent with viroid infection, whereas control plants remained asymptomatic. CDVd was consistently detected at 18 months but not at earlier stages, suggesting delayed systemic accumulation. Although Closterovirus tristezae (citrus tristeza virus, CTV) was present in the source plants, it was not detected in indicator plants at either time point, possibly associated with the higher temperatures required for viroid symptom development during bioindexing. The frequent occurrence of mixed infections highlights the epidemiological relevance of viroid interactions and supports the need for reinstatement and strengthening of the national citrus certification program.