The detection of avocado sunblotch viroid in avocado using a real-time reverse transcriptase polymerase chain reaction

The sunblotch disease is one of the diseases that affects avocado (Persea americana Mill) crops in particular, causing serious crop losses. The causal agent is the avocado sunblotch viroid (ASBVd), a circular RNA-based pathogen built of 247 nucleotides, which causes stem discoloration, bleaching, variegation, and distortion on the leaves and sunken yellow or red blotches on the fruits. In this study, a novel SYBR green-based real-time RT-PCR (RT-qPCR) assay specific for ASB viroid was developed. The assay was followed by a standard curve and a melting curve analysis for the detection, identification, and quantification of the full-length ASB viroid. The assay’s lower limit of detection in the samples of avocado leaves was 8.8 copies/μl. The RT-qPCR was 100 times more sensitive to ASBVd than the conventional RT-PCR. The melting curve analysis showed a variation in the specific melting temperature (Tm) between positive samples, probably due to the alteration in the nucleotide sequences of viroids. In conclusion, the use of RT-qPCR assays established in this study, as a routine diagnostic method in breeding programs, could decrease the viroid incidence in avocado crops and its further accumulation in plants.