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

Abstract

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.

Global transcriptomic analysis in avocado nursery trees reveals differential gene expression during asymptomatic infection by avocado sunblotch viroid (ASBVd)

“…Nested real-time PCR was carried out using TaqMan™ Gene Expression Master Mix from Applied Biosystems™ (Thermo Fisher Scientific) with the internal viroid primers ASBVd2qPCR-F and ASBVd2qPCR-R designed by Morey-León et al. (2018) and the ASBTM probe modified from Geering et al.…”

The Avocado Sunblotch Viroid: An Invisible Foe of Avocado

“…With the development of protocols to obtain preparations enriched in low-molecular-weight RNAs and the characterization of ASBVd [16,18], new and faster techniques such as polyacrylamide gel electrophoresis (PAGE) were developed [33,60]. Nevertheless, this technique was not completely reliable due to inconsistent results because known positives were often missed [71]. Improved and more reliable sensitive molecular techniques such as Reverse Transcription Polymerase Chain Reaction (RT-PCR) and dot blot hybridization have been recently adapted as better techniques to diagnose ASBVd-infected avocado plants.…”

“…RT-PCR performed in one [18] or two steps [24,76] has long been used as a routine laboratory test for ASBVd. More recently, a more sensitive method based on real-time RT-PCR was developed [71]. Non-destructive massive detection of avocado trees showing symptoms of sunblotch disease was performed using satellite spectral imaging [68] with an accuracy of 70% of actual ASBVd infection with respect to parallel analyses performed using RT-PCR [68].…”

The hunt for plant viruses in South Africa: The early days

“…Other molecular techniques have been developed more recently; for example, a highly sensitive novel SYBR green-based method based on real-time reverse transcription (RT)-PCR was reported. 31 The RT-qPCR is 100 times more sensitive to ASBVd than conventional RT-PCR. Infected asymptomatic trees play an important role in the epidemiology of this disease, and avocado nurseries need to be certified to ensure they provide pathogen-free avocado material.…”