Validation of Propidium Monoazide-qPCR for Assessing Treatment Effectiveness against ‘Candidatus Liberibacter asiaticus’ in Citrus


Citation
Yang, Ancona (2022). Agronomy 12 (11)
Names (1)
Subjects
Agronomy and Crop Science
Abstract
Huanglongbing (HLB) is an important citrus disease associated with the phloem-limited, uncultured bacterium ‘Candidatus Liberibacter asiaticus’(CLas). Effective treatments against CLas have to be validated in the field, however, methods for the field assessment of treatment effectiveness are time-consuming, in part because DNA-based assays, including quantitative PCR (qPCR), cannot differentiate between live and dead bacterial DNA. The aim of this study was to develop a method for rapid the evaluation of HLB therapies in field experiments. To this aim, a DNA extraction method from citrus leaf tissues with propidum monoazide (PMA), a dye that binds covalently to dsDNA making it unavailable for amplification in subsequent qPCR reactions, was optimized. The results indicated that the efficacy of PMA-qPCR was highly dependent on the primer set used. Primers targeting the 16S region of CLas showed a clear distinction between qPCR from PMA-treated and non-treated samples, while the RNR and LJ900 primers did not show significant differences between the DNA extraction methods. The PMA-qPCR viability analysis of CLas from citrus cuttings treated with different ampicillin (Amp) concentrations showed that all concentrations reduced CLas titers significantly starting 4 days after the initial treatment, unlike the water treatment, which did not show any change. This method was used for assessing the antibacterial activity of Amp, Streptomycin, Oxytetracycline (OTC), and a water control in field tests. The PMA-qPCR results indicated that Amp and OTC displayed significant antibacterial activity against CLas by 8 days post-injection, which was not detected in the non-PMA qPCR analysis. This method could allow the rapid validation of treatments against CLas in field experiments and facilitate the implementation of effective management strategies against HLB.
Authors
Publication date
2022-11-09
DOI
10.3390/agronomy12112783

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