Development of a multiplex PCR system for efficient pathogen detection in alfalfa plants

Abstract

A multiplex PCR protocol was developed for the simultaneousdetection of major pathogens affecting alfalfa (Medicago sativaL.), including oomycetes and fungi (Phytophthora medicaginis,Xylaria sp., Fusarium oxysporum forma specialis (f. sp.) medicaginis,Rhizoctonia solani, Peronospora trifoliorum, Athelia rolfsii,Pseudopeziza medicaginis, Uromyces striatus, Diaporthe sp. andPhoma medicaginis) and bacteria (Xanthomonas alfalfae subsp.alfalfae and Pseudomonas syringae). DNA for validation wasextracted from 15 alfalfa samples collected throughout Argentina,and Normalised Difference Vegetation Index (NDVI) data fromOneSoil were used to analyse and normalise collection sites.Results showed that Rhizoctonia solani, Athelia rolfsii, Diaporthesp. and Xanthomonas alfalfae were present in 100% of samples,providing insight into the prevalence of these pathogens. Xylariasp. and Pseudopeziza medicaginis were detected in only 30% ofthe samples, suggesting that corky root rot and common leafspot had low prevalence in the region during the study period.Overall, this multiplex protocol allows efficient, global pathogendetection in alfalfa, supporting better disease management andcrop improvement. The results highlight the potential to guiderural producers in mitigating key diseases regionally and globally.