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

A multiplex PCR protocol was developed for the simultaneous detection of major pathogens affecting alfalfa (<i>Medicago sativa</i> L.), including oomycetes and fungi (<i>Phytophthora medicaginis</i>, <i>Xylaria</i> sp., <i>Fusarium oxysporum</i> forma specialis (f. sp.) <i>medicaginis</i>, <i>Rhizoctonia solani</i>, <i>Peronospora trifoliorum</i>, <i>Athelia rolfsii</i>, <i>Pseudopeziza medicaginis</i>, <i>Uromyces striatus</i>, <i>Diaporthe</i> sp. and <i>Phoma medicaginis</i>) and bacteria (<i>Xanthomonas alfalfae</i> subsp. <i>alfalfae</i> and <i>Pseudomonas syringae</i>). DNA for validation was extracted from 15 alfalfa samples collected throughout Argentina, and Normalised Difference Vegetation Index (NDVI) data from OneSoil were used to analyse and normalise collection sites. Results showed that <i>Rhizoctonia solani</i>, <i>Athelia rolfsii</i>, <i>Diaporthe</i> sp. and <i>Xanthomonas alfalfae</i> were present in 100% of samples, providing insight into the prevalence of these pathogens. <i>Xylaria</i> sp. and <i>Pseudopeziza medicaginis</i> were detected in only 30% of the samples, suggesting that corky root rot and common leaf spot had low prevalence in the region during the study period. Overall, this multiplex protocol allows efficient, global pathogen detection in alfalfa, supporting better disease management and crop improvement. The results highlight the potential to guide rural producers in mitigating key diseases regionally and globally.