Detection and characterization of Xylella fastidiosa in Iran: first report in alfalfa (Medicago sativa)

Bacterial pathogens, especially Xylella fastidiosa (Xf), are significant threats to agricultural productivity, affecting economically important crops. The recent detection of Xf in Europe and the Middle East, including Iran, has emphasized the urgency for comprehensive surveillance to assess and understand the genetic diversity and distribution of this pathogen. A comprehensive survey from 2019 to 2022 was carried out in Iran to investigate Xf occurrence. A total of 403 samples were collected from alfalfa, almond, citrus, cherry, grapevine, olive, and pistachio plantations. Using serological (DAS-ELISA) and molecular (PCR) techniques, Xf was detected in nine samples from grapevine, five from almond, and 18 from alfalfa, and these include the first records Xf infections in alfalfa in Iran. Multiprimer-PCR assays carried out on Xf-infected plants, using ALM1/ALM2, XF2542-L/XF2542-R, and XF1968-L/XF1968-R primers for subspecies and strain differentiation, showed that the isolates from almond were Xf subsp. multiplex, and those from alfalfa were Xf subsp. fastidiosa. The Xf subsp. multiplex infecting almonds belonged to Xf genotype II. Pathogenicity tests carried out using Xf subsp. multiplex and fastidiosa isolates showed that the pathogen caused symptoms on Nicotiana benthamiana plants within 20 d post-inoculation. This study emphasizes the requirement for continuous monitoring, to mitigate the impacts of Xf on Iranian agriculture, and to prevent widespread outbreaks of this pathogen in multiple crop types.