Detection of Viable Xanthomonas fragariae Cells in Strawberry Using Propidium Monoazide and Long-Amplicon Quantitative PCR

Xanthomonas fragariae causes angular leaf spot in strawberry. The pathogen's association with its host tissue is thought to be a condition for its survival. Consequently, transmission of the pathogen to field production sites occurs almost exclusively through the movement of contaminated planting stock. The aim of this study was to develop a propidium monoazide (PMA)-quantitative PCR (qPCR) protocol for specific detection of viable X. fragariae cells. The qPCR procedure was developed for two different primer pairs: one producing a long amplicon (863 bp) and the other a short amplicon (61 bp). Both pairs were tested on mixtures of viable and heat-killed bacteria cells, bacteria-spiked strawberry petiole samples, and petioles collected from symptomatic, inoculated plants. The results showed that long-amplicon PMA-qPCR enabled specific and sensitive detection of X. fragariae with a detection limit of 10(3) CFU/ml, and it significantly improved PMA efficiency in differentiating viable from dead bacterial cells relative to short-amplicon PMA-qPCR. Based on the delta threshold cycle (C-t) values (i.e., the difference in C-t values between PMA-treated and nontreated samples), the long-amplicon PMA-qPCR was able to suppress the detection of dead X. fragariae cells 1.9- to 3.1-fold across all petiole samples tested. The quantification results from PMA-qPCR for mixtures of viable and dead cells were highly correlated with the predicted bacterial concentrations in a linear relationship (R-2 = 0.981). This assay can be useful for identifying inoculum sources in the strawberry production cycle, which may lead to improved disease management strategies.