Improved seed health tests for Xanthomonas axonopodis pv. phaseoli in common bean

Xanthomonas axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans are important seedborne pathogens of Phaseolus vulgaris. In order to maintain seed quality and meet phytosanitary requirements, accurate seed health testing methods are critical. Currently employed selective-media-based methods for these pathogens include several variations in extraction procedures. In order to optimize pathogen extraction from P. vulgaris seeds, we assessed the influence of different extraction steps on the sensitivity of X. axonopodis pv. phaseoli detection, including incubation time/temperature, vacuum extraction and centrifugation of seed extract. The results showed that vacuum extraction and centrifugation of seed extracts increased sensitivity, and the highest sensitivity was obtained with the 3-hour vacuum extraction at room temperature followed by centrifugation. These results were confirmed on seventy 1000-seed subsamples from 14 different naturally infested seedlots. Our results suggest that a 3-hour vacuum extraction followed by centrifugation would be a valuable modification of the current method approved by the International Seed Testing Association (ISTA).Based on DNA sequence information from RAPD fragments generated from X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans, real-time PCR methods were developed for detection and quantification of the pathogens in or on seeds. Assay specificity was tested against DNA of several Xanthomonas species and pathovars of X. axonopodis. None of the closely related Xanthomonas strains were amplified using this PCR assay. The detection limit of the TaqMan assay for purified DNA and cells was 20 fg and 20 CFU per 25 mul PCR reaction mixture, respectively. A linear model was developed for seed contamination level in relation to amplification cycles based on sensitivity tests on seed samples spiked with inoculated seeds. On naturally infested Phaseolus vulgaris seedlots, real-time PCR detection was more sensitive than the selective medium assay. Real-time PCR should be useful for rapid, highly sensitive and specific detection of these seedborne pathogens to ensure seed quality control and meet phytosanitary regulations. To my knowledge this is the first published real-time PCR assay developed for X. axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans.Seed transmission frequency and survival in storage were compared between both common blight pathogens from different geographic origins. Xanthomonas axonopodis pv. phaseoli var. fuscans isolates had a higher percentage of seed-seedling transmission than X. axonopodis pv. phaseoli isolates (P<0.001). Both variants reduced seedling emergence compared to the noninoculated control, and X. axonopodis pv. phaseoli var. fuscans isolates reduced seedling emergence more than X. axonopodis pv. phaseoli (P<0.001). The incidence of bacterial blight symptoms was higher in seedlings from X. axonopodis pv. phaseoli var. fuscans inoculated seeds than in seedlings from seeds inoculated with X. axonopodis pv. phaseoli (P<0.001). Real-time PCR showed that a higher percentage of seedlings were infected with X. axonopodis pv. phaseoli var. fuscans than X. axonopodis pv. phaseoli. PCR results also revealed symptomless infection of seedlings. Survival (population size) of both bacteria on stored seeds was monitored over time using real-time PCR. Survival did not differ significantly between the two variants and real-time PCR gave higher population size estimates than a culture plating test after three months in storage (P<0.05).