Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Abstract

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media-based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10 1 to 10 5 CFU per seed; one contaminated seed was mixed into 1000-seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).

Rapid Detection and Typing of Xanthomonas campestris pv. vesicatoria by Pyrosequencing

“…campestris pv. campestris , were previously shown to be infeasible to be differentiated by conventional technologies (He and Munkvold ). Using the pyrosequencing primers, strain‐specific sequences between 571 and 610 bases for respective Xanthomonas species were generated around an hour.…”

“…holcicola, X. badrii and X. campestris pv. campestris, were previously shown to be infeasible to be differentiated by conventional technologies (He and Munkvold 2012).…”

“…vesicatoria but pathovars of X . campestris as well as non‐pathogenic strains of Xanthomonas display a positive signal in this technique (He and Munkvold ). Enzyme‐linked immunosorbent assays (ELISA) and DNA amplification have been developed to detect X. campestris pv.…”

“…The current detection and inspection methods, for instance semi-selective media, include two enrichment steps and subsequent biochemical identification; however, not only X. campestris pv. vesicatoria but pathovars of X. campestris as well as non-pathogenic strains of Xanthomonas display a positive signal in this technique (He and Munkvold 2012). Enzyme-linked immunosorbent assays (ELISA) and DNA amplification have been developed to detect X. campestris pv.…”