مقايسه روش هاي مختلف رديابي باكتري Xanthomonas axonopdis pv. phaseoli در بذر لوبيا

COMPARISON OF DIFFERENT METHODS FOR DETECTION OF Xanthomonas axonopodis PV. phaseoli IN BEAN SEEDS

In recent years, bean common blight as a destructive disease has caused serious yield losses in Iran. Infected seed is known as the major source of primary inoculum and serves as a pathogen to disseminate elsewhere. Therefore, using pathogen-free seed is an important factor in disease management. In this study, capability of several detection methods including Indirect ELISA, Direct PCR, Bio-PCR and Ic-PCR were compared for monitoring Xanthomonas axonopodis pv. phaseoli in bean seeds collected from Markazi province fields. The extractions of seed samples soaked in washing buffer and distilled water separately were used for Indirect ELISA and PCR amplification with species specific pairs of primer X4c\X4e. In Ic-PCR, seed extracts were loaded in wells which were coated with immunoglobulin and the sample DNA after boiling was subjected to PCR. For Bio-PCR assay, aliquots of the extracts were plated onto semi-selective modified nutrient broth yeast extract agar. The growing colonies were suspended in sterile distilled water after boiling was applied for PCR amplification. The results indicated that sensitivity of Indirect ELISA was low and at least 105 cfu/ml was needed as a detection threshold. The results for direct PCR were not reproducible and Ic-PCR was found an expensive method. The Bio-PCR technique was considered as a reliable and specific method which was able to detect as little as 1 cfu/ml in seed extracts plated on semi-selective media. Comparing the results indicated that the Bio-PCR assay is suitable for sensitive and routine testing of seed samples of beans for the presence of X. axonopodis pv. phaseoli