Abstract
A real-time PCR assay (qPCR) based on TaqMan chemistry was developed to quantify Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybeans. Specific primers and probes were designed based on the sequences of the rpoD gene. In the assay, the gene from 61 X. axonopodis pv. glycines strains was amplified, but not from any other Xanthomonas species tested, various reference strains derived from soybeans, or 30 indigenous bacteria isolated from soybeans in Fukui Prefecture. For absolute quantification, a calibration curve was constructed based on a recovery test (Sawada et al., Plant Protect. 62: 611-617, 2008): a series of 1 g soybean seeds was spiked with 10-fold dilutions of the bacterial suspensions, and DNA from each 1-g series with a 10-fold dilution was extracted and subjected to qPCR. A strong linear relationship was found between the threshold cycle value and the bacterial density from 6×102 to 6×108 cfu per 1 g of soybean seeds; the detection limit might be close to 6×101 cfu per 1 g of seeds. This qPCR experimental system in conjunction with the following sampling procedure proved to be a reliable method for estimating the degree of contamination in seeds: (1) crush 1 kg of test seeds and mix well, (2) sample 1 g of these crushed seeds, and repeat this procedure 10 times, (3) extract DNA from every 1 g of crushed seeds sampled and perform the qPCR experiment three times to derive quantitative values, (4) average the 30 quantitative values obtained, and use this value to determine the degree of contamination of the original seeds tested.
