Development of a PCR-based method for monitoring the status of Alcaligenes species in the agricultural environment

Abstract

To analyze the status of the genus Alcaligenes in the agricultural environment, we developed a PCR method for detection of these species from vegetables and farming soil. The selected PCR primers amplified a 107-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 1.06 pg of pure culture DNA, corresponding to DNA extracted from approximately 23 cells of Alcaligenes faecalis. Meanwhile, PCR primers generated a detectable amount of the amplicon from 2.2×102 CFU/ml cell suspensions from the soil. Analysis of vegetable phylloepiphytic and farming soil microbes showed that bacterial species belonging to the genus Alcaligenes were present in the range from 0.9×100 CFU per gram (or cm2) (Japanese radish: Raphanus sativus var. longipinnatus) to more than 1.1×104 CFU/g (broccoli flowers: Brassica oleracea var. italic), while 2.4×102 to 4.4×103 CFU/g were detected from all soil samples. These results indicated that Alcaligenes species are present in the phytosphere at levels 10-1000 times lower than those in soil. Our approach may be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.