SAWADA, H.1 * (2015). Simultaneous identification of phytopathogenic Rhizobium species (former Agrobacterium species) using multiplex colony-direct PCR. Jpn. J. Phytopathol. 81: 332-340.The genus Rhizobium includes two plant pathogens, crown gall and hairy root bacteria. Among the existing species of the genus, the following six species have at least either of these two pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Of these six species, four species (R. radiobacter species complex, R. rhizogenes, R. vitis, and R. rubi) were the focus of this study to develop a method for their simultaneous identification by means of a multiplex colony-direct PCR. Respective species-specific primer sets were designed based on sequences of the recA or pyrG genes. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as an identification tool was then validated using the Rhizobium species containing pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from species-specific amplifications (R. radiobacter species complex [245 bp], R. vitis [319 bp], R. rubi [420 bp], or R. rhizogenes [503 bp]) and the other (780-784 bp) from 16S rDNA as the internal control, were stably obtained from all the strains belonging to the targeted four species. When the other strains were used, only the fragment derived from 16S rDNA was amplified. Therefore, this method is useful for the rapid identification of the four Rhizobium species (R. radiobacter species complex, R. rhizogenes, R. vitis, and R. rubi), which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies.