PCR primers for identification of opine types of Agrobacterium tumefaciens in Japan

Abstract

The polymerase chain reaction (PCR) is a rapid, precise method for detecting and identifying pathogenic bacteria. In addition to the published primers for identification of Agrobacterium tumefaciens up to species level, two sets of primers were designed to identify the nopaline and octopine types of Agrobacterium tumefaciens. The RBF-RBR primer set designed based on the nopaline type T-DNA right border detected the nopaline type A208 and R225f strains, and the ocsF-ocsR primer set derived from the ocs gene of the octopine type A. tumefaciens detected the octopine type A348 strain. After polymerase Chain reaction (PCR) amplification by the RBF-RBR primers, the A208 and R225f strains could be differentiated from each other by restriction fragment length polymorphism digestion using the restriction enzymes DraI and XbaI. Multiple colonies can be screened at one time in a single PCR tube with satisfactory efficiency, thereby allowing rapid detection of pathogenic A. tumefaciens. Following a rough screening by classical biovar medium and α-methyl-d-glucoside medium, the developed PCR system was introduced to identify isolates collected from soil and crown gall samples. Of 42 isolates determined to be A. tumefaciens, 7 were found to be octopine type; all the rest were R225f type.