Detection and Identification of Brenneria nigrifluens, the Causal Agent of the Shallow Bark Canker of Walnut by, PCR Amplification

A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1–C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens . Several strains of B. nigrifluens were assessed with F1–C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies.