Enhanced detection and isolation of the walnut pathogen Brenneria rubrifaciens, causal agent of deep bark canker

Deep bark canker (DBC) of walnut is caused by the bacterium Brenneria rubrifaciens which produces the red pigment rubrifacine. This disease of English walnut trees, is characterized by deep vertical cankers which exude sap laden with B. rubrifaciens. Although DBC is not observed on young trees, it is hypothesized that B. rubrifaciens is present in host tissue years before symptom development. Therefore, a sensitive technique would be useful in detecting B. rubrifaciens in asymptomatic trees. Tn5 mutants deficient in rubrifacine production (pig(-)) were generated and DNA sequences from pig mutants were used to design two primer sets; GSP1F-GSP1R and GSP2F-GSP2R. A third primer pair, BR1-BR3 was designed from the 16S rRNA gene. The three primer pairs did not amplify the diagnostic bands from members of the following bacterial genera: Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Rhizobium. In addition, no amplification was observed using DNA from the following Brenneria species, alni, nigrifluens, quercina, or salicis. All three DNA primer sets detected B. rubrifaciens in spiked greenhouse soil and infiltrated walnut leaf tissue. PCR detection limits for BR, GSP1, and GSP2 primer pairs were 254, 254, and 2.54 x 10(4) colony forming units (CFU) respectively. Real-time PCR detection limit for BR primers was 8 CFU. The differential medium, yeast extract dextrose calcium carbonate agar (YDCA) was amended with novobiocin, and bacitracin, to enhance isolation from environmental samples. The improved detection and isolation methods described here will facilitate examination of B. rubrifaciens ecology under both nursery and orchard conditions.