Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2008
Publication Date: 5/1/2008
Citation: Mcclean, A.E., Sudarshana, P., Kluepfel, D.A. 2008. Enhanced detection and isolation of the walnut pathogen brennaria rubrifaciens: causal agent of deep bark canker. European Journal of Plant Pathology. 10.1007/s 10658-008-9308
Technical Abstract: 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 younger 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 primers sets; GSP1F-GSP1R and GSP2F-GSP2R. A third primer pair, BR1-BR3 was designed from the 16S rRNA gene. No amplification was observed using the three primer pairs with species from the following bacterial genera: Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Rhizobium. In addition, no amplification was observed with Brenneria alni, Brenneria nigrifluens, Brenneria quercina, or Brenneria salicis. All three DNA primer sets detected B. rubrifaciens in spiked sterile soil and infiltrated walnut leaf tissue. PCR detection limits for BR, GSP1, and GSP2 primer pairs were 254, 254, and 2.54 x 104 colony forming units (CFU) respectively. Real-time PCR detection limit for BR primers was 20 fg and 8 CFU. The differential medium, yeast extract dextrose calcium carbonate agar 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.