Submitted to: Walnut Research Conference
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2006
Publication Date: August 1, 2006
Citation: Mcclean, A.E., Sudarshana, P., Kluepfel, D.A. 2006. Development of a pcr-based method for the detection of brennaria rubrifaciens; the causal agent of deep bark canker of walnut. Walnut Research Conference. Technical Abstract: Deep Bark Canker (DBC), caused by the bacterium Brenneria rubrifaciens (previously known as Erwinia rubrifaciens), afflicts English walnut cultivars and is characterized by late onset of symptoms in trees greater than 15 years old. These symptoms include deep bleeding vertical cankers throughout the tree that exude a bacterial-laden reddish brown sap. When cultured in artificial media, B. rubrifaciens produces a unique water-soluble red pigment of unknown function called rubrifacine. Given the unique nature of this pigment we choose to develop PCR primers specific to one of the genes involved in rubrifacine biosynthesis. Twenty-two transposon mutants deficient in rubrifacine production were generated from a pool of 603 mutants. Gene specific primers (GSP1) were designed to amplify a 233 bp region around the transposon insertion site in pigment-minus mutant 61. GSP1 primers failed to generate an amplification product from the genomic DNA isolated from closely related Erwinia and Brenneria species or from the 15 species in 6 different plant-associated bacterial genera that were examined. The limits of PCR-based detection, with GSP1 primers, was determined using both pure culture isolates of B. rubrifaciens and walnut leaves infiltrated with B. rubrifaciens. Genomic DNA from pure culture B. rubrifaciens was serially diluted and used as target DNA with GSP1 primers. The limit of PCR detection was 0.27 'g of B. rubrifaciens DNA which represents approximately 61 B. rubrifaciens cells. GSP1 primers also were able to detect as few as 14 B. rubrifaciens cells in 1 mg of bacteria-infiltrated walnut leaf tissue. These primers provide a new specific and sensitive tool for the detection of B. rubrifaciens strains in both sap and walnut plant tissue.