Submitted to: Walnut Research Conference
Publication Type: Proceedings
Publication Acceptance Date: 1/1/2007
Publication Date: 1/1/2007
Citation: Mcclean, A.E., Sudarshana, P., Kluepfel, D.A. 2007. Real-time pcr detection and development of a bioassay for the deep bark canker pathogen, brennaria rubrifaciens. Walnut Research Conference.
Technical Abstract: Deep Bark Canker (DBC), caused by the bacterium Brennaria 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 along the trunk and larger branches that exude a bacterial-laden reddish brown sap. B. rubrifaciens produces a unique water-soluble red pigment called rubrifacine when cultured in the laboratory. Here we describe the new primer pair, BR-1 and BR-3 that amplify a unique 409bp region of the 16S rDNA sequence that facilitates the sensitive and specific detection of B. rubrifaciens. Using these primers in a realtime-PCR system we were able to detect as few as 8 B. rubrifaciens colony forming units (CFU). A survey of 11 antibiotics revealed that B. rubrifaciens is resistant to erythromycin and novobiocin at 10 mg/L and 30 mg/L respectively. Amending the cultivation medium with these antibiotics has improved the semi-selective cultivation of B. rubrifaciens on solid media. Both walnut cultivars, Hartley and Chandler, grown in tissue culture are susceptible to infection by B. rubrifaciens. With in 21 days after inoculation Hartley shoots turned necrotic and died. Chandler shoos exhibited a similar phenotype 10wk after inoculation. This latter finding will be useful in our search for Brennaria genes involved in pathogenesis and the identification of walnut genotypes resistant to deep bark canker.