Copper sensitivity in Tasmanian populations of the walnut blight pathogen Xanthomonas arboricola pv juglandis

Walnut blight, caused by Xanthomonas arboricola pv juglandis (Xaj), is a major concern for the walnut industry world wide, including Australia. The disease has been responsible for significant yield loss of more than 50%, especially in early leafing varieties of walnut in Tasmania. The disease causes necrotic lesions on all above ground parts of the tree that are green and succulent. Walnut blight is currently controlled in Tasmania by the application of copper containing bactericides as protective sprays at 7- 14 days intervals from bud burst. Copper is sometimes applied up to ten times per growing season, and this spray program does not always provide a satisfactory level of disease control. The major objective of this study was to estimate the range of copper sensitivity among a broad sample of Xaj isolated from blight lesions on walnut fruit from Tasmania. To achieve this objective, the identity of each bacterial isolate was determined by their phenotype on semi-selective media and by analysing their fatty acid profile by GCFAME (Gas Chromatograph Analysis of Fatty Acids Methyl Esters). The identity and genetic variation among bacterial isolates was investigated further by application of a DNA fingerprinting technique called rep-PCR. According to GC-F AME analyses, 32 of 45 isolates of bacteria from symptomatic walnut fruit appeared to be Xanthomonas arboricola. The frequent isolation of Xanthomonas arboricola from blight lesions on walnut fruit suggested that this organism was associated with the disease. The phenotypic characters of 67 strains of bacteria isolated from symptomatic walnut fruit were studied on two semiseletive media: Brilliant Cresyl Blue Starch (BS) medium and Succinate Quinate (SQ) medium. The semiselective nature of these media was revealed because some non Xanthomonads gave a positive reaction on both media and two isolates of Xanthomonas, putatively X. arboricola, did not appear to metabolise quinate on SQ media. Genetic variation was observed among isolates of Xaj from Tasmania and there was limited evidence to suggest these isolates could be distinguished from non-Xanthomonads using rep-PCR. Copper sensitivity was quantified for 37 isolates of Xanthomonas arboricola (putatively Xaj), isolated from blight lesions on walnut fruit from commercial orchards and home gardens in Tasmania. Each bacterial isolate was exposed to copper sulphate in vitro, at varying concentrations up to 1.2 mM or 76 ˜í¬¿g Cu2+/ml of liquid medium. All three isolates of Xanthomonas arboricola from home gardens and five isolates from commercial walnut orchards were highly sensitive to copper, with strain mortality occurring at :'.S 0.2 mM copper sulphate. Application of a quadratic model to the copper dose response curve enabled estimation of the lethal dose of copper sulphate for 50% mortality of bacterial cells (LD 50) and the minimum lethal dose (MLC). At least two isolates of Xanthomonas arboricola from commercial walnut orchards were considered copper tolerant with an MLC that was greater than 0.5 mM or 32 ˜í¬¿g Cu2+/ml copper sulphate. This is the first report of copper tolerant strains of the walnut blight bacterium from Australia and the southern hemisphere. The results highlight the urgent need to reduce inputs of copper in commercial walnut production by development of more sustainable solutions for managing bacterial blight.