Prevalence of Copper Resistance among Foliar Bacterial Pathogens of Tomato in Tennessee

Foliar bacterial diseases are among the most important diseases of tomato. Bacterial spot is caused by four species: Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri, hereafter referred to as Xanthomonas spp. Bacterial speck is caused by Pseudomonas syringae pv. tomato and bacterial canker is caused by Clavibacter michiganensis subsp. michiganensis. Fixed copper products are relied upon extensively for control, due to a lack of effective and economical alternatives. Copper resistance in bacterial spot and speck pathogens has been reported worldwide. Copper resistance quite likely exists in Tennessee, but the extent has never been determined. The objectives of this study were to (i) optimize the conditions for in-vitro determinations of the level of copper sensitivity in Xanthomonas spp., P. syringae pv. tomato, and C. michiganensis subsp. michiganensis cultures in a protocol that would be practical for processing large sample numbers, and validation with bioassays; (ii) determine the incidence and distribution of copper resistance in Tennessee populations of these pathogens. Parameters selected for in-vitro copper resistance testing were sucrose peptone agar adjusted to pH 6.8 (Xanthomonas spp. and C. michiganensis subsp. michiganensis) or 6.2 (P. syringae pv. tomato), amended with 2-(N-morpholino)ethanesulfonic acid (20 mM) and CuSO4·5H2O (200 µ[micro]g/ml), and amended with 200 µ[micro]g/ml CuSO4·5H2O. After solidifying, media were promptly streaked with cells produced on solid agar, and incubated for three days. Bacterial growth was visually assessed on a 0 to 10 scale. For rapid identification, a multiplex PCR protocol was developed and used successfully to detect the three pathogens from symptomatic plants. When plants were co-inoculated with P. syringae pv. tomato and C. michiganensis subsp. michiganensis, the latter could not be detected. As a result, alternative methods of pathogen identification were employed in the field survey. A survey of 19 fields representing 195 acres in six Tennessee counties during the 2010 and 2011 growing seasons, 162 were identified as Xanthomonas spp., none as P. syringae pv. tomato, and four as C. michiganensis subsp. michiganensis. Copper resistance was detected in 95.1% of Xanthomonas spp. isolates and 4.9% were sensitive. All C. michiganensis subsp. michiganensis isolates were sensitive