Characterization of resistance response to pathogens is a fundamental strategy in plant breeding programmes. Thus, in this study, we developed an effective inoculation method for resistance tests of bacterial halo blight (BHB), caused by Pseudomonas syringae pv. garcae on coffee. Firstly, aggressiveness of eight selected bacterial strains as well as seven mixed strains was assessed on coffee seedlings of Mundo Novo IAC 376‐4 cultivar. Two experiments were conducted comparing three inoculation techniques: initially, we compare the methods of sprinkling and multiple needles on four cultivars of Coffea arabica; later, four cultivars and seven genotypes were compared for the multiple needles method and the abrasion technique. Severity was evaluated according a disease rating scale (DRS), considering either leaf surface area affected for sprinkling, or affected inoculated area for inoculation by multiple needles and abrasion. The area under the disease progress curve of disease (AUDPC) was calculated considering weekly evaluations of disease from seven until 42 days postinoculation (DPI). The standard deviation (SD), coefficient of variation (CV) and confidence interval (CI) variables were used to denote the effectiveness of the methods. According to the results, strain IBSBF 1197 was the most aggressive, and three other strains showed high aggressiveness. All inoculation methods were able to discriminate the resistance response to BHB, wherein the sprinkling method was less efficient than multiple needles and abrasion technique was more efficient than multiple needles. In addition, disease evaluations at 14 DPI showed a high correlation coefficient with area under AUDPC at 42 DPI, validating the early selection to BHB. Results of inoculation methods indicated the abrasion on first pair of leaves, together with evaluations on fourteenth DPI, as the most promising technique for early selection on coffee breeding to BHB.

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In 2005, a sample of carrot seeds var. Kikuyo imported from Japan was submitted to the Instituto Biológico for bacteriological phytosanitary certification. Circular, convex and creamy colored colonies were obtained. These isolates produced a green fluorescent pigment on King's B medium and incited a hypersensitivity reaction in tobacco leaves. They were oxidase and arginine dihydrolase negative, did not produce levan but caused soft rot in potato slices and were identified as Pseudomonas viridiflava. Artificial inoculations by infiltration of bacterial suspensions at the crown area or by placing a drop of bacterial suspension on this area and pricking with a sterile needle, produced light to dark brown areas around the injured points 5 days after inoculation. The bacterium was then re-isolated. Although this bacterium could be an opportunistic pathogen and was isolated from asymptomatic samples, eventual losses in the field and postharvest storage is possible.

Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 -Phytobacteria Culture Collection of Instituto Biológico -IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl 2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test. KEYWORDS

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