Bacterial leaf spot of coffee caused by Pseudomonas syringae pv. tabaci in Brazil

Abstract

Coffee (Coffea arabica) is widely planted and an economically important crop in Brazil both for domestic consumption and export. During 2006 a new disease affected coffee seedlings cv. Catuai in a commercial nursery located in Arandu county, State of São Paulo. Disease incidence was estimated to be about 1–2% of seedlings. Initially small brown lesions were observed on leaves and then becoming black and angular; alternatively, there were irregular lesions surrounded by a large yellow halo, which sometimes coalesced. These symptoms were similar to those caused by Pseudomonas syringae pv. garcae or Burkholderia andropogonis, pathogens of coffee (Amaral et al., 1956; Rodrigues-Neto et al., 1981). From diseased tissues fluorescent pseudomonad bacteria were isolated on King’s B medium. Colonies were creamy white, rounded, with irregular margins. The isolates selected (IBSBF 2240, 2241 and 2249) were positive for levan production and tobacco hypersensitivity; and negative for oxidase, protopectinase and arginine dihydrolase (LOPAT group 1a) (Lelliott et al., 1966). Also, the bacterial strains produced acid from D-mannitol, inositol, D-sorbitol and erytritol but not from adonitol. D(-) tartrate and L-lactate were not utilised. Ice nucleation was negative. For comparison, the type and reference strains of P. syringae pv. garcae (IBSBF 248P), P. syringae pv. syringae (IBSBF 281T) and P. syringae pv. tabaci (IBSBF 1972P) were included in the identification assays. Biochemical tests were applied according to Braun-Kiewnick & Sands (2001) and molecular tests were based on PCR-RFLP of the hrp L gene of P. syringae pv. syringae. Pathogenicity of the three strains was confirmed by spraying bacterial suspensions (approximately 108 cfu mL−1) on healthy leaves of coffee seedlings previously wounded with a sterile needle and then covered with transparent plastic bags for 3 days. Control plants were treated with sterile distilled water. After 2 weeks, lesions developed only on inoculated leaves and identical bacteria to those inoculated were re-isolated. In molecular tests, the hrp gene was employed using the primers pshrp 1F (5′-CTCAGA/GGCGTTCATC/TCA-3′) and pshrp 2R (5′-TCAGGCC/TAACGGGTCA/TATCT-3′). In PCR-RFLP using AluI, HaeIII, HinfI, HpaII and TaqI restriction endonucleases, the isolated strains showed identical profiles with P. syringae pv. tabaci. Based on biochemical and molecular tests the pathogen was identified as P. syringae pv. tabaci. This is the first report of this species causing disease on coffee.