For this experiment, plants were inoculated with inoculum concentration of OD540 = 0.05 (Exp. 1) and 0.1 (Exp. 2). Immediately after sampling, leaf

fragments were weighed and kept in a refrigerator (4°C) until analysis. The fragments were surface sterilized by dipping them in a 50% alcohol solution for 1 min, 2% sodium hypochlorite solution for 40 s, followed by three AG 14699 rinses in sterile deionized water. Leaf segments were ground using a sterile pestle and mortar with 0.85% saline solution (1 : 10, w/v). A total of 10 μl of each leaf macerate was serially diluted in glass vials containing 990 μl of sterile saline solution at 0.85% prepared with 0.02% Tween 80. Aliquots of 10 μl from each dilution series were spread on XTS-agar media (agar, 23 g/l; glucose, 5 g/l; cyclohexamide, 100 mg/ml; gentamycine, 10 mg/ml; and cephalexin, 10 mg/ml) (Schaad et al., 2001). The Petri dishes were kept in a growth chamber at 28°C for 4 days. The number of bacterial colonies was counted in each dilution series and data were transformed to log5 CFU g per leaf. In a separate experiment, the EL was determined according to the methodology described by Lima et al. (2002) with a few modifications. A total of 23 discs (≈8 mm in diameter) were collected from the fourth and fifth leaves from the main tiller from plants of each replication for each

treatment at 0, 1, 2, 3, 4, 6, 8, and 10 d.a.i. For this experiment, plants were inoculated with inoculum concentration of OD540 = 0.05 (Exp. 1) and 0.1 (Exp. 2). Leaf discs were thoroughly washed in deionized sterile water, and then left to float on 60 ml of deionized water in sealed glass vials for 4 h at 25°C. selleck kinase inhibitor The EL was estimated using a conductivity meter Sitaxentan (Tecnopon mCA-150, MS Tecnopon Instrumenação Científica, Brazil) and the values were expressed as the percentage of total conductivity, which was obtained after placing the glass vials in an oven at 90°C for 2 h. In a separate experiment, samples from the second, third, and fourth leaves from plants of each replication for each treatment were collected at 0, 3, 6, 9, and 12 d.a.i. Plants were inoculated with an inoculum concentration of OD540 = 0.05. Leaves were kept in liquid nitrogen

during sampling and then stored at −80°C until further analysis. A representative sample of 0.1 g of leaf sample from each replication and treatment was ground into a fine powder in a pestle and mortar with liquid nitrogen. The fine powder was transferred to a microcentrifuge tube, homogenized with 1.5 ml of 80% methanol, and extracted overnight on a rotary shaker (150 r.p.m.) at room temperature. The homogenate solution was protected from light oxidation by covering the microcentrifuge tube with aluminum foil. The dark-green methanolic extract was centrifuged at 12 000 g for 5 min, the supernatant was transferred to a new microcentrifuge tube and stored at −20°C. The residue was kept at −20°C for further determination of lignin and lignin-like phenolic polymers.