MATERIAL AND METHODS
Endophytic bacteria
Isolates from leaves and branches (Nunes, 2004), of Coffea arabica and Coffea robusta plants from Pedreira, Mococa, and Pindorama counties, State of São Paulo, Brazil (Table 1), were maintained in the culture colletion of the Laboratório de Microbiologia Ambiental, Embrapa Meio Ambiente, in sterile distilled water (Castellani, 1967). Forty bacterial isolates were evaluated regarding their capacity to inhibit the germination of H. vastatrix urediniospores, and 44 isolates were used to control coffee leaf rust in leaf discs, detached leaves, and seedlings of C. arabica, cv. Mundo Novo.
Urediniospore germination
The endophytic bacteria isolates were cultivated on nutrient agar medium (Peptone 5 g; meat extract 3 g; agar 15 g; distilled water 1000 mL) for 24 hours at 28 ± 2ºC, and then transferred with to slants containing sterilized, distilled water. Samples were then shaken vigorously to obtain a homogeneous cell suspension, which was standardized to an optical density of A550 = 0.1. Urediniospores of H. vastatrix, race II, were collected from coffee leaves containing lesions obtained from plants in "Centro de Café e Plantas Tropicais, Instituto Agronômico de Campinas", and stored in a container with sodium dichromate (relative humidity 52%; 7 ± 2ºC). Urediniospores were suspended in water at a concentration of 1.0 mg mL-1 using a magnetic stirrer for 5 minutes. A 15.0 µL aliquot of this suspension, and a 15.0 µL aliquot of the endophytic bacteria suspension were then transferred to microscope slide, mixed and enclosed within plastic boxes containing a layer of foam saturated with water, and sealed with glass plates to maintain high relative humidity. After incubation for six hours (22 ± 1ºC) in the dark, the germination was interrupted by adding 15.0 µL of lactophenol cotton blue dye onto each droplet, and examined under light microscope. The percentage of germinated urediniospores (10 fields at 200 ´ magnification) was calculated. Urediniospores with germ tubes of at least one half of the length of their larger diameter were considered germinated. Trials were set up in a completely randomized experimental design (n = 4). The experiment was repeated and the means were used for statistical analysis. Sterilized water and propiconazole (TiltÒ CE; 1.2 µL of the commercial product per mL of water) were used as controls.
Leaf discs
Discs of young and completely developed leaves of C. arabica cv. Mundo Novo plants were removed with a 2.0 cm diameter cork punch and placed into plastic boxes, abaxial surface facing up, over a layer of foam saturated with water (Eskes, 1989). Using a micro-pipet, 25.0 µL of the endophytic bacteria suspension were applied on the leaf discs, 72 and 24 hours before, after, and simultaneously with the same volume of H. vastatrix urediniospores suspension (1.0 mg mL-1). After inoculation, boxes were covered with glass plates and incubated in the dark for 24 hours. Then the boxes were maintained under 12h photoperiod, 500-1000 lux, 22 ± 2ºC, and approximately 100% relative humidity. The experiment was set on completely randomized design (n = 3), represented by nine leaf discs each. Severity of the disease was evaluated 30 days after inoculation, using a rating scale from 1 to 5, according to the percentage of leaf area with lesions (1 = 0%; 2 = 1-25%; 3 = 26-50%; 4 = 51-75%; and 5 > 75% of leaf area with lesions). The Waller-Duncan (a = 0.05) test was used to compare the lowest and the highest mean values of lesions percentage for each treatment.
Detached leaves
Ten isolates of endophytic bacteria used in this experiment were selected according to results observed in the two previous tests. The treatments consisted of a bacterial suspension (A550 = 0.1) sprayed on completely developed coffee leaves (C. arabica cv. Mundo Novo), 72 and 24 hours before, after and simultaneously with the inoculation of the urediniospore suspension. The coffee leaves were placed in plastic boxes, abaxial surface facing up, over a layer of foam saturated with water, covered with a glass plate and incubated as described. The experimental design was randomized blocks (n = 3), each replicate consisting of three leaves. Inoculation was performed using a sprayer attached to a compressor, pressure 10 lb in-2. Following inoculation, the boxes were covered and placed in the dark for 24 hours, at 22 ± 2ºC. Treatments were evaluated 21 days after inoculation, by counting the number of lesions per leaf. Means were compared by Tukey test (a = 0.05).
Coffee plants
The same endophytic bacterial isolates used in the detached leaves were used in this study. Coffee seedlings (C. arabica cv. Mundo Novo) susceptible to all H. vastatrix strains were obtained from "Centro de Café e Plantas Tropicais, Instituto Agronômico de Campinas", and transplanted into plastic pots containg 5 L of Red Yellow Latosol, sifted through 1.0 cm2 mesh sieve and mixed with 2.0 kg of lime, 5.0 kg of simple superphosphate, and 0.5 kg of potassium chloride per m3 of soil. The bacterial suspensions (A550 = 0.1) were manually sprayed to the foliage until runoff. The urediniospore suspension was applied with a sprayer attached to a compressor, pressure 10 lb in-2. After inoculation with the H. vastatrix urediniospore suspension (1.0 mg mL-1), plants were incubated in the dark for 48 hours at 22 ± 2ºC, 100% relative humidity, and then transferred to a greenhouse. Plants were irrigated daily and after 30 days the number of lesions per inoculated leaf was evaluated. Sterilized water was used as control. Trial was set up in a randomized blocks design (n = 3), with two plants per pot and, means compared by Tukey test (a = 0.05).
The most effective isolates were identified based on cell membrane fatty acid contents, analyzed in a gas chromatograph, using microbial identification software (MIDI, Sherlock® TSBA Library version 5.0, Microbial ID, Newark, DE, USA). Isolates with a similarity index of 0.6 or higher were considered positively identified.
Answers to all your Biology Questions
