Pseudomonas strains were isolated from roots of pepper, strawberry, cabbage, cauliflower, broccoli, broad bean and radish fields in the main cultivation areas of Aydin province of Turkey. For bacterial isolations, plant roots were washed under running tap water, and 3 g of roots from each plant were blended in a stomacher (BagMixer^®, Interscience) in 27 mL of physiological saline (0.9% NaCl, pH 7.0) for 1 min. Aliquots of suspensions were plated on King’s B medium (King et al., 1954) and incubated at 25 °C for 48 h. Single bacterial colonies were transferred to King’s B medium for purity, and cultures were cryopreserved with 15% glycerol at −80 °C.

Pseudomonads isolates were tested for their ability to produce antifungal substances against P. capsici using a dual-culture in vitro assay on potato dextrose agar (PDA) plates. Standardized bacterial suspensions (3 × 10⁸ cells/mL) were prepared from overnight cultures of Pseudomonas isolates on King’s B medium. One 10 μL droplet of bacterial suspension was applied to each of four points symmetrically placed on sites at equal distances (2.5 cm) from the center of each PDA plate. After 24 h of incubation at 24 °C, a single 10 mm diameter mycelial disc was placed in the center of the plate. As a control, a disc of P. capsici was grown on a PDA plate. The diameter of each fungal colony was measured along two perpendicular axes after a 10 day incubation at 24 °C in darkness, and the percentage inhibition of mycelial growth was calculated.

Antagonistic Pseudomonads were later assayed for biosurfactant production with the mineral salt-CTAB-methylene blue agar plate method originally described by Siegmund and Wagner (1991). Ten microliters of bacterial suspension (10⁸ cells/mL) was pipetted onto the center of one square (1.5 × 1.5 cm) of the 4 × 4 grid drawn on the bottom of the plate containing mineral salt-CTAB-methylene blue agar. The plates were then incubated at 30 °C for 48 h. Biosurfactant activity was evaluated by measuring the diameters of the blue halos around the colonies after incubating the plates at 4 °C for 24 h. The Pseudomonas strains selected in this work and relevant information on their identities, based on fatty acid analysis (FAME) are listed in Table 1.

The ability of selected Pseudomonas strains to solubilize tricalcium orthophosphate (TCP) was tested on TCP medium (De Freitas et al., 1997). Ten μL of bacterial suspension (10⁸ cells/mL) from each of 11 Pseudomonas isolates was dropped onto the center of one square (1.5 × 1.5 cm) of the 4 × 4 grids drawn on the bottoms of the plates containing TCP. After incubation at 25 °C for 7 days, the presence of a clear zone around the colony indicated phosphate-solubilizing activity of the bacterial isolates, and the diameters of the clear halos were measured in millimeters.

Production of indolic compounds by bacteria was examined using a colorimetric method described by Patten and Glick (2002). Pseudomonas isolates was first cultured in 5 mL tryptic soy broth at 25 °C overnight. To synchronize the cells of each isolate, two consecutive 24 h transfers were made with 20 μL of inoculum, and then 20 μL aliquots were transferred into 5 mL of DF salts minimal media (Dworkin and Foster, 1958), supplemented with the following concentrations of L-tryptophan 0, 0.1, 0.5, 1, 5, 10, 50 and 100 μg/mL. After incubation for 42 h, the bacterial cells were then removed from the culture medium by centrifugation at 5,500 g for 10 min. A 1 mL aliquot of each supernatant was mixed vigorously with 4 mL of Salkowski’s reagent (Gordon and Weber, 1951) and allowed to stand at room temperature for 20 min before the absorbance at 535 nm was measured. The concentration of indolic compounds in each culture medium was determined by comparison with a standard curve.

Production of hydrogen cyanide (HCN) was determined using a modification of the procedure of Bakker and Schippers (1987). The Pseudomonas isolates were grown in screw-cap test tubes containing 5 mL of King’s B broth supplemented with 4.4 g/L of glycine, with filter paper strips soaked in picric acid solution (0.5% picric acid and 2.0% Na₂CO₃) placed in the cap of each tube. The cap of the tubes were sealed with Parafilm^® and incubated in an orbital shaker at 20 °C for 96 h. Microbial production of cyanide was determined by a color shift from yellow to brown in the filter paper (Castric, 1975). Reactions were scored as weak (yellow to light brown), moderate (brown), or strong (reddish brown) for each of the Pseudomonas strains.

Pseudomonas strains were grown on universal siderophore detection medium CAS agar plates (Schwyn and Neilands, 1987). Droplets of 10 μL of bacterial suspensions (10⁸ cells/mL) were spotted onto CAS agar plates with 4 × 4 grid patterns. Siderophore production was calculated as the diameter of the orange halo around the colonies as measured after 72 h of incubation at 25 °C.

The ability of the Pseudomonas strains to produce antibiotics was analyzed by PCR amplification of a phlD-related gene using the primer pair B2BF and BPR4 (McSpadden Gardener et al., 2001). Template DNA was prepared by boiling 200 μL of bacterial suspension in MilliQ water for 13 min followed by immediate chilling on ice, and then centrifugation at 13,000 g for 3 min at 4 °C. Two microliters of the supernatants was used directly in 20 μL PCR mix. The generation of a 629 bp product indicated the presence of the phlD gene.

The abilities of individual Pseudomonas strains to produce extracellular lytic enzymes, such as chitinase, protease and cellulase, were determined by spot inoculation of 10 μL of bacterial suspension prepared from 24 h old nutrient agar cultures on substrates containing appropriate media (Berg et al., 2000). Chitinase activity was assayed by inoculating chitin-agar plates. The clear halos, indicating enzymatic degradation of chitin, were measured after five days of incubation at 30 °C. Screening for cellulase producers was done on carboxymethylcellulose agar (Kasana et al., 2008). After incubation at 28 °C for 48 h, the plates were flooded with Gram’s iodine for 3 to 5 min, and they were observed for zones of clearance around the colonies. Protease activity, indicated by casein degradation, was determined from clearing zones in skim milk agar after 5 days of incubation at 20 °C (Krechel et al., 2002).

For greenhouse assays, Pseudomonas strains were applied as a soil drench. Bacterial suspensions from 24 h old bacterial streak cultures adjusted to 1×10⁹ cfu/mL with sterilized distilled water were used for inoculation. One highly virulent single-spore culture of P. capsici strain Pc-101 isolated from pepper plants in Aydin province was used in all experiments. The fungus was routinely stored as agar plugs in sterile distilled water at room temperature and inoculum was prepared using a method of Kurze et al. (2001). Eight, 6 mm-diameter mycelial plugs from one-week-old cultures of P. capsici were used to inoculate a 250 mL mixture of wheat bran: vermiculite (1:1) containing 30 g of soybean flour and 84 mL of distilled water. This mixture was incubated at 22 °C for 28 days and incorporated into an autoclaved soil:sand: peat (1:1:1) mixture at a final concentration of 1/100 (v/v).

Two experiments were performed to determine the effectiveness of the selected Pseudomonas strains for suppression of Phytophthora blight of pepper with artificial inoculation under controlled greenhouse conditions by comparing the efficiency of acibenzolar-S-methyl (ASM) (Bion^® 50 WG, Syngenta Crop Protection Inc.) and acibenzolar-S-methyl (4%) + mefenoxam (40%) (Bion MX^® 44 WG, Syngenta Crop Protection Inc.). Two-week-old pepper (cv. California Wonder) seedlings were transplanted into 10 cm-diameter (500 ml) plastic pots containing 380 ml of autoclaved soil:sand:peat (1:1:1) mixture. Plants were incubated at 25 ± 2 °C with a 14-h photoperiod and fertilized once a week with liquid fertilizer (Sheffer 16-8-24 N-P-K plus micronutrients).

In the first experiment, the six Pseudomonas strains (6L10, 6ba6, 3ss9, mbj, 6ba2 and 3k9) were selected from 11 biosurfactant-producing Pseudomonas isolates by comparing their biocontrol activities. Twenty-five milliliters of bacterial suspension, prepared as described above, was applied by soil drench at the base of the stem of each pepper plant immediately after transplanting. Chemicals were applied three weeks after transplanting by thoroughly spraying pepper plants with 25 mL of a solution of acibenzolar-S-methyl (0.1 μg a.i./mL) and acibenzolar-S-methyl + mefenoxam (0.2 mg product/mL) with the excess fungicide running into the potting mix. Plants were also treated with 25 mL of sterile distilled water by soil drench for the untreated control. After 3 days of chemical treatments, when the plants were about 5-week-old, 4 mL of Phytophthora inoculum, prepared as described above, was mixed homogeneously with 20 mL of autoclaved soil:sand:peat (1:1:1) mixture, and the resulting inoculum was mixed thoroughly and carefully with the upper 1−2 cm of soil in each pot of all treatment plots, avoiding injury to lateral roots of the plants. The pots were watered immediately after oomycete inoculation. Three weeks after pathogen inoculation, the severity of symptoms was assessed in terms of the mean disease severity index (DSI) of plants with a scale where 0 = healthy plant; 1 = older leaves wilted, with brownish lesions beginning to appear on stems with slight stunting; 2 = all leaves wilted except top two or three, with 50% stunting; 3 = all leaves wilted and turned light tan, with 51 to 80% stunting; and 4 = leaves defoliated and whole stem turned dark brown and withered, with severe stunting (dead plant). Reduced DSI compared to untreated control plants was used as an indicator of biological or chemical control efficacy.

In the second experiment, four Pseudomonas strains (6L10, 6ba6, 3ss9 and mbj) that caused a significant reduction in Phytophthora blight severity in the first experiment were applied for alone and in combination with olive oil by following the same procedure as in the first experiment. The treatments consisted of four bacterial inoculations with and without olive oil, ASM and ASM + mefenoxam, olive oil and uninoculated control. In the case of olive oil, 5 mL was applied by soil drench at the base of the stem of each pepper plant and then bacterial inoculum was applied as described above. The second experiment was evaluated in the same manner as in the first.

In vitro tests including inhibition of mycelial growth of P. capsici, production of biosurfactants, indolic compounds, HCN, siderophore, extracellular enzyme activities and phosphate solubilization of bacteria were performed in a completely randomized design with 3 replicates. The pot experiments were established in a randomized complete block design with three replicates. Each treatment has ten pots in each replicate block. All the tests and in vivo experiments were repeated at least once. Data were subjected to analysis of variance with the general linear models procedure of JMP statistical software (Mac version 9.0.2, SAS Institute Inc., Cary, NC, USA). Mean separations were determined by Tukey’s HSD (honestly significant difference) test at P = 0.05.

From the one hundred one isolates obtained from the roots of different plants, only twenty four Pseudomonas isolates showed significant inhibition of vegetative growth of P. capsici in vitro. Fifteen of these isolates had significantly higher activity, with more than 50% inhibition on PDA plates while the remaining isolates had between 3.6% and 49.3% inhibition. Of the 24 Pseudomonas isolates, eleven produced biosurfactants on CTAB-methylene blue indicator plates containing glycerol as the carbon source (Table 1). Among the 11 isolates with biosurfactant-producing ability, strains 6ba6, 6ba2, 3ss9, 6L10 and 3k9 showed high production of biosurfactants, while the other six strains (6ba3, 6t14, ke, 6L14, mbj and 3tg8) demonstrated low biosurfactant production. Fatty acid methyl esters (FAME) analysis identified strains as Pseudomonas fluorescens (6ba6, 3ss9, 6L10, ke, 3k9, 6L14 and mbj) and Pseudomonas sp. (6ba2, 6ba3, 6t14 and 3tg8) with a similarity index of 0.80 or higher (Table 1).

Of the 11 Pseudomonas isolates assessed for inorganic phosphate solubilization, only 10 solubilized Ca₃(PO₄)₂ in the qualitative P-solubilization plate method, and 11 isolates produced indolic compounds from L-tryptophan in vitro. Among the 11 strains of Pseudomonas tested for HCN production, only strains 6ba2, 3ss9, 6L10, ke, 3k9, 6L14 and mbj showed high production of HCN. The other four strains (6ba6, 6ba3, 6t14, 3tg8) produced less HCN. All isolates produced siderophores on CAS medium, and the capacity for siderophore production was found to be highly dependent on the antagonistic potential of each Pseudomonas isolate as in Table 1. Maximum siderophore production was recorded in Pseudomonas isolates 6ba6, 6ba2, 6ba3 and 6t14. Eleven isolates produced a 629-bp DNA product in a PCR-based assay using a primer set that amplifies the phlD gene, which encodes the antifungal agents. Of the eleven isolates, four of which had strong protease activity (6ba6, 6ba2, 6L10 and 6L14), and 5 of which had weak protease activity (6ba3, 3ss9, ke, 3k9 and mbj) while the remaining two (6t14 and 3tg8) exhibited no protease activity in an in vitro plate assay. None of the biosurfactant producing Pseudomonas isolates showed cellulase or chitinase activity in vitro.

The five high biosurfactant-producing strains (6ba6, 6ba2, 3ss9, 6L10 and 3k9) and mbj were selected for evaluation of biocontrol activity in the greenhouse.

Pepper plants in the first and second experiments began showing symptoms of infection, including wilt, stunting and lesions at the bases of the stems, seven days after inoculation with P. capsici. In both experiments, all treatment groups (ASM, ASM + mefenoxam, and all tested bacterial isolates with and without olive oil) significantly reduced disease severity compared to the untreated control (Table 2). When analyzing the results obtained in both experiments, it was found that the most effective treatment for inhibiting the development of lesions on pepper plants inoculated with P. capsici was foliar spraying of ASM + mefenoxam at 0.2 mg product/ml.

In the first experiment, the six biosurfactant producing bacterial isolates applied by soil drenching significantly (P < 0.05) reduced disease severity of Phytophthora blight on pepper compared to the untreated control (Table 2). Among these six isolates, isolates 6L10, 6ba6 and 3ss9 provided significant control of the disease, by 61.3, 61.3 and 48.4%, respectively. The other three isolates (mbj, 6ba2 and 3k9), including foliar application of ASM, were less effective, with the control ranging from 29 to 38.7%. Foliar applications of ASM at 0.2 mg a.i./mL were phytotoxic, causing the edges of leaves to curl up. Pseudomonas isolates 6L10, 6ba6, 3ss9 and mbj were selected as potentially effective antagonists for the second greenhouse experiment.

In the second experiment, Pseudomonas isolates 6L10, 6ba6, 3ss9 and mbj were evaluated separately or in combination with olive oil for efficacy against P. capsici under greenhouse conditions. When compared with the untreated control, the treatments of the four bacterial isolates applied as a soil drench or in combination with olive oil significantly reduced the severity of the disease. However, the treatments with individual bacterial isolates combined with olive oil consistently resulted in significantly (P > 0.05) lower disease severity than the same isolates applied alone. Among the treatments that included olive oil, isolates 6ba6, 6L10 and 3ss9, and to a lesser extent mbj, significantly reduced disease severity by 81.1, 78.4, 75.7, and 56.8%, respectively (Table 2). The same bacterial isolates applied without olive oil, as well as the treatment with olive oil alone, also had a significant but smaller effect on disease reduction compared to the untreated control. Similarly, foliar application of ASM (0.2 mg a.i./mL) was less effective, with control efficiencies of 29.7%, but the same phytotoxicity symptoms recurred in the second experiment.