Pathogenic bacterium
The virulent isolate of C. michiganensis subsp. michiganensis (Cmm7) was obtained from the Department Plant Pathology, Assiut University, Egypt. This bacterium was previously isolated and identified, using biochemical methods (Holt et al. 1994). It was grown on yeast peptone glucose agar medium (YPGA) at 27 ± 1 °C for 4 days, (YPGA; [l−1) 10 g peptone, 5 g yeast extract, 20 g dextrose, 15 g agar). For inoculation of tomato plants, Cmm7 cells were cultured in YPG liquid medium at 27 ± 1 °C for 72 h with shaking at 150 rpm. The bacterial cell suspensions were centrifuged (10,000×g for 8 min), the bacterial cells re-suspended in a tap water, and density adjusted to 2 × 108 cfu ml−1.
Antagonistic bacteria
Ten bacterial isolates (BCAs) were recovery from the rhizosphere of healthy tomato plants grown in a field at Assiut, Egypt. The BCAs were maintained on slopes of King’s B medium (KB; [l−1] 20 g proteose peptone, 1.5 g K2HPO4, 1.5 g Mg2SO4.7H2O, 20 g agar) and Luria-Bertani broth (LB; [l−1] 10 g tryptone, 5 g yeast extract, 10 g NaCl, 20 g agar), respectively, stored at 4 °C. The activity of 10 BCAs against Cmm7 was tested, using the agar well diffusion method, after Abo-Elyousr and El-Hendawy (2008). The cell suspension of Cmm7 was grown over the surface of KB medium. The Petri dishes left to dry and then 0.05 ml of an overnight culture of the isolated bacteria was placed into 9 mm wells. The Petri dishes were then incubated for 2 days at 27 ± 1 °C before examination for inhibition zones. The experiment was repeated twice with four replicates per treatment. The bacterial isolates, which showed a high antagonistic ability against the pathogenic bacterium, were selected and identified according to their morphological and cultural biochemical tests (Schaad 1988 and Holt et al. 1994).
Treatment of tomato plant and disease evaluation under greenhouse conditions
Tomato plants
Tomato seeds cultivar Super Marmalade were provided by the Department of Vegetable, Assiut University, Egypt. The seeds were sown in 20-cm-diameter clay pots, containing a mixture of sand and soil (1.4 w w−1) at 2 kg pot−1. All pots were placed in greenhouse at 30 ± 5 °C, 68–80% RH, with 11 h of natural light and 13 h of darkness daily, and the plants were watered as required.
Preparation of talc-based formulation of BCAs
Four bacterial isolates were selected to be formulated, based on their activity against the pathogen in vitro. To prepare the bacterial control agent (BCA) formulation, the method of Vidhyasekaran and Muthamilan (1995) was adopted. Briefly, the isolate of BCA was grown on a liquid KB medium at 27 ± 1 °C on a rotary shaker at 150 rpm for 48 h. A 400-ml aliquot of the bacterial suspension containing 108 cfu ml−1 was mixed with 1 kg of the talc powder (dry-sterilized at 105 °C for 12 h), 15 g CaCO3 (to adjust the pH to 7), and 10 g carboxymethylcellulose. After drying the formulation overnight under sterile conditions, it was packed into polypropylene bags.
Inoculation of tomato plant and disease evaluation
Tomato plants were removed from the pots, and the roots of plants were removed at 3 cm from the tips, using sterilized scissors. Roots with the adhering soil were placed in a suspension of Cmm7 (2 × 108 cfu ml−1) for 5 min. The plants in control treatments were treated with a tap water at the same time. After that, the plants were planted again in the original pots. The inoculated plants were placed under greenhouse conditions and watered with a tap water when needed. One month after inoculation, the vascular browning was evaluated, using a rating system from 0 to 3 according to Hassan and Buchenauer (2008).
The vascular browning index was calculated as follows:
$$ \mathrm{Vascular}\ \mathrm{browning}\ \mathrm{index}\ \left(\%\right)=\frac{\mathrm{Sum}\ \mathrm{of}\ \mathrm{ratings}\ \left(0-3\right)\ \mathrm{scale}}{\mathrm{Maximum}\ \mathrm{possible}\ \mathrm{score}\times \mathrm{number}\ \mathrm{of}\ \mathrm{interlodes}\ \mathrm{evaluated}}\times 100 $$
Effect of BCAs on seedling vigor and seed germination in vitro
Tomato seeds were immersed in a bacterial suspension containing 1 × l08 cfu ml− 1 of each BCA and, as the controls; seeds were treated by the pathogen and water, respectively. All tubes were gently shaken for 1 h and then blot-dried, plated on wet blotters, and the germination tested by the filter paper method. A set of each treatment (containing 50 seeds) was replicated 3 times. The Vigor Index of the seeds calculated according to Abdul Baki and Anderson (1973):
$$ \mathrm{Vigor}\ \mathrm{Index}=\left(\mathrm{mean}\ \mathrm{shoot}\ \mathrm{length}+\mathrm{mean}\ \mathrm{root}\ \mathrm{length}\right)\times \mathrm{percentage}\ \mathrm{of}\ \mathrm{germination} $$
Effect of treatment with BCAs as a suspension or formulation on disease severity
Treatment with BCAs
Four bacterial control agents (BCAs) (Bacillus subtilis, B. amyloliquefaciens, Pseudomonas fluorescens, and P. aeruginosa) were used at the concentration of 2 × 108 cfu ml−1, and the formulation was used at 15 g per kg−1 seeds. Pathogen inoculation was carried out as mentioned above in “inoculation of tomato plants and disease evaluation.” All pots with the treated and inoculated seedlings were placed under the greenhouse conditions as described previously, and the disease severity was recorded 1 month after inoculation. The experiment was repeated twice with four replicates. Each replicate consisted of two seedlings. Also, soil drenching with each BCA at 50 ml pot−1, individually, were applied to tomato plants (four true-leaf stages).
Determination of the bacterial pathogen in plants
For the dilution plating method, samples of the lower stem internodes of the different treatments were taken 4 weeks after inoculation. Once the fresh weight was measured, the stem tissue was washed with tap water, surface-sterilized with 70% ethanol and again washed with a sterile water. Plates were incubated for 5 days at 26 °C, and the number of bacterial colonies was counted (Abo-Elyousr and El-Hendawy 2008).
Fresh and dry weight determination
Tomato seedlings from each treatment were removed and washed by water to remove any soil then blotted with tissue paper, and the fresh weight of shoots was determined. To determine the dry weight the shoots, they were dried at 60 °C for 72 h.
Production of siderophores, hydrogen cyanide, and indole acetic acid by the bacterial control agents
Siderophore production
The production of siderophores by PGPR species was assayed by the plate assay method, as described by Schwyn and Neilan (1987). After growing overnight, PGPR isolates (10 μl) were spotted on Chrome Azurol S blue agar plates and incubated at 28 ± 2 °C for 48 h. A yellow-orange zone around the spotted colony was taken as a positive indication of siderophore production. The extent of siderophore biosynthesis was measured as the diameter of the zone developed (Alexander and Zuberer 1991).
HCN production
The potential of the efficient bioagents to produce hydrogen cyanide (HCN) was assessed, as per the method of von Rohr et al. (2009). Whatman No. 1 filter paper pads were placed on the lids of the Petri plates, and the plates were sterilized. Tryptic soy agar medium amended with glycine (4.4 g l−1) was sterilized and poured into the sterile plates. Each isolate (24 h old) were streaked onto the medium. The filter paper padding in each plate was soaked by 2 ml of sterile picric alkaline solution (2% sodium carbonate in 0.5% picric acid). The inoculated plates were sealed with parafilm to contain the gaseous metabolite produced by the antagonistic bacteria and to allow a chemical reaction with picric acid on the top. After incubation at 28 ± 2 °C for 1 week, a color change of the filter paper was noticed, and the HCN production potential of the antagonistic bacteria was assessed.
IAA production
The production of indole acetic acid (IAA) by the BCAs was determined, as detailed elsewhere (Egamberdieva et al. 2008). Briefly, the tested isolates were inoculated in KB medium supplemented with 1.5% NaCl and Mg2SO4 and incubated at 28 ± 2 °C at 150 rpm. After cultivation for 3 days, aliquots of bacterial cultures were centrifuged at 13,000 rpm for 10 min. A 2-ml aliquot of the supernatant was transferred to a tube with 2 drops of orthophosphoric acid and 4 ml of Salkowski reagent. The mixture was incubated at room temperature for 25 min and recorded as positive, if a pink color developed.
Statistical analysis
Four replicates per treatment were used and the experiments were performed twice with a completely randomized design. Because the analyses showed insignificant interaction between the two experiments, results were combined for final analysis. The data were evaluated by analysis of variance, using SPSS 10.0 (SPSS, Chicago, IL, USA). Differences between treatment means were determined, using the least significant difference (LSD) test (P < 0.05).
