Bacterial isolates and root-knot nematode, M. incognita
Fourteen B. subtilis isolates were isolated during 2002–03 from 5 locations in Egypt (El-Malaha, El-Amria, El-Nobaria, Abo-Homos, and El-Sharkia) by Dr. Abo-Zaid, G.A., Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), Egypt. All the isolates were identified according to the morphological, biochemical, and physiological tests recommended by Sneath et al. (1986) and Collee et al. (1996). Seven B. subtilis isolates; B1, B4, B7, B8, B10, B11, and B14 were identified based on the sequencings of 16S rRNA gene. The root-knot nematode, M. incognita used in this study was provided by Plant Pathology Department, Faculty of Agriculture, Alexandria University, Egypt.
Cultivation in shake-flask
Growth of the 14 B. subtilis cultures was conducted in shake flasks under constant controlled conditions of temperature, pH, and agitation. Bacterial colonies of B. subtilis isolates were inoculated into a nutrient liquid medium (peptone, 5 g/l and beef extract, 3 g/l) and incubated overnight (16 h) at 30 °C, with a constant shaking at 200 rpm. 1 ml of each culture was transferred into a 250 ml Erlenmeyer flask containing 49 ml of number 3 medium of which each 1 L contained 10 g peptone, 10 g glucose, 1 g KH2PO4 and 0.5 g MgSO4. 7H2O in distilled water and pH was adjusted to 7 (Asaka and Shoda 1996). Cultures were incubated overnight at 30 °C and shacked at 200 rpm until reaching 2 × 109 cfu/ml. 20 ml of each culture was centrifuged at 5590×g for 20 min, and its cell free supernatant was collected and passed through a syringe filter (0.2 μ). Also, cell pellet of each isolate was collected, washed, and centrifuged at 5590×g for 20 min. Cell pellet of each culture was suspended in 20 ml of sterile distilled water. The culture broth, cell-free supernatant, and cell pellet of these bacterial isolates were used for treatments in dilutions of their original concentrations, namely, 1:1 (50%), 1:4 (25%), and 1:10 (10%).
In vitro suppressive effect of B. subtilis isolates against M. incognita
Culture broth, cell pellet suspension, and cell-free supernatant of each B. subtilis isolate were examined for its ability to inhibit egg hatching and juvenile (J2) activity. Approximately 50 eggs and 50 juveniles (J2) were transferred into each well of a 12-well plate. Afterwards, culture broth, cell pellet suspension, and cell-free supernatant of the 14 B. subtilis isolates were added separately at a concentration of 50%. Culture broth, cell pellet suspension and cell-free supernatant of the five B. subtilis isolates; B4, B5, B7, B8 and B10 were added at the concentrations of 25 and 10%. The control treatment included medium free from B. subtilis isolates. The plates were covered and incubated at 25 °C for 72 h. Three replicates/treatment were analyzed. The percentages of hatched eggs and survival of juveniles were counted under microscope (Huang et al. 2016).
Fermentation experiment
Bioreactor (fermentor)
Batch fermentation was performed, according to Matar et al. (2009), in a working volume of 4 L in a 10 L bench-top bioreactor (Cleaver, Saratoga, USA), prepared with three 6-bladed disc-turbine impeller and four baffles, and joined to a digital control unit. The process was automated through a control unit provided with control panel of 10.4ˮ color touch-screen interface and storage program up to 59,994 programs for different kinds of conditions. Temperature and pH were adjusted at 30 °C and 7, correspondingly. pH was controlled by automatic feed of 2 N NaOH and 2 N HCl. Sterilized air passed through sterile filter was supplied, originally at 0.5 VVM (air volume per medium volume per minute). Agitation rate ranged between 200 and 400 rpm to maintain the dissolved oxygen level above 20%. METTLER TOLEDO electrodes were used to determine the dissolved oxygen level and pH values on-line.
Batch fermentation
Two batch fermentation runs of B. subtilis isolate B10 (Accession No. EF583055), which gave the lowest percentage of hatched eggs and the highest percentage of juvenile mortality of M. incognita, were started in a fermentor, with an optical density of 0.2 and 0.5 at 550 nm, using number 3 medium (Asaka and Shoda 1996). Shake flask pre-cultured seed was prepared as follows: a single colony of B. subtilis isolate B10 was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of production medium of N3 medium. Bacterial isolate was cultured overnight at 30 °C and shacked at 200 rpm. During the time of the two batch fermentation, several samples of culture were taken and cell number was determined by measuring culture optical density at 550 nm. Glucose level was estimated enzymatically (Matar et al. 2009), using (GOD-PAD) colorimetric kit (Diamond Diagnostic, Egypt). Ability of culture broth of B. subtilis isolate B10 to antagonize and inhibit juvenile (J2) activity of M. incognita at a concentration of 5% (1:20) was tested (Huang et al. 2016). Dry cell weight was estimated by centrifuging 10 ml sample at 894×g for 10 min, and the pellet was re-suspended, washed, and centrifuged again as before. Pellets were then dried overnight in a dry-air oven at 80 °C (Van Dam-Mieras et al. 1992).
Suppressive effect of B. subtilis isolate B10 against M. incognita under greenhouse conditions
A pot experiment was carried out under greenhouse conditions, using tomato plants as a host plant. Twenty four pots (25 × 25 cm in diameter) were filled by 5 kg mixture of sterilized clay and sand (2:1 v/v), and the nematode eggs were applied at the rate of 1000 eggs/pot, 3 days after transferring tomato seedlings (30 days old) to the pots. Culture broth, cell pellet suspension, and cell-free supernatant of B. subtilis isolate B10 were applied at a concentration (10 ml/pot, 2 × 109 cfu/ml) twice, 3 days after nematode inoculation and 1 month after the first treatment as a soil drench. Each treatment was accomplished by three replicates. Eight treatments were performed as follows: (1) M. incognita as a check treatment; (2) untreated control; (3) culture broth; (4) cell pellet suspension (5) cell-free supernatant; (6) M. incognita + culture broth; (7) M. incognita + cell pellet suspension; and (8) M. incognita + cell-free supernatant. The plants were up-rooted after 60 days then root galls and egg masses were determined. Also, the fresh and dry weight of shoots and roots were determined. The roots were stained for 15 min in an aqueous solution of Phloxine B stain (0.15 g/l water), then washed with running tap water to remove residual stain and detect the presence of nematode egg masses (Holbrook et al. 1983).
Formulation experiment and preparation of B. subtilis isolate B10
Talc powder (TP) was used for preparing a bioformulation. The culture broth containing 2.0 × 109 cfu/m1 of B. subtilis isolate B10 was used for the preparation. To 400 ml of culture broth, 2 g of glucose and 10 g of carboxymethylcellulose (CMC) were added as additives. Glucose served as carbon source for keeping the cells viable, while CMC acted as an adhesive. The culture broth and additives were mixed uniformly on a vortex mixer. 15 g of calcium carbonate was added to 1 kg of talc powder (TP) and mixed well for adjusting the pH to 7.0. Four hundreds ml of culture broth with additives were mixed with 1 kg of the talc powder. The formulation was shade-dried to reduce the moisture content to ~ 20% and then packed in UV-sterilized polythene bags and sealed (Vidhyasekaran and Muthamilan 1995).
Effect of formulated B. subtilis isolate B10 on M. incognita under greenhouse conditions
A pot experiment was carried out under greenhouse conditions, using tomato plants as a host plant. Twenty seven pots (15 × 15 cm in diameter) were filled by 3 kg mixture of clay and sand (2:1 v/v), and the nematode eggs were applied at the rate of 1000 eggs/pot, 3 days after transferring tomato seedlings (30 days old) to pots. Formulation contained B. subtilis isolate B10 was applied at a concentration of 0.02, 0.06, and 0.1 g/pot twice, 3 days after nematode inoculation and 1 month after the first treatment, as a soil drench. Each treatment was accomplished by three replicates. Nine treatments were performed as follows: (1) M. incognita as a check treatment; (2) untreated control; (3) formulation 0.02 g/pot; (4) formulation 0.06 g/pot (5) formulation 0.1 g/pot; (6) M. incognita + formulation 0.02 g/pot; (7) M. incognita + formulation 0.06 g/pot; (8) M. incognita + formulation 0.1 g/pot; and (9) M. incognita + Vydete (2 ml/l). The plants were up-rooted after 50 days, and then root galls and egg masses were determined. The fresh and dry weight of shoots and roots were determined as mentioned before (Holbrook et al. 1983).
Statistical analysis
All data obtained from laboratory bioassay and pots experiments were analyzed using analysis of variance (ANOVA). The significant differences among treatments were determined according to the least significant differences (LSD) at P < 0.05 level of probability, using the CoStat software.