- Open Access
Biocontrol of the root-knot nematode, Meloidogyne incognita, using an eco-friendly formulation from Bacillus subtilis, lab. and greenhouse studies
© The Author(s) 2018
- Received: 16 June 2018
- Accepted: 31 October 2018
- Published: 20 November 2018
Culture broth, cell pellet suspension and cell free supernatant of 14 Bacillus subtilis isolates obtained from different Egyptian locations were checked for their ability to repress egg hatching and juvenile (J2) activity of root-knot nematode, Meloidogyne incognita under laboratory environment. Treatments using culture broth of B. subtilis isolates B10 and B8, at a concentration of 50%, recorded lowest percentages of hatched eggs reaching 44.7 and 46.3%, respectively. Culture broth of B. subtilis isolate B10 at the same concentration showed a higher percentage of juvenile mortality reached 99.7%. Batch fermentation was completed, using 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, for maximizing biomass production and suppression effects of culture broth. Batch fermentation no. 2, which started in a bioreactor with optical density of 0.5, was the best process that achieved a higher cell biomass and percentage of juvenile mortality of 4.52 g/l and 74.3%, respectively, using culture broth of 5%. Under greenhouse conditions, culture broth, cell pellet suspension, and cell-free supernatant of B. subtilis isolate B10 were used to test their potential for reducing number of galls and egg masses in the roots of tomato plants. The treatment of using culture broth at a concentration 10 ml/pot, 2 × 109 cfu/ml in a soil infested with M. incognita, was highly significant in decreasing number of galls and egg masses reaching the average of 9.3 and 6.7, with reduction percentage of 81.1 and 89.5%, respectively, compared with the control treatment of M. incognita only. In addition, B. subtilis isolate B10 was formulated and applied as bionematicide to test its efficiency in reducing number of galls and egg masses. Treatment with bioformulation at a concentration of 0.1 g/pot was more significant than the other concentrations in reducing number of galls and egg masses, reaching the average of 12 and 7 with a reduction percentage of 69.7 and 71.2%, correspondingly.
- Bacillus subtilis
- Meloidogyne incognita
The root-knot nematodes, Meloidogyne spp., are one of the highly important soil-borne pathogens that cause great economic damages to horticultural and field crops, and considered one of the most dangerous plant-parasitic nematodes, which can infect approximately all of the world’s main crop plants (Oka et al. 2000). Nematodes generally habitat the soil and usually attack the underground parts of plants; therefore, their management is a very complex (Stirling 1991). Different methods can be utilized for controlling Meloidogyne spp. such as cultural practices, resistant cultivars, and chemical nematicides which are easy for application and more effective. Concerns about public health and environmental safety are pushing for reducing chemical nematicides used in controlling of Meloidogyne spp. Thus, improvement of new alternative of management is needed, especially environmentally friendly methods. Management of Meloidogyne spp. can be achieved, using biocontrol agents (Siddiqui 2002 and Khan et al. 2008). Huang et al. (2016) reported that the highest ovicidal activity against M. incognita documented, using a combination of Syncephalastrum racemosum and Paecilomyces lilacinus at a concentration of 50%, reduced 70% of egg hatching than the control. Moreover, the treatment with S. racemosum, at a concentration of 50%, showed a best larvicidal activity, where the rate of mortality reached its highest value of 96.7%. Bacillus spp. can produce lytic enzymes, cyclic lipopeptides (Gray et al. 2006), and many secondary metabolites that play an essential role in biocontrol of Meloidogyne spp. Many lytic enzymes are produced by B. cereus, including chitinase and glucanse (Csuzi 1978). These lytic enzymes have been demonstrated to be antagonistic to M. javanica (Wepuhkhulu et al. 2011). Cyclic lipopeptides of surfactin and iturin produced by B. subtilis were more able to inhibit egg hatching of M. incognita and play an imperative role in increasing the percentage of juvenile mortality (Kavitha et al. 2012). The main goal of fermentation process is scaling-up production of cell biomass and the biological products. Matar et al. (2009) maximized cell biomass of B. subtilis isolate G-GANA7, as a bioagent against some plant pathogens, using batch fermentation strategy that achieved a cell biomass of 3.2 g/l. Scaling-up production of surfactin (lipopeptide) from B. subtilis to be used as a bioagent was performed in an innovative bioreactor, maximum concentration of surfactin achievement was 6.45 g/l (Yeh et al. 2006). Population densities and galls of root-knot nematodes, Meloidogyne spp. decreased by using the treatments with Pseudomonas aeruginosa, B. subtilis, and antagonistic fungus P. lilacinus (Prakob et al. 2009). Also, B. cereus X5 was effective in suppression of root-knot nematodes, hence number of galls of tomato roots decreased compared to the control (Tong-Jian et al. 2012). Burkett-Cadena et al. (2008) reported that formulations (contained strains of bacilli) of Equity® (multiple strains), BioYield® (two strains), and RhizoVital® (strain FZB42) reduced M. incognita eggs per gram root, juvenile nematodes per gram of soil, and galls per plant on tomato.
The main objective of this study was to evaluate the antagonistic and suppression effects of culture broth, cell pellet suspension, and cell free supernatant of B. subtilis isolates on egg hatching and juvenile (J2) activity of M. incognita under laboratory conditions. In addition, to maximize both biomass production and suppression effects of the promising B. subtilis isolate, using batch fermentation. As well, to evaluate their efficacy to reduce number of galls and egg masses in the roots of tomato plants under greenhouse conditions.
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).
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.
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).
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.
Effect of culture broth, cell pellet, and supernatant of Bacillus subtilis isolate B10 on number of galls and egg masses of Meloidogyne incognita occurrence in the roots of tomato plants
Number of galls
49.3 ± 3.1a*
64 ± 2.6a
0 ± 0e
0 ± e
0 ± 0e
0 ± e
0 ± 0e
0 ± e
0 ± 0e
0 ± e
N + CU
9.3 ± 1.5d
6.7 ± 1.2d
N + P
18 ± 2c
10 ± 1.7c
N + S
27 ± 2.6b
21 ± 2.6b
Effect of culture broth, cell pellet, and supernatant of Bacillus subtilis isolate B10 on fresh and dry weight of shoots and roots of tomato plants
Fresh weight of Shoots (g)
Dry weight of shoots (g)
Fresh weight of roots (g)
Dry weight of roots (g)
26.4 ± 1.9f*
6.1 ± 0.8e
14.3 ± 0.4e
6.4 ± 0.4f
69.2 ± 3.6b
12 ± 1.4bc
18.4 ± 0.4b
7.9 ± 0.2bcd
74.3 ± 1.4a
14 ± 0.2a
21.6 ± 0.8a
9.5 ± 0.3a
63.5 ± 2c
13.6 ± 0.1a
17.8 ± 0.7bc
8.5 ± 0.2b
57 ± 1.5d
11.8 ± 0.4c
15.4 ± 0.3d
7.3 ± 0.3de
N + CU
65.9 ± 1.3bc
13 ± 0.6ab
17.4 ± 0.6c
8.2 ± 0.9bc
N + P
58.4 ± 1.8d
11.1 ± 0.3c
15.6 ± 1d
7.5 ± 0.4cde
N + S
49.9 ± 2.3e
9.1 ± 0.5d
14.9 ± 0.2de
6.9 ± 0.2ef
Effect of formulated Bacillus subtilis isolate B10 on number of galls and egg masses of Meloidogyne incognita occurrence in the roots of tomato plants
Number of galls
39.7 ± 2.1a*
24.3 ± 1.2a
0 ± 0f
0 ± f
0 ± 0f
0 ± f
0 ± 0f
0 ± f
0 ± 0f
0 ± f
N + F1
24.3 ± 1.2b
14.3 ± 1.2b
N + F2
16.3 ± 0.6c
10.3 ± 0.6c
N + F3
12 ± 1d
7 ± 1d
N + Vydate
4.7 ± 0.6e
2.7 ± 0.6e
In conclusion, biocontrol agents can be used as an alternative approach of the chemical nematicides for controlling various nematodes. B. subtilis is recommended for the management of root-knot nematode, M. incognita. However, further investigations are required to determine the formulation that can achieve the best results.
The authors thank Prof. Ibrahim, I.K.A. and Prof. El-Saeedy, A. at Plant Pathology Department, Faculty of Agriculture, Alexandria University, El-Shatby, Alexandria, Egypt. Also, Prof. Hafez, E.E. and Dr. Kenawy, A.M. at City of Scientific Research and Technological Applications, Alexandria, Egypt for their critical reading and revision.
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