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Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery
Journal of Invertebrate Pathology 100 (2009) 94–99
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Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery Metasebia Terefe, Tadele Tefera *, P.K. Sakhuja Haramaya University, College of Agriculture, Department of Plant Sciences, P.O. Box 138, Dire Dawa, Ethiopia
a r t i c l e
i n f o
Article history: Received 18 July 2008 Accepted 7 November 2008 Available online 14 November 2008 Keywords: Bacillus firmus Biological control Meloidogyne incognita Tomato
a b s t r a c t Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha1 was effective in reducing the number of galls (75–84%), and increased shoot height (29–31%) and weight (20–24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Root-knot nematodes cause extensive damage to a wide variety of economically important crops, including tomato. Three species of root-knot nematode, Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria are considered the most important on the basis of their worldwide geographical distribution and large host range (Sassser et al., 1982). They are also involved with fungi and bacteria in many disease complexes and can also break down plant resistance to pathogens (Taylor and Brown, 1976). A number of methods for the management of root-knot nematode such as chemical control, organic amendments, resistant varieties, soil solarization and biological control have been tried with different levels of successes for the protection of tomato plants (Randhawa et al., 2001; Sakhuja and Jain, 2001). Chemical management is effective, but expensive and may lead to residue and soil pollution problems. Tomato varieties resistant to root-knot nematode have been developed in some countries, but are not very popular due to their lower yields. An environmentally safe and economically feasible root-knot nematode control practices needs to be available. Biological control * Corresponding author. E-mail address: [email protected] (T. Tefera). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.11.004
is free from residual and adverse environmental effects (Sumeet and Mukerji, 2000). Fungi such as Paecilomyces lilacinus, Pochonia chlamydosporium, Hirsutella rhossiliensis, Dactylella oviparasitica and Cylindrocarpon destructans are well known parasites nematodes (Anastasiadis et al., 2008; Walia and Vats, 2000). A few bacteria such as Pasteuria penetrans directly parasitize nematodes, while many bacteria including of Bacillus, Agrobacterium, Azotobacter, Pseudomonas and Clostridium produce toxins that kill nematodes (Walia et al., 2000). BioNem WP has been developed as a commercial BioNematicide by Agro-green, Minrav, Ashdod, Israel. It contains the bacteria Bacillus firmus and has been reported effective against, M.incognita, Meloidogyne hapla, Heterodera spp., Tylenchulus semipenetrans, Xiphinema index and Ditylenchus dipsaci (Keren-Zur et al., 2000). BioNem is currently introduced to Ethiopia to be used against rootknot nematode in vegetables. In order to utilize a microbial pathogen for practical pest control, registration as a microbial pesticide is necessary. In Ethiopia, a set of efficacy data following the national guidelines should be submitted to register a microbial pesticide. Therefore, this study was conducted to generate empirical information on the efficacy of BioNem against the root-knot nematode M. incognita by testing the effects of the bacterium on nematode egg hatching, juvenile motility, gall formation, population density; and the effect on growth of tomato plants in green house and in field conditions.
M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99
2. Materials and methods 2.1. Establishment of root-knot nematodes culture Tomato plants infected with root-knot nematodes were collected from a vegetable farm in the Haramaya University (HU), Ethiopia. Egg masses were picked off infected roots using forceps and a needle and then allowed to hatch. Juveniles were inoculated around the roots of tomato seedlings (cv. Marglobe) raised in sterilized soil in pots. This was sub cultured to maintain sufficient numbers of root-knot nematodes for subsequent experiments. 2.2. Experiment-I: effect of BioNem on egg hatching, juvenile mobility and recovery of M. incognita 2.2.1. Effect of BioNem on egg hatching About 60-day-old tomato (cv. Marglobe) roots that were heavily infected with root-knot nematodes were collected from the HU vegetable farm on October 8, 2007. Two hours after collection, roots were brought to the Plant Pathology Laboratory of HU, and washed gently using tape water. Using a sterile dissecting needle and forceps the egg masses were separated from the roots, and kept in refrigerator at 10 °C for one day to prevent hatching before application of treatments. Five concentrations of BioNem, i.e., 0.5%, 1%, 1.5%, 2% and 2.5% were prepared by weighing the powdered form of BioNem. About 99.5, 99, 98.5, 98 and 97.5 ml sterile water were added to beakers containing the five respective concentrations of BioNem. The suspension was shaken by hand and kept at room temperature (20– 23 °C) in the laboratory for 10 h. The suspension was filtered using muslin cloth. About 5 ml of the BioNem suspension was added to a sterile Petri dish (9 cm diameter). Five equal sized egg masses were randomly picked using sterile forceps and were placed in each Petri dish containing the suspension. Egg masses kept in sterile water were used as a control. The Petri dish containing the suspension and the egg masses were kept at room temperature on the laboratory bench. The experiment was laid out in completely randomized design (CRD) with three replications. Mean average maximum and minimum temperature were 19.6, 23.5 and 15.6 °C, respectively. Seventy-two hours after treatment application, the Petri dishes were shaken, the suspension containing the nematodes was transferred to a counting dish, nematode juveniles were counted using stereoscopic microscope at magnification of 50. Fresh BioNem suspension was added to the egg masses and kept for hatching again at room temperature. This was done every 72 h, till hatching ceased in the control. The number of nematodes was log-transformed to normalize variances. The transformed data were subjected to an analysis of variance (ANOVA). Treatment means were separated using least significant differences (LSDs). 2.2.2. Effect of BioNem on juvenile mobility About 90-day-old tomato (cv. Marglobe) roots that were infected with root-knot nematodes collected from a vegetable farm in Dire Dawa (Ethiopia) and washed gently under running water. Using dissecting needle and forceps the egg masses were separated from the roots, and kept in the laboratory bench at room temperature (20–23 °C) for three days until juveniles hatched. Six concentrations of BioNem, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% were prepared as described above. The suspension was shaken and kept at room temperature (20–23 °C) in the laboratory for 12 h. The suspension was separated using centrifugation at 3000 rpm for 10 min. About 3-ml suspension of the BioNem was added to a sterile Petri dish (3 cm diameter). Three milliliter suspension containing mean number of 58, 46, 50, 53, 48 and 60 juveniles (J2) of root-knot nematodes were added to the Petri dishes
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with the BioNem suspension at the rates of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, respectively. Three-milliliter suspension containing an average of 56 juveniles, M. incognita, kept in a Petri dish with 3-ml sterile water used as a control. The experiment was laid out in a completely randomized design (CRD) with three replications. Mean, maximum and minimum temperatures were 19.8, 24.2 and 15.7 °C, respectively. The number of motile juveniles was recorded 24, 36 and 48 h after treatment application, using stereoscopic microscope (X50). Juveniles were considered paralyzed (non-mobile) if they did not move when probed with a fine needle. Percentage mobility of the juveniles was arc-sine transformed. The transformed data were subjected to an ANOVA. Treatment means were separated using LSD. 2.2.3. Effect of BioNem on recovery of paralyzed juveniles Recovery of juveniles from the toxic effect of BioNem was checked by randomly taking 10, 11, 11, 14 and 13, paralyzed juveniles from the preceding (mobility test) experiment from the respective concentrations of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%. The paralyzed juveniles were kept in fresh water for 48 h. The experiment was laid out in a completely randomized design with three replications. The number of mobile juveniles was counted 24 and 48 h after keeping the paralyzed juveniles in fresh water using stereomicroscope microscope (50). Percentage recovery of juveniles was log-transformed. The transformed data were subjected to an ANOVA. Treatment means were separated using LSD. 2.3. Experiment-II: effect of BioNem on nematode infestation and growth of tomato plants in the greenhouse Seeds of tomato (cv. Marglobe) were sown in sterile soil in plastic trays in the greenhouse. A 1-month-old seedling was transplanted into each pot containing 1200 cc of sterilized soil with 1:2:3 proportions of sand, compost and clay, respectively. A week after transplanting, the soil around the seedlings was inoculated with M. incognita at 1000 juveniles per plant. Five treatments of BioNem were used. These were 1, 2, 4, 8 and 16 g powder formulation of the BioNem. Each treatment was applied to a tomato seedling in a pot a week after transplanting. There were also two controls: uninoculated control and inoculated control. In uninoculated control, neither the nematode nor the BioNem was applied; whilst in inoculated control, the nematode was applied but the BioNem was not. The experiment was laid out in a randomized complete block design (RCBD) with four replications. Seedlings were watered as needed. No fertilizers were used. The following data were collected 50-days after treatment application. The number of galls per plant was counted manually. Average of 10 egg masses per plant were randomly taken using sterile forceps and dissecting needle. The egg masses were vigorously shaken with 5% sodium hypochlorite in stoppered flasks for 2 min based on the procedure of Hussey and Barker (1973). From the total suspension, a 3-ml suspension was pipetted into a counting dish and the numbers of eggs were counted using a stereomicroscope at a magnification of 50. The length of the plant was measured for each pot from the soil line to the tip of the stem and expressed in cm. The plants were cut at the crown level and the weight was measured using electronic balance. The plant roots were removed from the soil and gently washed with distilled water to remove soil particles. The weight of the roots was recorded using an electronic balance. The fresh shoot, which was brought to a laboratory from the green house using a paper bag, was kept in an oven at 105 °C for 24 h, and the dry shoot weight was recorded. The soil samples were collected from each pot using a labeled paper bags
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and taken to a laboratory. Final population density of nematode was determined based on the Baermann funnel technique (Southey, 1970). The data on plant height, fresh shoot weight, fresh root weight, dry shoot weight, number of galls, number of eggs/egg masses and final nematode population/pot were log-transformed to normalize variances. The transformed data were subjected to an analysis of variance (ANOVA) using Statistical Analysis System (SAS) (version 6.12, SAS, Institute Inc., Cary, NC, USA). Treatment means were separated using the least significant differences (LSDs). Back transformed values were presented. 2.4. Experiment-III: effect of BioNem on root-knot nematodes infestation and the growth of tomato plant in nursery The experiment was conducted at Dire Dawa (Ethiopia) to evaluate the effectiveness of the BioNem under nursery conditions in 2007 cropping season. The site is located at 9°60 N and 41°80 E longitude, 1197 m above sea level, receives 520 mm mean annual rainfall, 28.1–34.6 and 14.5–21.6 °C mean maximum and minimum temperature ranges, respectively. The soil type is Eutric Regosol. Raised nursery beds (20 cm high) were prepared. The plot size was 0. 25 m2 (0.5 m 0.5 m). Prior to establishment of treatments, of finely chopped 8 kg roots of egg plant (Solanum melongena L.) severely infested with root-knot nematode was incorporated into the prepared beds in a uniform manner to make nematode sick plots. Seeds were planted 1–2 cm deep in rows on October 28, 2007. Rows were 10 cm apart and the distance between plots (beds) was 50 cm and between blocks was 1 m. The fertilizer, DAP was applied at the rate of 150 kg ha1 at the time of sowing. A total of four treatments 50, 100, 200 and 400 kg ha1 powder formulation of BioNem were used. There were also treated and untreated controls. In the treated control, the nematicide, phenamiphos (Nemacur 10 G) was used. Whilst in untreated control, neither BioNem nor nematicide was used. The treatments and the nematicide were applied at the time of sowing. The treatments were applied manually with the help of sand as a carrier. The experiment was laid out in a randomized complete block design (RCBD) with four replications. The nursery beds were watered and weeded as required. Plant height, number of galls per plant, biomass (fresh shoot and root weight) was recorded from 15 seedlings collected at random from the 3 middle rows of each plot, 45-days after planting, as described above. Plant height and biomass were subjected to ANOVA using Statistical Analysis System (SAS) (version 6.12, SAS, Institute Inc., Cary, NC, USA). The number of galls was log-transformed transformed to normalize variances. The transformed number of galls was subjected to an analysis of variance (ANOVA). The treatment means were separated using the least significant differences (LSDs). Back transformed values were presented. 3. Results 3.1. Experiment-I: effect of BioNem on egg hatching, juvenile mobility and recovery 3.1.1. Effect of BioNem on egg hatching Application of BioNem at all concentrations significantly inhibited egg hatching (Table 1 and Fig. 1). Some egg hatching occurred to some extent in BioNem treatments in the first 3 days, but it declined drastically in the following 3 days and then stopped completely, while in the control egg hatching continued for up to 24 days. Percent inhibition of egg hatching ranged from 98% to
100%. Application of BioNem at 2.5% concentration caused 100% inhibition. 3.1.2. Effect of BioNem on inhibition and recovery of juvenile mobility There were significant differences (P < 0.05) in mobility of juveniles between the treatments (Table 2). BioNem at 2.5% and 3% concentrations caused 100% inhibition in motility of juveniles 24 h after treatment application. Similarly, none of the juveniles recovered from the paralysis caused by BioNem at 2.5% and 3% concentrations, 24 and 48 h after treatment with water. 3.2. Experiment-II: effect of BioNem on nematode infestation and growth of tomato plants in the greenhouse Application of BioNem significantly (P < 0.05) reduced gall formation (Table 3). The mean number of galls ranged from 99 to 226 in BioNem treated plants compared to 1084 galls in nematode-inoculated control plants. The greatest reduction (91%) in gall formation was recorded from BioNem applied at the rate of 8 g/pot, while the least reduction (79%) in gall formation was obtained at 1 g/pot. However, BioNem applied at 16 g/pot was phytotoxic and resulted in seedling mortality. There were significant differences (P < 0.05) in population density between. Treatment with BioNem at 8 g/pot caused the greatest reduction in nematode populations (75 per 100 cc soil) compared to 306 in the control (Table 3). The percent reduction in final nematode population ranged from 65% to 76% compared to the nematode-inoculated control. There were significant differences (P < 0.05) between treatments in number of eggs per egg mass (Table 3). BioNem applied at different rates adversely affected egg formation. Treatment with the BioNem at 8 g/pot resulted in the least number of eggs (360) compared to 653 eggs being produced in the inoculated control. However, application of BioNem at 1, 2 and 4 g had 44, 429 and 388 eggs, respectively. There were significant differences (P < 0.05) in treatments in affecting plant height and biomass (Table 4). The tallest plant (60 cm) was recorded from the uninoculated control and the shortest from the inoculated control (31 cm). Plant height in the BioNem treatments ranged from 50 to 53 cm which represented a 60–71% increase in height over the inoculated control. Application of BioNem significantly (P < 0.05) improved shoot weight over the inoculated control (Table 4). Heavier (92–110 g) shoot weight was recorded from treatments with BioNem at 2 g and 4 g/pot than in the inoculated control (89 g). There were significant differences (P < 0.05) between treatments in fresh root weight (Table 4). BioNem applied at 1, 2, 4 and 8 g/pot resulted in 32, 30, 29 and 27 g mean fresh root weight, respectively. The smallest fresh root weight (26 g) was recorded from the uninoculated control plants, whilst the largest fresh root weight (52.5 g) was recorded from the inoculated control. 3.3. Experiment-III: effect of BioNem on root-knot nematode infestation and growth of tomato plants in the nursery There were significant differences (P < 0.05) between treatments in gall formation, plant height and plant biomass (Table 5). With increasing concentration of BioNem, gall formation decreased. The least number of galls was formed after treatment with phenamiphos (treated control), followed by BioNem applied at 400 and 200 kg ha1. The tallest plant height (21.9–22.9 cm) was recorded from treatments with 200, 400 kg ha1 and the nematicide treated control as compared to the untreated control (15.7 cm). The largest
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M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99 Table 1 Effect of different concentrations of B. firmus on cumulative hatchability of egg masses of M. incognita 24-days after treatment application. Concentration (%)
No. of juveniles hatched/5 egg masses
% Inhibition of hatching over control
0.5 1.0 1.5 2.0 2.5 Control (0%)
4.86 ± 0.35b 1.71 ± 0.22b 0.71 ± 0.13b 0.29 ± 0.07b 0 ± 0b 279.33 ± 0.91a
98.3 99.4 99.7 99.9 100 —
Means within the same column with a common letter are not significantly different (P < 0.05).
450 400
No. of eggs hatched
350 300 250 200 150 100 50 0 3
6
9
12
15
18
21
24
Time (days) 0%
2%
1%
2.50%
1.50%
0.50%
Fig. 1. Effect of different concentration of BioNem on rate of egg hatching in M. incognita.
biomass (17.5–18.4 g) was recorded from treatment with BioNem at 200 and 400 kg ha1, and nematicide treated control. 4. Discussion In vitro studies have clearly revealed that BioNem inhibits egg hatching of M. incognita. Interfering with the life cycle of root-knot nematode by managing egg masses prior to hatching is important. From a single egg mass up to 500 juveniles can hatch and infect the same root or roots nearby and start a new life cycle (Dropkin,
1989). Three fungi namely Fusarium equiseti, F. oxysporum and Rhizoctonia solani have been reported to produce toxic substances in culture medium, which inhibit the hatching of M. incognita significantly (Sakhuja et al., 1978). Certain strains of Bacillus sphaericus and Bacillus laterosporus (Bone, 1988; Bone, 1991) and Bacillus chitinosporus (Marrone et al., 1998) which inhibit egg hatch of animal parasitic nematodes in their host animals have been patented. Although such observations have been made following growth of the bacteria on rich media in vitro, there is no evidence that such mechanisms are of importance in the rhizosphere where nutrients are much less abundant. Saxena et al. (2000) reported that production of different types of antibiotics and enzymes is nature of Bacillus species, which are strongly antagonistic to several pathogens such as root-knot nematode. Nematode egg shells consists of three layers namely, vitelline, chitinous and lipid layers (Zdarska et al., 2001). It might be possible that B. firmus secretes some toxins, which damages the egg shell of root-knot nematode. During the initial few days hatching, though less, but occurred. However, later it stopped completely and during this period bacteria might have produced enough toxins to damage the egg shell or the juveniles within the eggs. The hatch inhibitory and egg disintegrating effect of the BioNem might be due to certain toxic substances present in the product or secreted by B. firmus that possess ovicidal property. Mendoza et al. (2008) reported pure culture filtrates of B. firmus reduced M. incognita egg hatching. Al-Banna and Khyami-Horani (2004) isolated local strains of Bacillus thuringiensis from various locations and showed their lethality to eggs and second-stage juveniles (J2) of root-knot nematodes under laboratory conditions. Deny-another commercial biocontrol product for nematodes is based on a natural isolate of the bacterium Burkholderia cepacia, which also reduces egg hatching and juvenile mobility (Meyer and Roberts, 2002). Rhizobacteria reduce nematode populations mainly by production of toxins (Siddiqui and Mahmood, 1999), induction of systemic resistance (Hasky-Gunther et al., 1998), changing nematode behavior (Sikora and Hoffmann-Hergarter, 1993) and interfering with plant recognition (Oostendorp and Sikora,1990) and promoting plant growth (El-Nagdi and Youssef, 2004). A commercial transplant mix (Bio yieldTM, Gustafson LLC) containing Paenobacillus macerans and Bacillus amylolique faciens has been developed to control plant parasitic nematodes on tomato, bell pepper and straw berry in USA (Meyer, 2003). BioNem significantly reduced nematode population and root infestation, from their extensive testing on vegetable crops including tomato, cucumber, pepper, garlic and some herbs (Giannakou and Prophetou-Athanasiadou, 2004; Giannakou et al. 2007). Treatment with BioNem adversely affected juvenile mobility. Giannakou and Prophetou-Athanasiadou (2004) reported that the biocontrol effects of BioNem could be partially attributed to the
Table 2 Effect of B. firmus on inhibition of M. incognita juvenile mobility in vitro. Concentration (%)
0.5 1.0 1.5 2.0 2.5 3.0 Control
Inhibition of mobility in B. firmus suspension (%)
Recovery of mobility in water (%)
24 h
36 h
48 h
24 h
48 h
52.3 ± 5.6c 54.8 ± 4.8c 60.7 ± 6.4c 82.6 ± 2b 100 ± 0a 100 ± 0a 2.0 ± 1d
55.7 ± 5c 59.2 ± 4.5c 66.9 ± 8.8c 86.3 ± 2b —* — 2.0 ± 1d
62.6 ± 5.3c 73 ± 9.8cd 80 ± 6.7bc 92.5 ± 0.9ab — — 2.0 ± 1d
34 ± 6.8a 31 ± 5.7ab 19 ± 7.5b —** — — —
47 ± 11.7a 31.7 ± 5.7ab 19 ± 7.5b — — — —
Means within the same column with a common letter are not significantly different (P < 0.05). * No mobility recorded as all juveniles were inhibited 24 h after treatment application. ** No recovery recorded as at 2.5% and 3% concentrations.
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Table 3 Effect of B. firmus (BioNem) WP on infestation of M. incognita in tomato plant in the greenhouse. BioNem/pot (g)
Galls/root
Reduction in gall over IC (%)
Final nematode population
Reduction in final population over IC (%)
Number of eggs/egg mass
Reduction in number of eggs over IC (%)
1 2 4 8 IC UC
226 ± 2.2b 164 ± 1.6c 123 ± 2.9d 99 ± 3e 1084 ± 5.4a —*
79 85 89 91 — —
106 ± 2.7b 95 ± 2bc 89.5 ± 2c 75 ± 1.7d 306 ± 5a —
65 69 71 76 — —
444 ± 2.2b 429 ± 3.5c 388 ± 2.7d 360 ± 2e 653 ± 3a —
32 38.6 41 45 — —
Means within the same column with a common letter are not significantly different (P < 0.05). IC, inoculated control. UC, uninoculated control. * No nematode was recorded in uninoculated control.
Table 5 Effect of B. firmus WP on M. incognita infestation and growth of tomato plants in nursery. BioNem (kg ha1)
Galls/plant
Reduction in galls over UC (%)
Plant height (cm)
Increase in height over UC (%)
Plant biomass (g)
Increase in biomass over UC (%)
50 100 200 400 TC UC
2.5 ± 0.2b 1.85 ± 0.1bc 1.25 ± 0.1cd 0.83 ±2.1d 0.78 ± 0.1d 5.5 ± 0.64a
53.87 64.63 75.68 84.00 85.40 —
20.1 ± 0.47b 20.9 ± 0.24b 22.3 ± 0.19a 22.9 ± 0.26a 21.95 ± 0.4a 15.7 ± 0.44c
21.6 24.6 29.3 31.4 28.3 —
15.5 ± 0.44b 16.1 ± 0.74b 17.5 ± 0.66a 18.4 ± 0.53a 17.5 ± 0.86a 13.4 ± 0.24c
10.6 13.3 20.9 24.4 20.1 —
TC, treated control (phenamiphos at 11 kg a.i./ha). UC, untreated control. Means within the same column with a common letter are not significantly different (P < 0.05).
Table 4 Effect of B. firmus WP on growth of tomato plant in M. incognita infested soil in greenhouse. BioNem/pot (g)
Plant height (cm)
Increase in height over IC (%)
Fresh shoot weight (g)
Increase in fresh shoot weight over IC (%)
Fresh root weight (g)
1 2 4 8 IC UC
50.0 ± 0.95c 53.1 ± 0.4b 53.0 ± 0.65b 53.6 ± 0.41b 31.3 ± 0.75d 60.0 ± 1.8a
59.74 69.74 69.32 71.24 – 92.26
87.0 ± 0.8c 92.4 ± 1b 92.9 ± 0.7b 89 ± 0.7c 59.0 ± 2d 110 ± 1.7a
47.45 56.61 57.45 50.84 — 86.44
32.0 ± 0.8b 30.0 ± 0.8c 29.0 ± 1.5c 27.0 ± 0.4d 52.5 ± 1a 26.0 ± 0.4d
IC, inoculated control. UC, uninoculated control. Means within the same column with a common letter are not significantly different (P < 0.05).
stimulating effect that the animal and plant additives contained in the BioNematicide formulation. Cayrol et al. (1989) found that culture filtrates of P. lilacinus were lethal to juveniles of root-knot nematode. Olubunmi and Rajani (2004) have also reported 100% mortality of juveniles of M. incognita with culture filtrates of Trichoderma. Siddiqui et al. (2008) also reported that Pseudomonas and Bacillus spp. inhibited hatching and penetration of M. incognita. The inhibition of motility by BioNem might be due to certain nematostatic and or nematicidal substances present in the product or secreted by B. firmus. Application of the BioNem to M. incognita reduced the number of galls, number of eggs, and final nematode population in tomato plants both in the greenhouse and under field condition. With the increase in application rate there was corresponding reduction in the number of galls and final population density. BioNem contains 3% lyophilized B. firmus spores and 97% additives (plant and animal extracts) (Giannakou and Prophetou-Athanasiadou, 2004). The mortality of tomato seedlings when treated with BioNem at 16 g/ pot is probably due to the high concentration of the additives. Increases in plant growth are due to the reduction in nematode attack as evidenced by the low final nematode population density. Formation of galls on the roots and colonization of root tissue by this nematode deprives plants of nutrients (Bird, 1974). The damage also occurs due to devitalization of root tips, which may stop
their growth or cause excessive branching of roots. Olubunmi and Rajani (2004) also reported that the galls on the root system disturb important root functions such as the uptake and transport of water and nutrients. The root weight of inoculated control plants was greater than BioNem applied plants. The difference in root weight was not related to the weight of the shoot. This could be due to the formation of large number of galls on inoculated plants. Wong and Mai (1973) also reported that differences in root weight may be explained by gall development; gall mass being heavier than an equivalent linear length of similar non-galled roots. In the present study, application of BioNem at sowing reduced infestation on seedlings and improved plant growth in the nursery. In contrast to the manufacturers recommendation for tomato (http://www.agrogreen.co.il/bionem-product.asp), (80 kg ha1) however, application of BioNem at 200 kg ha1 provided 75% reduction in galling, increased seedling height by 29% and weight by 21% over untreated control. Previous studies have also shown that Bacillus thuringensis (Bt) strain CR-371 applied as a soil drench led to a 53% reduction of tomato root galling caused by M. incognita (Zuckerman et al., 1993). Oostendorp and Sikora, (1990) also reported significant suppression of nematode multiplication by Pseudomonas fluorescens, which was due to its capability of altering root exudates, which could alter nematode behavior and suppress nematode populations in the root system. The shorter height of seedlings of untreated controls
M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99
might be due to the stunting action of M. incognita. This type of stunting or reduced heights caused by root-knot nematode has been reported earlier (Thimann, 1960; Jinfa et al., 2006). In conclusion, BioNem WP provides effective control that favorably compared with nematicides applications. A single application of BioNem at planting was sufficient for the entire season. This indicates the unique ability of BioNem to be used in the integrated management of root-knot nematode in tomato. Acknowledgment The General Chemical Trading (PLC), Addis Abeba, is acknowledged for sponsoring this work as part of BioNem Testing Project. References Al-Banna, L., Khyami-Horani, H., 2004. Nematicidal activity of two Jordanian strains of Bacillus thuringiensis on root-knot nematodes. Nematol. Mediterr. 32, 41–45. Anastasiadis, I.A., Giannakou, I.O., Prophetou-Athanasiadou, D.A., 2008. The combined effect of the application of a biocontrol agent Paecilomyces lilacinus, with various practices for the control of root-knot nematodes. Crop Prot. 27, 352–361. Bird, A.F., 1974. Plant responses to root-knot nematodes. Ann. Rev. Phytopathol. 12, 69–85. Bone, W.L., 1988. Control of parasitic nematode ova with Bacillus sphaericus. U.S. Patent No. 4781922. Bone, W.L., 1991. Control of parasitic nematode ova/larvae with a Bacillus laterosporus. U.S. Patent 5045314. Cayrol, J., Djian, C., Pijarowski, L., 1989. Study of the nematicidal properties of the culture filtrate of the nematophagus fungus Paecilomyces lilacinus. Rev. Nematol. 12, 331–336. Dropkin, V.H., 1989. The genera of phytonematodes. In: Introduction to Plant Nematology. Wiley-Interscience Publication, pp. 89–217. El-Nagdi, W.M.A., Youssef, M.M.A., 2004. Soaking faba bean seed in some bio-agent as prophylactic treatment for controlling Meloidogyne incognita root-knot nematode infection. J. Pestic. Sci. 77, 75–78. Giannakou, I.O., Anastasiadis, I.A., Gowen, S.R., Prophetou-Athanasiadou, D.A., 2007. Effects of a non-chemical nematicide combined with soil solarization for the control of root-knot nematodes. Crop Prot. 26, 1643–1644. Giannakou, I.O., Prophetou-Athanasiadou, D., 2004. A novel non-chemical nematicide for the control of root-knot nematodes. Appl. Soil Ecol. 26, 69–79. Hasky-Gunther, K., Hoffmann-Hergarten, S., Sikora, R.A., 1998. Resistance against the potato cyst nematode Globodera pallida systematically induced by the rhizobacteria Agrobacterium radiobacter (G12) and Bacillus sphaericus (B43). Fund. Appl. Nematol. 21, 511–517. Hussey, R.S., Barker, K.R., 1973. A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Dis. Rep. 57, 1025–1028. Jinfa, Z., Waddell, C., Sengupta, G.C., Potenza, C., Cantrell, R.G., 2006. Crop Sci. 46, 1581–1586. Keren-Zur, M., Antonov, J., Bercovitz, A., Feldman, K., Husid, A., Kenan, G., Markov, N., Rebhun, M., 2000. Bacillus firmus formulations for the safe control of rootknot nematodes. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, vol. 2A, pp. 47–52. Marrone, P.G., Manker, D.C., Heins, S.D., Jimenez, D.R., 1998. Nematicidal Bacillus strain and metabolite and methods of thereof. U.S. Patent 5733544. Mendoza, A.R., Kiewnick, S., Sikora, R.A., 2008. In vitro activity of Bacillus firmus against the borrowing nematode Radopholus similis the root-knot nematode Meloidogyne incognita and the stem nematode Ditylenchus dipsaci. Biocontrol Sci. Technol. 18, 377–389.
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Meyer, S.L.F., 2003. United States Department of Agriculture–Agricultural Research Service research programs on microbes for management of plant-parasitic nematodes. Pest Manag. Sci. 59, 665–670. Meyer, S.L.F., Roberts, D.P., 2002. Combinations of biocontrol agents for management of plant-parasitic nematode and soilborne plant-pathogenic fungi. J. Nematol. 34, 1–8. Olubunmi, O.F., Rajani, S.N., 2004. Biological control of root-knot nematodes (Meloidogyne spp.) on tissue culture banana (Dwarf cavendish var. Basarai). Acta Horticult. 635, 183–186. Oostendorp, M., Sikora, R.A., 1990. In-vitro interrelationships between rhizosphere bacteria and Heterodera schachtil. Rev. Nematol. 13, 269–274. Randhawa, N., Sakhuja, P.K., Singh, I., 2001. Management of root-knot nematode Meloidogyne incognita in tomato with organic amendments. Plant Dis. Res. 16, 274–276. Sakhuja, P.K., Singh, I., Sharma, S.K., 1978. Effect of some fungal filtrates on the hatching of Meloidogyne incognita. Indian Phytopathol. 31, 376–377. Sakhuja, P.K., Jain, R.K., 2001. Nematode diseases of vegetable crops and their management. In: Thind, T.S. (Ed.), Diseases of Fruits and Vegetables and their Management. Kalyani Pub., Ludhiana, India. Sassser, J.N., Carter, C.C., Taylor, A.L., 1982. A Guide to the Development of a Plant Nematology Program. Coop. Publ. Dept. Plant Pathol., North Carolia State Univ., and the U.S. Agency Int. Dev. Raleigh, N.C., pp. 21. Saxena, A.K., Pal, K.K., Tilak, K.V.B.R., 2000. Bacterial biocontrol agents and their role in plant disease management. In: Upadhyay, R.R., Mukerji, K.G., Chamola, B.P. (Eds.), Biocontrol Potential and its Exploitation in Sustainable Agriculture: Crop Diseases, Weeds, and Nematodes, vol. 1. Kluwer Academy Plenum, New York. Southey, J.F., 1970. Laboratory methods for work with plant and soil nematodes. H.M.S. Office, London. Siddiqui, Z.A., Mahmood, I., 1999. Role of bacteria in the management of plant parasitic nematodes: a review. Bioresour. Technol. 69, 167–179. Siddiqui, I.A., Haas, D., Heeb, S., 2008. Extracellular protease of Pseudomonas fluorescens CHAO, a biocontrol factrol with activity against the root-knot nematode Meloidogyne incognita. Appl. Environ. Microbiol. 71, 5646–5649. Sikora, R.A., Hoffmann-Hergarter, S., 1993. Biological control of plant parasitic nematodes with plant-health promoting rhizobacteria. In: Lumsden, P.D., Vaugh, J.L. (Eds.), Biologically Based Technology, ACS Symposium Series, USA, pp. 166–172. Sumeet, S., Mukerji, K.G., 2000. Exploitation of protoplast fusion technology in improving biocontrol potential. In: Upadhyay, R.R., Mukerji, K.G., Chamola, B.P. (Eds.), Biocontrol Potential and its Exploitation in Sustainable Agriculture: Crop Diseases Weeds and Nematodes, vol. 1. Kluwer Academy Plenum, New York, pp. 39–48. Taylor, E.C., Brown, F.J.D., 1976. The geographical distribution of Xiphinema and Londgidorus nematodes in the British Isles and Ireland. Ann. Appl. Biol. 84, 383– 402. Thimann, K.F., 1960. The auxins. In: Wilkins, M.W. (Ed.), The Physiology of Plant Growth and Development. McGraw Hill, New York, pp. 2–45. Walia, R.K., Vats, R., 2000. Fungal antagonists of Phytonematodes. In: Upadhyay, R.R., Mukerji, K.G., Chamola, B.P. (Eds.), Biocontrol Potential and its Exploitation in Sustainable Agriculture: Crop Diseases, Weeds, and Nematodes, vol. 1. Kluwer Academy Plenum, New York. Walia, R.K., Sharma, S.B., Vats, R., 2000. Bacterial antagonists of Phytonematodes. In: Upadhyay, R.R., Mukerji, K.G., Chamola, B.P. (Eds.), Biocontrol Potential and its Exploitation in Sustainable Agriculture: Crop Diseases, Weeds, and Nematodes, vol. 1. Kluwer Academy Plenum, New York. Wong, T.K., Mai, W.F., 1973. Pathogenicity of Meloidogyne hapla to lettuce as affected by bionoculum level, plant age at inoculation and temperature. J. Nematol. 5, 126–129. Zdarska, Z., Huffman, D.G., Moravec, F., Nebesarova, J., 2001. Egg shell ultrastructure of the fish nematode, Huffmanela huffmani (Trichosomoididae). Folia Parasitol. 48, 231–234. Zuckerman, B.M., Dicklow, M.B., Acosta, N., 1993. A strain of Bacillus thuringiensis for the control of plant-parasitic nematodes. Biocontrol Sci. Technol. 3, 41–46.
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery Metasebia Terefe, Tadele Tefera *, P.K. Sakhuja Haramaya University, College of Agriculture, Department of Plant Sciences, P.O. Box 138, Dire Dawa, Ethiopia
a r t i c l e
i n f o
Article history: Received 18 July 2008 Accepted 7 November 2008 Available online 14 November 2008 Keywords: Bacillus firmus Biological control Meloidogyne incognita Tomato
a b s t r a c t Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha1 was effective in reducing the number of galls (75–84%), and increased shoot height (29–31%) and weight (20–24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Root-knot nematodes cause extensive damage to a wide variety of economically important crops, including tomato. Three species of root-knot nematode, Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria are considered the most important on the basis of their worldwide geographical distribution and large host range (Sassser et al., 1982). They are also involved with fungi and bacteria in many disease complexes and can also break down plant resistance to pathogens (Taylor and Brown, 1976). A number of methods for the management of root-knot nematode such as chemical control, organic amendments, resistant varieties, soil solarization and biological control have been tried with different levels of successes for the protection of tomato plants (Randhawa et al., 2001; Sakhuja and Jain, 2001). Chemical management is effective, but expensive and may lead to residue and soil pollution problems. Tomato varieties resistant to root-knot nematode have been developed in some countries, but are not very popular due to their lower yields. An environmentally safe and economically feasible root-knot nematode control practices needs to be available. Biological control * Corresponding author. E-mail address: [email protected] (T. Tefera). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.11.004
is free from residual and adverse environmental effects (Sumeet and Mukerji, 2000). Fungi such as Paecilomyces lilacinus, Pochonia chlamydosporium, Hirsutella rhossiliensis, Dactylella oviparasitica and Cylindrocarpon destructans are well known parasites nematodes (Anastasiadis et al., 2008; Walia and Vats, 2000). A few bacteria such as Pasteuria penetrans directly parasitize nematodes, while many bacteria including of Bacillus, Agrobacterium, Azotobacter, Pseudomonas and Clostridium produce toxins that kill nematodes (Walia et al., 2000). BioNem WP has been developed as a commercial BioNematicide by Agro-green, Minrav, Ashdod, Israel. It contains the bacteria Bacillus firmus and has been reported effective against, M.incognita, Meloidogyne hapla, Heterodera spp., Tylenchulus semipenetrans, Xiphinema index and Ditylenchus dipsaci (Keren-Zur et al., 2000). BioNem is currently introduced to Ethiopia to be used against rootknot nematode in vegetables. In order to utilize a microbial pathogen for practical pest control, registration as a microbial pesticide is necessary. In Ethiopia, a set of efficacy data following the national guidelines should be submitted to register a microbial pesticide. Therefore, this study was conducted to generate empirical information on the efficacy of BioNem against the root-knot nematode M. incognita by testing the effects of the bacterium on nematode egg hatching, juvenile motility, gall formation, population density; and the effect on growth of tomato plants in green house and in field conditions.
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2. Materials and methods 2.1. Establishment of root-knot nematodes culture Tomato plants infected with root-knot nematodes were collected from a vegetable farm in the Haramaya University (HU), Ethiopia. Egg masses were picked off infected roots using forceps and a needle and then allowed to hatch. Juveniles were inoculated around the roots of tomato seedlings (cv. Marglobe) raised in sterilized soil in pots. This was sub cultured to maintain sufficient numbers of root-knot nematodes for subsequent experiments. 2.2. Experiment-I: effect of BioNem on egg hatching, juvenile mobility and recovery of M. incognita 2.2.1. Effect of BioNem on egg hatching About 60-day-old tomato (cv. Marglobe) roots that were heavily infected with root-knot nematodes were collected from the HU vegetable farm on October 8, 2007. Two hours after collection, roots were brought to the Plant Pathology Laboratory of HU, and washed gently using tape water. Using a sterile dissecting needle and forceps the egg masses were separated from the roots, and kept in refrigerator at 10 °C for one day to prevent hatching before application of treatments. Five concentrations of BioNem, i.e., 0.5%, 1%, 1.5%, 2% and 2.5% were prepared by weighing the powdered form of BioNem. About 99.5, 99, 98.5, 98 and 97.5 ml sterile water were added to beakers containing the five respective concentrations of BioNem. The suspension was shaken by hand and kept at room temperature (20– 23 °C) in the laboratory for 10 h. The suspension was filtered using muslin cloth. About 5 ml of the BioNem suspension was added to a sterile Petri dish (9 cm diameter). Five equal sized egg masses were randomly picked using sterile forceps and were placed in each Petri dish containing the suspension. Egg masses kept in sterile water were used as a control. The Petri dish containing the suspension and the egg masses were kept at room temperature on the laboratory bench. The experiment was laid out in completely randomized design (CRD) with three replications. Mean average maximum and minimum temperature were 19.6, 23.5 and 15.6 °C, respectively. Seventy-two hours after treatment application, the Petri dishes were shaken, the suspension containing the nematodes was transferred to a counting dish, nematode juveniles were counted using stereoscopic microscope at magnification of 50. Fresh BioNem suspension was added to the egg masses and kept for hatching again at room temperature. This was done every 72 h, till hatching ceased in the control. The number of nematodes was log-transformed to normalize variances. The transformed data were subjected to an analysis of variance (ANOVA). Treatment means were separated using least significant differences (LSDs). 2.2.2. Effect of BioNem on juvenile mobility About 90-day-old tomato (cv. Marglobe) roots that were infected with root-knot nematodes collected from a vegetable farm in Dire Dawa (Ethiopia) and washed gently under running water. Using dissecting needle and forceps the egg masses were separated from the roots, and kept in the laboratory bench at room temperature (20–23 °C) for three days until juveniles hatched. Six concentrations of BioNem, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% were prepared as described above. The suspension was shaken and kept at room temperature (20–23 °C) in the laboratory for 12 h. The suspension was separated using centrifugation at 3000 rpm for 10 min. About 3-ml suspension of the BioNem was added to a sterile Petri dish (3 cm diameter). Three milliliter suspension containing mean number of 58, 46, 50, 53, 48 and 60 juveniles (J2) of root-knot nematodes were added to the Petri dishes
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with the BioNem suspension at the rates of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, respectively. Three-milliliter suspension containing an average of 56 juveniles, M. incognita, kept in a Petri dish with 3-ml sterile water used as a control. The experiment was laid out in a completely randomized design (CRD) with three replications. Mean, maximum and minimum temperatures were 19.8, 24.2 and 15.7 °C, respectively. The number of motile juveniles was recorded 24, 36 and 48 h after treatment application, using stereoscopic microscope (X50). Juveniles were considered paralyzed (non-mobile) if they did not move when probed with a fine needle. Percentage mobility of the juveniles was arc-sine transformed. The transformed data were subjected to an ANOVA. Treatment means were separated using LSD. 2.2.3. Effect of BioNem on recovery of paralyzed juveniles Recovery of juveniles from the toxic effect of BioNem was checked by randomly taking 10, 11, 11, 14 and 13, paralyzed juveniles from the preceding (mobility test) experiment from the respective concentrations of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%. The paralyzed juveniles were kept in fresh water for 48 h. The experiment was laid out in a completely randomized design with three replications. The number of mobile juveniles was counted 24 and 48 h after keeping the paralyzed juveniles in fresh water using stereomicroscope microscope (50). Percentage recovery of juveniles was log-transformed. The transformed data were subjected to an ANOVA. Treatment means were separated using LSD. 2.3. Experiment-II: effect of BioNem on nematode infestation and growth of tomato plants in the greenhouse Seeds of tomato (cv. Marglobe) were sown in sterile soil in plastic trays in the greenhouse. A 1-month-old seedling was transplanted into each pot containing 1200 cc of sterilized soil with 1:2:3 proportions of sand, compost and clay, respectively. A week after transplanting, the soil around the seedlings was inoculated with M. incognita at 1000 juveniles per plant. Five treatments of BioNem were used. These were 1, 2, 4, 8 and 16 g powder formulation of the BioNem. Each treatment was applied to a tomato seedling in a pot a week after transplanting. There were also two controls: uninoculated control and inoculated control. In uninoculated control, neither the nematode nor the BioNem was applied; whilst in inoculated control, the nematode was applied but the BioNem was not. The experiment was laid out in a randomized complete block design (RCBD) with four replications. Seedlings were watered as needed. No fertilizers were used. The following data were collected 50-days after treatment application. The number of galls per plant was counted manually. Average of 10 egg masses per plant were randomly taken using sterile forceps and dissecting needle. The egg masses were vigorously shaken with 5% sodium hypochlorite in stoppered flasks for 2 min based on the procedure of Hussey and Barker (1973). From the total suspension, a 3-ml suspension was pipetted into a counting dish and the numbers of eggs were counted using a stereomicroscope at a magnification of 50. The length of the plant was measured for each pot from the soil line to the tip of the stem and expressed in cm. The plants were cut at the crown level and the weight was measured using electronic balance. The plant roots were removed from the soil and gently washed with distilled water to remove soil particles. The weight of the roots was recorded using an electronic balance. The fresh shoot, which was brought to a laboratory from the green house using a paper bag, was kept in an oven at 105 °C for 24 h, and the dry shoot weight was recorded. The soil samples were collected from each pot using a labeled paper bags
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and taken to a laboratory. Final population density of nematode was determined based on the Baermann funnel technique (Southey, 1970). The data on plant height, fresh shoot weight, fresh root weight, dry shoot weight, number of galls, number of eggs/egg masses and final nematode population/pot were log-transformed to normalize variances. The transformed data were subjected to an analysis of variance (ANOVA) using Statistical Analysis System (SAS) (version 6.12, SAS, Institute Inc., Cary, NC, USA). Treatment means were separated using the least significant differences (LSDs). Back transformed values were presented. 2.4. Experiment-III: effect of BioNem on root-knot nematodes infestation and the growth of tomato plant in nursery The experiment was conducted at Dire Dawa (Ethiopia) to evaluate the effectiveness of the BioNem under nursery conditions in 2007 cropping season. The site is located at 9°60 N and 41°80 E longitude, 1197 m above sea level, receives 520 mm mean annual rainfall, 28.1–34.6 and 14.5–21.6 °C mean maximum and minimum temperature ranges, respectively. The soil type is Eutric Regosol. Raised nursery beds (20 cm high) were prepared. The plot size was 0. 25 m2 (0.5 m 0.5 m). Prior to establishment of treatments, of finely chopped 8 kg roots of egg plant (Solanum melongena L.) severely infested with root-knot nematode was incorporated into the prepared beds in a uniform manner to make nematode sick plots. Seeds were planted 1–2 cm deep in rows on October 28, 2007. Rows were 10 cm apart and the distance between plots (beds) was 50 cm and between blocks was 1 m. The fertilizer, DAP was applied at the rate of 150 kg ha1 at the time of sowing. A total of four treatments 50, 100, 200 and 400 kg ha1 powder formulation of BioNem were used. There were also treated and untreated controls. In the treated control, the nematicide, phenamiphos (Nemacur 10 G) was used. Whilst in untreated control, neither BioNem nor nematicide was used. The treatments and the nematicide were applied at the time of sowing. The treatments were applied manually with the help of sand as a carrier. The experiment was laid out in a randomized complete block design (RCBD) with four replications. The nursery beds were watered and weeded as required. Plant height, number of galls per plant, biomass (fresh shoot and root weight) was recorded from 15 seedlings collected at random from the 3 middle rows of each plot, 45-days after planting, as described above. Plant height and biomass were subjected to ANOVA using Statistical Analysis System (SAS) (version 6.12, SAS, Institute Inc., Cary, NC, USA). The number of galls was log-transformed transformed to normalize variances. The transformed number of galls was subjected to an analysis of variance (ANOVA). The treatment means were separated using the least significant differences (LSDs). Back transformed values were presented. 3. Results 3.1. Experiment-I: effect of BioNem on egg hatching, juvenile mobility and recovery 3.1.1. Effect of BioNem on egg hatching Application of BioNem at all concentrations significantly inhibited egg hatching (Table 1 and Fig. 1). Some egg hatching occurred to some extent in BioNem treatments in the first 3 days, but it declined drastically in the following 3 days and then stopped completely, while in the control egg hatching continued for up to 24 days. Percent inhibition of egg hatching ranged from 98% to
100%. Application of BioNem at 2.5% concentration caused 100% inhibition. 3.1.2. Effect of BioNem on inhibition and recovery of juvenile mobility There were significant differences (P < 0.05) in mobility of juveniles between the treatments (Table 2). BioNem at 2.5% and 3% concentrations caused 100% inhibition in motility of juveniles 24 h after treatment application. Similarly, none of the juveniles recovered from the paralysis caused by BioNem at 2.5% and 3% concentrations, 24 and 48 h after treatment with water. 3.2. Experiment-II: effect of BioNem on nematode infestation and growth of tomato plants in the greenhouse Application of BioNem significantly (P < 0.05) reduced gall formation (Table 3). The mean number of galls ranged from 99 to 226 in BioNem treated plants compared to 1084 galls in nematode-inoculated control plants. The greatest reduction (91%) in gall formation was recorded from BioNem applied at the rate of 8 g/pot, while the least reduction (79%) in gall formation was obtained at 1 g/pot. However, BioNem applied at 16 g/pot was phytotoxic and resulted in seedling mortality. There were significant differences (P < 0.05) in population density between. Treatment with BioNem at 8 g/pot caused the greatest reduction in nematode populations (75 per 100 cc soil) compared to 306 in the control (Table 3). The percent reduction in final nematode population ranged from 65% to 76% compared to the nematode-inoculated control. There were significant differences (P < 0.05) between treatments in number of eggs per egg mass (Table 3). BioNem applied at different rates adversely affected egg formation. Treatment with the BioNem at 8 g/pot resulted in the least number of eggs (360) compared to 653 eggs being produced in the inoculated control. However, application of BioNem at 1, 2 and 4 g had 44, 429 and 388 eggs, respectively. There were significant differences (P < 0.05) in treatments in affecting plant height and biomass (Table 4). The tallest plant (60 cm) was recorded from the uninoculated control and the shortest from the inoculated control (31 cm). Plant height in the BioNem treatments ranged from 50 to 53 cm which represented a 60–71% increase in height over the inoculated control. Application of BioNem significantly (P < 0.05) improved shoot weight over the inoculated control (Table 4). Heavier (92–110 g) shoot weight was recorded from treatments with BioNem at 2 g and 4 g/pot than in the inoculated control (89 g). There were significant differences (P < 0.05) between treatments in fresh root weight (Table 4). BioNem applied at 1, 2, 4 and 8 g/pot resulted in 32, 30, 29 and 27 g mean fresh root weight, respectively. The smallest fresh root weight (26 g) was recorded from the uninoculated control plants, whilst the largest fresh root weight (52.5 g) was recorded from the inoculated control. 3.3. Experiment-III: effect of BioNem on root-knot nematode infestation and growth of tomato plants in the nursery There were significant differences (P < 0.05) between treatments in gall formation, plant height and plant biomass (Table 5). With increasing concentration of BioNem, gall formation decreased. The least number of galls was formed after treatment with phenamiphos (treated control), followed by BioNem applied at 400 and 200 kg ha1. The tallest plant height (21.9–22.9 cm) was recorded from treatments with 200, 400 kg ha1 and the nematicide treated control as compared to the untreated control (15.7 cm). The largest
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M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99 Table 1 Effect of different concentrations of B. firmus on cumulative hatchability of egg masses of M. incognita 24-days after treatment application. Concentration (%)
No. of juveniles hatched/5 egg masses
% Inhibition of hatching over control
0.5 1.0 1.5 2.0 2.5 Control (0%)
4.86 ± 0.35b 1.71 ± 0.22b 0.71 ± 0.13b 0.29 ± 0.07b 0 ± 0b 279.33 ± 0.91a
98.3 99.4 99.7 99.9 100 —
Means within the same column with a common letter are not significantly different (P < 0.05).
450 400
No. of eggs hatched
350 300 250 200 150 100 50 0 3
6
9
12
15
18
21
24
Time (days) 0%
2%
1%
2.50%
1.50%
0.50%
Fig. 1. Effect of different concentration of BioNem on rate of egg hatching in M. incognita.
biomass (17.5–18.4 g) was recorded from treatment with BioNem at 200 and 400 kg ha1, and nematicide treated control. 4. Discussion In vitro studies have clearly revealed that BioNem inhibits egg hatching of M. incognita. Interfering with the life cycle of root-knot nematode by managing egg masses prior to hatching is important. From a single egg mass up to 500 juveniles can hatch and infect the same root or roots nearby and start a new life cycle (Dropkin,
1989). Three fungi namely Fusarium equiseti, F. oxysporum and Rhizoctonia solani have been reported to produce toxic substances in culture medium, which inhibit the hatching of M. incognita significantly (Sakhuja et al., 1978). Certain strains of Bacillus sphaericus and Bacillus laterosporus (Bone, 1988; Bone, 1991) and Bacillus chitinosporus (Marrone et al., 1998) which inhibit egg hatch of animal parasitic nematodes in their host animals have been patented. Although such observations have been made following growth of the bacteria on rich media in vitro, there is no evidence that such mechanisms are of importance in the rhizosphere where nutrients are much less abundant. Saxena et al. (2000) reported that production of different types of antibiotics and enzymes is nature of Bacillus species, which are strongly antagonistic to several pathogens such as root-knot nematode. Nematode egg shells consists of three layers namely, vitelline, chitinous and lipid layers (Zdarska et al., 2001). It might be possible that B. firmus secretes some toxins, which damages the egg shell of root-knot nematode. During the initial few days hatching, though less, but occurred. However, later it stopped completely and during this period bacteria might have produced enough toxins to damage the egg shell or the juveniles within the eggs. The hatch inhibitory and egg disintegrating effect of the BioNem might be due to certain toxic substances present in the product or secreted by B. firmus that possess ovicidal property. Mendoza et al. (2008) reported pure culture filtrates of B. firmus reduced M. incognita egg hatching. Al-Banna and Khyami-Horani (2004) isolated local strains of Bacillus thuringiensis from various locations and showed their lethality to eggs and second-stage juveniles (J2) of root-knot nematodes under laboratory conditions. Deny-another commercial biocontrol product for nematodes is based on a natural isolate of the bacterium Burkholderia cepacia, which also reduces egg hatching and juvenile mobility (Meyer and Roberts, 2002). Rhizobacteria reduce nematode populations mainly by production of toxins (Siddiqui and Mahmood, 1999), induction of systemic resistance (Hasky-Gunther et al., 1998), changing nematode behavior (Sikora and Hoffmann-Hergarter, 1993) and interfering with plant recognition (Oostendorp and Sikora,1990) and promoting plant growth (El-Nagdi and Youssef, 2004). A commercial transplant mix (Bio yieldTM, Gustafson LLC) containing Paenobacillus macerans and Bacillus amylolique faciens has been developed to control plant parasitic nematodes on tomato, bell pepper and straw berry in USA (Meyer, 2003). BioNem significantly reduced nematode population and root infestation, from their extensive testing on vegetable crops including tomato, cucumber, pepper, garlic and some herbs (Giannakou and Prophetou-Athanasiadou, 2004; Giannakou et al. 2007). Treatment with BioNem adversely affected juvenile mobility. Giannakou and Prophetou-Athanasiadou (2004) reported that the biocontrol effects of BioNem could be partially attributed to the
Table 2 Effect of B. firmus on inhibition of M. incognita juvenile mobility in vitro. Concentration (%)
0.5 1.0 1.5 2.0 2.5 3.0 Control
Inhibition of mobility in B. firmus suspension (%)
Recovery of mobility in water (%)
24 h
36 h
48 h
24 h
48 h
52.3 ± 5.6c 54.8 ± 4.8c 60.7 ± 6.4c 82.6 ± 2b 100 ± 0a 100 ± 0a 2.0 ± 1d
55.7 ± 5c 59.2 ± 4.5c 66.9 ± 8.8c 86.3 ± 2b —* — 2.0 ± 1d
62.6 ± 5.3c 73 ± 9.8cd 80 ± 6.7bc 92.5 ± 0.9ab — — 2.0 ± 1d
34 ± 6.8a 31 ± 5.7ab 19 ± 7.5b —** — — —
47 ± 11.7a 31.7 ± 5.7ab 19 ± 7.5b — — — —
Means within the same column with a common letter are not significantly different (P < 0.05). * No mobility recorded as all juveniles were inhibited 24 h after treatment application. ** No recovery recorded as at 2.5% and 3% concentrations.
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M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99
Table 3 Effect of B. firmus (BioNem) WP on infestation of M. incognita in tomato plant in the greenhouse. BioNem/pot (g)
Galls/root
Reduction in gall over IC (%)
Final nematode population
Reduction in final population over IC (%)
Number of eggs/egg mass
Reduction in number of eggs over IC (%)
1 2 4 8 IC UC
226 ± 2.2b 164 ± 1.6c 123 ± 2.9d 99 ± 3e 1084 ± 5.4a —*
79 85 89 91 — —
106 ± 2.7b 95 ± 2bc 89.5 ± 2c 75 ± 1.7d 306 ± 5a —
65 69 71 76 — —
444 ± 2.2b 429 ± 3.5c 388 ± 2.7d 360 ± 2e 653 ± 3a —
32 38.6 41 45 — —
Means within the same column with a common letter are not significantly different (P < 0.05). IC, inoculated control. UC, uninoculated control. * No nematode was recorded in uninoculated control.
Table 5 Effect of B. firmus WP on M. incognita infestation and growth of tomato plants in nursery. BioNem (kg ha1)
Galls/plant
Reduction in galls over UC (%)
Plant height (cm)
Increase in height over UC (%)
Plant biomass (g)
Increase in biomass over UC (%)
50 100 200 400 TC UC
2.5 ± 0.2b 1.85 ± 0.1bc 1.25 ± 0.1cd 0.83 ±2.1d 0.78 ± 0.1d 5.5 ± 0.64a
53.87 64.63 75.68 84.00 85.40 —
20.1 ± 0.47b 20.9 ± 0.24b 22.3 ± 0.19a 22.9 ± 0.26a 21.95 ± 0.4a 15.7 ± 0.44c
21.6 24.6 29.3 31.4 28.3 —
15.5 ± 0.44b 16.1 ± 0.74b 17.5 ± 0.66a 18.4 ± 0.53a 17.5 ± 0.86a 13.4 ± 0.24c
10.6 13.3 20.9 24.4 20.1 —
TC, treated control (phenamiphos at 11 kg a.i./ha). UC, untreated control. Means within the same column with a common letter are not significantly different (P < 0.05).
Table 4 Effect of B. firmus WP on growth of tomato plant in M. incognita infested soil in greenhouse. BioNem/pot (g)
Plant height (cm)
Increase in height over IC (%)
Fresh shoot weight (g)
Increase in fresh shoot weight over IC (%)
Fresh root weight (g)
1 2 4 8 IC UC
50.0 ± 0.95c 53.1 ± 0.4b 53.0 ± 0.65b 53.6 ± 0.41b 31.3 ± 0.75d 60.0 ± 1.8a
59.74 69.74 69.32 71.24 – 92.26
87.0 ± 0.8c 92.4 ± 1b 92.9 ± 0.7b 89 ± 0.7c 59.0 ± 2d 110 ± 1.7a
47.45 56.61 57.45 50.84 — 86.44
32.0 ± 0.8b 30.0 ± 0.8c 29.0 ± 1.5c 27.0 ± 0.4d 52.5 ± 1a 26.0 ± 0.4d
IC, inoculated control. UC, uninoculated control. Means within the same column with a common letter are not significantly different (P < 0.05).
stimulating effect that the animal and plant additives contained in the BioNematicide formulation. Cayrol et al. (1989) found that culture filtrates of P. lilacinus were lethal to juveniles of root-knot nematode. Olubunmi and Rajani (2004) have also reported 100% mortality of juveniles of M. incognita with culture filtrates of Trichoderma. Siddiqui et al. (2008) also reported that Pseudomonas and Bacillus spp. inhibited hatching and penetration of M. incognita. The inhibition of motility by BioNem might be due to certain nematostatic and or nematicidal substances present in the product or secreted by B. firmus. Application of the BioNem to M. incognita reduced the number of galls, number of eggs, and final nematode population in tomato plants both in the greenhouse and under field condition. With the increase in application rate there was corresponding reduction in the number of galls and final population density. BioNem contains 3% lyophilized B. firmus spores and 97% additives (plant and animal extracts) (Giannakou and Prophetou-Athanasiadou, 2004). The mortality of tomato seedlings when treated with BioNem at 16 g/ pot is probably due to the high concentration of the additives. Increases in plant growth are due to the reduction in nematode attack as evidenced by the low final nematode population density. Formation of galls on the roots and colonization of root tissue by this nematode deprives plants of nutrients (Bird, 1974). The damage also occurs due to devitalization of root tips, which may stop
their growth or cause excessive branching of roots. Olubunmi and Rajani (2004) also reported that the galls on the root system disturb important root functions such as the uptake and transport of water and nutrients. The root weight of inoculated control plants was greater than BioNem applied plants. The difference in root weight was not related to the weight of the shoot. This could be due to the formation of large number of galls on inoculated plants. Wong and Mai (1973) also reported that differences in root weight may be explained by gall development; gall mass being heavier than an equivalent linear length of similar non-galled roots. In the present study, application of BioNem at sowing reduced infestation on seedlings and improved plant growth in the nursery. In contrast to the manufacturers recommendation for tomato (http://www.agrogreen.co.il/bionem-product.asp), (80 kg ha1) however, application of BioNem at 200 kg ha1 provided 75% reduction in galling, increased seedling height by 29% and weight by 21% over untreated control. Previous studies have also shown that Bacillus thuringensis (Bt) strain CR-371 applied as a soil drench led to a 53% reduction of tomato root galling caused by M. incognita (Zuckerman et al., 1993). Oostendorp and Sikora, (1990) also reported significant suppression of nematode multiplication by Pseudomonas fluorescens, which was due to its capability of altering root exudates, which could alter nematode behavior and suppress nematode populations in the root system. The shorter height of seedlings of untreated controls
M. Terefe et al. / Journal of Invertebrate Pathology 100 (2009) 94–99
might be due to the stunting action of M. incognita. This type of stunting or reduced heights caused by root-knot nematode has been reported earlier (Thimann, 1960; Jinfa et al., 2006). In conclusion, BioNem WP provides effective control that favorably compared with nematicides applications. A single application of BioNem at planting was sufficient for the entire season. This indicates the unique ability of BioNem to be used in the integrated management of root-knot nematode in tomato. Acknowledgment The General Chemical Trading (PLC), Addis Abeba, is acknowledged for sponsoring this work as part of BioNem Testing Project. References Al-Banna, L., Khyami-Horani, H., 2004. Nematicidal activity of two Jordanian strains of Bacillus thuringiensis on root-knot nematodes. Nematol. Mediterr. 32, 41–45. Anastasiadis, I.A., Giannakou, I.O., Prophetou-Athanasiadou, D.A., 2008. The combined effect of the application of a biocontrol agent Paecilomyces lilacinus, with various practices for the control of root-knot nematodes. Crop Prot. 27, 352–361. Bird, A.F., 1974. Plant responses to root-knot nematodes. Ann. Rev. Phytopathol. 12, 69–85. Bone, W.L., 1988. Control of parasitic nematode ova with Bacillus sphaericus. U.S. Patent No. 4781922. Bone, W.L., 1991. Control of parasitic nematode ova/larvae with a Bacillus laterosporus. U.S. Patent 5045314. Cayrol, J., Djian, C., Pijarowski, L., 1989. Study of the nematicidal properties of the culture filtrate of the nematophagus fungus Paecilomyces lilacinus. Rev. Nematol. 12, 331–336. Dropkin, V.H., 1989. The genera of phytonematodes. In: Introduction to Plant Nematology. Wiley-Interscience Publication, pp. 89–217. El-Nagdi, W.M.A., Youssef, M.M.A., 2004. Soaking faba bean seed in some bio-agent as prophylactic treatment for controlling Meloidogyne incognita root-knot nematode infection. J. Pestic. Sci. 77, 75–78. Giannakou, I.O., Anastasiadis, I.A., Gowen, S.R., Prophetou-Athanasiadou, D.A., 2007. Effects of a non-chemical nematicide combined with soil solarization for the control of root-knot nematodes. Crop Prot. 26, 1643–1644. Giannakou, I.O., Prophetou-Athanasiadou, D., 2004. A novel non-chemical nematicide for the control of root-knot nematodes. Appl. Soil Ecol. 26, 69–79. Hasky-Gunther, K., Hoffmann-Hergarten, S., Sikora, R.A., 1998. Resistance against the potato cyst nematode Globodera pallida systematically induced by the rhizobacteria Agrobacterium radiobacter (G12) and Bacillus sphaericus (B43). Fund. Appl. Nematol. 21, 511–517. Hussey, R.S., Barker, K.R., 1973. A comparison of methods of collecting inocula of Meloidogyne spp. including a new technique. Plant Dis. Rep. 57, 1025–1028. Jinfa, Z., Waddell, C., Sengupta, G.C., Potenza, C., Cantrell, R.G., 2006. Crop Sci. 46, 1581–1586. Keren-Zur, M., Antonov, J., Bercovitz, A., Feldman, K., Husid, A., Kenan, G., Markov, N., Rebhun, M., 2000. Bacillus firmus formulations for the safe control of rootknot nematodes. In: Proceedings of the Brighton Crop Protection Conference on Pests and Diseases, vol. 2A, pp. 47–52. Marrone, P.G., Manker, D.C., Heins, S.D., Jimenez, D.R., 1998. Nematicidal Bacillus strain and metabolite and methods of thereof. U.S. Patent 5733544. Mendoza, A.R., Kiewnick, S., Sikora, R.A., 2008. In vitro activity of Bacillus firmus against the borrowing nematode Radopholus similis the root-knot nematode Meloidogyne incognita and the stem nematode Ditylenchus dipsaci. Biocontrol Sci. Technol. 18, 377–389.
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