US20150282488A1 - Compositions and methods for controlling plant-parasite nematode - Google Patents

Compositions and methods for controlling plant-parasite nematode Download PDF

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US20150282488A1
US20150282488A1 US14/426,806 US201314426806A US2015282488A1 US 20150282488 A1 US20150282488 A1 US 20150282488A1 US 201314426806 A US201314426806 A US 201314426806A US 2015282488 A1 US2015282488 A1 US 2015282488A1
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plant
biosurfactant
isolated
activity
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Jennifer Lynn Riggs
Eder Leonardo Sastoque Cala
Joseph W. Kloepper
Katheryn Kay Scott Lawrence
Juan David Castillo Russi
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Bayer CropScience LP
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Assigned to BAYER CROPSCIENCE LP reassignment BAYER CROPSCIENCE LP ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CALA, EDER LEONARDO SASTOQUE, KLOEPPER, JOSEPH W, CASTILLO-RUSSI, JUAN DAVID, LAWRENCE, KATHERYN KAY SCOTT, RIGGS, JENNIFER LYNN
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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    • A01N63/22—Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)

Definitions

  • the disclosure also provides for compositions comprising the same, methods of making the same, methods of controlling plant-parasite nematode, and methods of protecting plants.
  • Bacteria are an important group of natural antagonists of plant-parasitic nematodes. Bacteria are distributed broadly, have diverse modes of action, and have broad host ranges (Tian, B. et al.). They exhibit diverse modes of action against nematodes that include parasitism, production of toxins, antibiotics, or lytic enzymes; induce systemic resistance, and promote plant health (Aatlen, P. M. et al.; Kerry, B. R. 2000; Kerry, B. R. 1987; Siddiqui, Z. A. et al.; Stirling, G. R. 1991; Tian, B. et al.; Van Loon, L. C. et al.).
  • bacteria can be in direct contact with the entrance sites of the nematodes and influence root exudates that can affect the nematode development (Sikora, R. A. 1992).
  • the genera Pasteuria, Pseudomonas , and Bacillus have shown promising potential for nematode biocontrol (Meyer S. L. F; Siddiqui, Z. A. et al.; Stirling, G. R.; Tian, B. et al.).
  • Bacillus firmus strain GB-126 is a nematode biocontrol agent registered initially in a bionematicide in Israel under the trade name of BIONEM® WP (Blachinsky, D. et al.; Keren-Zur et al.). This formulation was shown to reduce galling index caused by Meloidogyne spp. on cucumber and tomato plots (Keren-Zur et al.). Also, under field conditions, suppression of Meloidogyne spp. was observed within 2 months of transplanting cucumbers and continued through the end of the experiments (Giannakou, O. I. et al., 2004). Control provided by B. firmus GB-126 was less effective than the soil fumigant dazomet.
  • B. firmus that contains seaweed extract (BIONEM® L) was able to reduce Helicotylenchus spp. and Tylenchorhynchus spp. in golf greens (Wick, R. L. 2006). Furthermore, synergism of B. firmus with other nematode biocontrol agents has been reported to improve nematode reduction (Mendoza, A. R. et al. 2009). In banana, B. firmus was evaluated against R. similis and applied in combination with F. oxysporum and P. lilacinus , which reduced the infection of this migratory endoparasitic nematode (Mendoza, A. R. et al. 2009).
  • An object of the present invention is to provide a novel entity having anti-nematodal activity derived from Bacillus firmus bacteria selected from the group consisting of isolated biosurfactant, isolated protease, isolated amylase, isolated lipase, and isolated cellulase, is disclosed. Another object is to provide for compositions comprising the same. Another object is to provide for methods of making the same.
  • a further object is to provide for methods of controlling plant-parasite nematode, and methods of protecting plants.
  • the disclosure provides for an a novel entity having anti-nematodal activity derived from Bacillus firmus bacteria selected from the group consisting of isolated biosurfactant, isolated protease, isolated amylase, isolated lipase, and isolated cellulose.
  • the disclosure provides for a novel composition having anti-nematodal activity comprising at least one entity derived from Bacillus firmus bacteria selected from the group consisting of an isolated biosurfactant, an isolated protease, an isolated amylase, an isolated lipase, and an isolated cellulase.
  • the disclosure provides for an extract of a Bacillus firmus culture having the following characteristics: anti-nematodal activity and presence of a biosurfactant.
  • the disclosure provides for an extract of a Bacillus firmus culture having the following characteristics: anti-nematodal activity and activity selected from the group consisting of protease activity, amylase activity, lipase activity, and cellulase activity.
  • the disclosure also provides for a method of making the same.
  • the disclosure also provides for a method of controlling plant-parasitic nematode by applying an entity or composition as described herein to to a plant, the seed material, or the area on which the plant grows.
  • the disclosure provides for a method of protecting a plant by applying an entity or composition as described herein to a plant, the seed material, or the area on which the plant grows.
  • the plant-parasitic nematode is Rotlyenchulus reniformis.
  • FIG. 1 sets forth the effects of B. firmus metabolites at 100% and 50% concentration on R. reniformis eggs as compared to water and media control.
  • FIG. 2 sets forth the effects of B. firmus metabolites at 100% and 50% concentration on R. reniformis second stage juveniles as compared to water and media control.
  • FIG. 3 sets forth tests of biosurfactant production of B. firmus GB-126.
  • A represents the positive emulsification of kerosene.
  • B represents halo formation due to cell lysis in blood agar.
  • C represents positive oil drop collapse test.
  • FIG. 4 sets forth the effect of biosurfactant produced by B. firmus on R. reniformis second stage juveniles after 30 minutes of inoculation (P ⁇ 0.05) as compared to BP media and water controls and B. firmis bacteria.
  • FIG. 5 sets forth the effect of purified biosurfactant produced by B. firmus at different concentrations (ppm) on R. reniformis second stage juveniles after 30 minutes of inoculation under in vitro conditions (P ⁇ 0.05).
  • FIG. 6 sets forth the photos of second stage juveniles of R. reniformis form (control and after application of 2 ppm B. firmis biosurfactant) observed under SEM in in vitro trial.
  • FIG. 7 sets forth photos of growth of cotton plants after systemic resistance trial of B. firmus GM-126 and S. maracescens against R. reniformis in cotton plants.
  • FIG. 8 sets forth the enzyme reaction test of B. firmus GB-126: (A) represents production of proteases, (B) represents production of amylases, (C) represents production of cellulases, and (D) represents production of chitinases.
  • FIG. 9 sets forth R. reniformis life stages with cotton seeds treated with B. firmus GB-126 at 7 ⁇ 10 6 cfu/seed under greenhouse conditions in autoclaved soil (P ⁇ 0.05).
  • the disclosure provides for an a novel entity having anti-nematodal activity derived from Bacillus firmus bacteria selected from the group consisting of isolated biosurfactant, isolated protease, isolated amylase, isolated lipase, and isolated cellulase.
  • Anti-nematodal activity is understood as meaning any activity useful in controlling or killing nematodes, such as reducing nematode egg hatching or paralyzing juveniles of nematodes. Testing for anti-nematodal activity is done both in-vitro and in-vivo through laboratory and greenhouse procedures. In-vitro testing can involve testing eggs and/or juvenile nematodes in selective media in small 96-well plates and measurements done by microscopic evaluations. Proper control treatments would be combined to monitor and compare activity. Under greenhouse studoes soil was autoclaved using two 90 minute cycles at 130° C. at 1.0 kg/cm 3 pressure with a 24 hour cool down between cycles to remove any natural competition for the microflora.
  • vermiform stages were extracted from the soil by the modified gravity screening and sucrose centrifugation-flotation. Eggs stages were extracted from cotton roots by shaking the root system in a 1% NaOCl solution for four minutes at 120 rpm. The nematode suspension was collected and rinsed with water through a 25 ⁇ m sieve. Females in roots were stained with acid fuschin to facilitate enumeration of the females invading the root. Variables measured included plant height, shoot and root weights, females and eggs per gram of root, and the number of vermiform life stages in soil.
  • the strain of Bacillus firmus is GB-126.
  • the entity described herein has anti-nematodal activity against Reniform nematode, Rotlyenchulus spp.; Dagger nematode, Xiphinema spp.; Lance nematode, Hoplolaimus spp.; Pin nematode, Paratylenchus spp.; Ring nematode, Criconemoides spp.; Rootknot nematode, Meloidogyne spp.; Sheath nematode, Hemicycliophora spp.; Spiral nematode, Helicotylenchus spp.; Stubbyroot nematode, Trichodorus spp.; Cyst nematode, Heterodera spp.; Sting nematode, Belonolaimus , spp.; and/or Stunt
  • the entity as described herein is a biosurfactant obtainable by a method comprising: obtaining a medium in which Bacillus firmus bacteria has been grown and precipitating the biosurfactant from the medium.
  • Biosurfactant is understood to mean microbially produced surface-active compounds. They are amphiphilic molecules with both hydrophilic and hydrophobic regions causing them to aggregate at the interfaces between fluids with different polarities such as water and hydrocarbons (Jennings E. R. et al., 2000).
  • B. firmus was grown under blood agar, where a positive production of biosurfactant is indicated by a transparent halo around the bacterial colony. Additional testing, B. firmus was cultured on nutrient broth at 30° C. for 24 hours. Subsequently, the living cells were recovered by centrifugation at 5,181 xg for 15 minutes, and cells were washed twice with NaCl 0 . 85 % (w/v) and later suspended in 5 ml of NaCl0.85% (w/v). They were used to inoculate 45 ml of saline Davis minimal broth with an inoculum ratio of 1% (v/v).
  • the composition was K 2 HPO 4 5.23 g/l, KH 2 PO 4 1.91 g/l, MgSO 4 0.09 g/l, (NH4)2SO 4 1 g/l, as well as 1 ml/l of trace elements solution (CoCl 3 20 mg/l, H 3 BO 3 30 mg/l, ZnSO 4 10 mg/l, Cu 2 SO 4 1 mg/l, Na 2 MoO 4 3 mg/l, FeSO 4 10 mg/l and MgSO 4 2.6 mg/l). Cultures were incubated at 30° C. ⁇ 2 at 150 rpm for 3 days. Again B. firmus living cells were separated from the supernatant by centrifugation (20 minutes at 4000 xg).
  • the supernatant was filtered through a Millipore filter 0.45-0.22 ⁇ m to obtain the final bacterial biosurfactant product.
  • This final product was autoclaved twice for 30 minutes at 120° C. at 1 kg/cm 3 pressure to kill all the bacterium's living cells and inactivate its enzymes.
  • emulsifying activity of the cell-free supernatant was evaluated by mixing 0.5 ml with 0.5 ml of kerosene and 4 ml of distilled water to a disposable culture tube (borosilicate glass 16 ⁇ 150 mm)
  • the negative control consisted of distilled water and kerosene
  • the positive control consisted of distilled water, kerosene, and Triton X-100 (100 mg/ml).
  • the medium in which Bacillus firmus bacteria has been grown is obtained by growing B. firmus bacteria aerobically in medium.
  • biosurfactant isolation bacterial cells are removed from the surfactant-containing medium by centrifugation, and the biosurfactant is precipitated from the supernatant by adding an acid. The acid precipitates are recovered by centrifugation and are extracted with a solvent. After precipitation with an acid, the crude fraction dissolved in the solvent is evaporated, and the final purified biosurfactant product is diluted in distilled water at specific concentrations.
  • the entity as described herein is an isolated protease.
  • Protease is understood to mean any polypeptides or complex of polypeptides or fragments of polypeptides having protease activity. Production of protease enzymes were evaluated by use of milk agar assay. A clear halo following 24 hr period of incubation is recorded as a positive indication for the production of proteases.
  • the entity as described herein is an isolated amylase.
  • Amylase is understood to mean any polypeptides or complex of polypeptides or fragments of polypeptides having amylase activity. Production of amylase enzymes were evaluated by use of starch agar assay. A clear halo following 24 hr period of incubation is recorded as a positive indication for the production of amylases.
  • the entity as described herein is an isolated lipase.
  • Lipase is understood to mean any polypeptides or complex of polypeptides or fragments of polypeptides having lipase activity. Production of lipase enzymes were evaluated by use of trybutirin agar assay. A clear halo following 24 hr period of incubation is recorded as a positive indication for the production of lipases.
  • the entity as described herein is an isolated cellulase.
  • Cellulase is understood to mean any polypeptides or complex of polypeptides or fragments of polypeptides having cellulase activity.
  • Production of cellulase enzymes were evaluated by use of carboximetil cellulose (CMC) agar agar assay. A clear halo following 24 hr period of incubation is recorded as a positive indication for the production of cellulases.
  • CMC carboximetil cellulose
  • the disclosure provides for a novel composition having anti-nematodal activity comprising at least one entity derived from Bacillus firmus bacteria selected from the group consisting of an isolated biosurfactant, an isolated protease, an isolated amylase, an isolated lipase, and an isolated cellulase.
  • the strain of Bacillus firmus is GB-126.
  • the composition described herein has anti-nematodal activity against Rotlyenchulus reniformis.
  • the composition described herein comprises an acid precipitate from a culture medium in which the Bacillus firmus bacteria was grown.
  • the acid precipitate described herein is obtainable by a method comprising: adding an acid to a medium in which a Bacillus firmus bacteria has been grown to generate an acid precipitate, and (b) isolating the acid precipitate from the medium.
  • the acid precipitate has anti-nemadodal activity.
  • the medium in which Bacillus firmus bacteria has been grown is obtained by growing B. firmus bacteria aerobically in medium.
  • biosurfactant isolation bacterial cells are removed from the surfactant-containing medium by centrifugation, and the biosurfactant is precipitated from the supernatant by adding an acid. The acid precipitates are recovered by centrifugation and are extracted with a solvent. After precipitation with an acid, the crude fraction dissolved in the solvent is evaporated, and the final purified biosurfactant product is diluted in distilled water at specific concentrations.
  • the composition as described herein comprises of an isolated biosurfactant.
  • the isolated biosurfactant concentration in the composition as described herein is greater than 0.5 ppm.
  • the isolated biosurfactant concentration in the composition described herein is at least 1 ppm.
  • the isolated biosurfactant concentration in the composition described herein is from 1 to 2 ppm.
  • the isolated biosurfactant concentration in the composition described herein is at least 2 ppm.
  • the isolated biosurfactant concentration in the composition described herein is from 1 to 5 ppm.
  • the isolated biosurfactant concentration in the composition described herein is from 2 to 5 ppm.
  • composition as described herein is an isolated protease.
  • composition as described herein is an isolated amylase.
  • composition as described herein is an isolated lipase.
  • composition as described herein is an isolated cellulase.
  • the disclosure provides for an extract of a Bacillus firmus bacteria having the following characteristics: anti-nematodal activity and presence of a biosurfactant.
  • the disclosure provides for an extract of a Bacillus firmus bacteria having the following characteristics: anti-nematodal activity and activity selected from the group consisting of protease activity, amylase activity, lipase activity, and cellulase activity.
  • Extract shall refer to any fraction extracted from reference material.
  • the disclosure also provides for a method of controlling plant-parasitic namatode by applying an entity or composition as described herein to a plant, the seed material (e.g. grains, seeds or vegetative propagation organs such as tubers or shoot parts with buds) or the area on which the plants grow (e.g. the area under cultivation).
  • the seed material e.g. grains, seeds or vegetative propagation organs such as tubers or shoot parts with buds
  • the area on which the plants grow e.g. the area under cultivation.
  • the disclosure provides for a method of protecting a plant by applying an entity or composition as described herein to a plant, the seed material, or the area on which the plant grows.
  • Plants are to be understood as meaning in the present context all plants and plant populations such as desired and undesired wild plants or crop plants (inclusive of naturally occurring crop plants).
  • Crop plants can be plants which can be obtained by conventional plant breeding and optimisation methods or by biotechnological and recombinant methods or by combinations of these methods, inclusive of the transgenic plants and inclusive of the plant varieties protectable or not protectable by plant breeders' rights.
  • Plant parts are to be understood as meaning all aerial and subterranean plant parts and organs of the plants such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, trunks, flowers, fruiting bodies, fruits, seeds, roots, tubers and rhizomes.
  • the plant parts also include vegetative and generative propagation material, for example cuttings, tubers, rhizomes, seedlings and seeds.
  • the plant selected from the group consisting of agronomic crops, corn (field, seed & popcorn), sorghum, wheat, barley, rye, oats, rice, forage grasses, soybeans, canola, peanuts, cotton, alfalfa, fruting vegetables (peppers & tomatoes), melons, squash, pumpkins, cucumbers and potatoes.
  • agronomic crops corn (field, seed & popcorn), sorghum, wheat, barley, rye, oats, rice, forage grasses, soybeans, canola, peanuts, cotton, alfalfa, fruting vegetables (peppers & tomatoes), melons, squash, pumpkins, cucumbers and potatoes.
  • the plant-parasitic nematode is Reniform nematode, Rotlyenchulus spp.; Dagger nematode, Xiphinema spp.; Lance nematode, Hoplolaimus spp.; Pin nematode, Paratylenchus spp.; Ring nematode, Criconemoides spp.; Rootknot nematode, Meloidogyne spp.; Sheath nematode, Hemicycliophora spp.; Spiral nematode, Helicotylenchus spp.; Stubbyroot nematode, Trichodorus spp.; Cyst nematode, Heterodera spp.; Sting nematode, Belonolaimus , spp.; and/or Stunt nematode, Tylenchorhynchus spp.
  • the plant is cotton. In an aspect the plant is agronomic crops. In an aspect, the plant is corn (field, seed & popcorn). In an aspect, the plant is sorghum. In an aspect, the plant is wheat. In an aspect, the plant is barley. In an aspect, the plant is rye. In an aspect, the plant is oat. In an aspect, the plant is rice. In an aspect, the plant is forage grasses. In an aspect, the plant is soybeans. In an aspect, the plant is canola. In an aspect, the plant is peanuts. In an aspect, the plant is cotton. In an aspect, the plant is alfalfa. In an aspect, the plant is fruting vegetables (peppers & tomatoes). In an aspect, the plant is melon. In an aspect, the plant is squash. In an aspect, the plant is pumpkin. In an aspect, the plant is cucumber. In an aspect, the plant is potato.
  • the eggs were placed in a modified Baerman dish on a slide warming tray at 27° C. Second stage juveniles were hatched after three days.
  • Bacillus firmus GB-126 was grown in 50 mL of Tryptic Soy Broth (TSB) (BACTOTM) for four days and then placed in 50 mL plastic tubes and centrifuged for 20 minutes at 4000x g. The supernatant was collected and filtered through a Millipore filter 0.45-0.22 ⁇ m to obtain the final bacterial metabolite product (CFE).
  • TAB Tryptic Soy Broth
  • Table 1 demonstrates the statistical significance of FIG. 1 .
  • Table 2 demonstrates the statistical significant of FIG. 2 .
  • B. firmus GB-126 Different enzymatic properties of B. firmus GB-126 were evaluated to test their capacity to degrade different media. Production of enzymes was evaluated as positive when a transparent halo was formed around the bacterium culture. Bacillus firmus GB-126 was grown for 24 hours on milk agar to test the production of proteases, starch agar for the production of amylases, carboximetil cellulose (CMC) agar for the production of cellulases, chitinase agar for the production of chitinases, and trybutirin agar for the production of lipases.
  • CMC carboximetil cellulose
  • the CMC agar and chitinase agar required the application of 5 ml of congo red to stain the media and a transparent halo after 24 hours of culturing the bacteria.
  • culture was stained with lugol.
  • the composition was K 2 HPO 4 5.23 g/l, KH 2 PO 4 1.91 g/l, MgSO 4 0.09 g/l, (NH 4 ) 2 SO 4 1 g/l, as well as 1 ml/l of trace elements solution (CoCl 3 20 mg/l, H 3 BO 3 30 mg/l, ZnSO 4 10 mg/l, Cu 2 SO 4 1 mg/l, Na 2 MoO 4 3 mg/l, FeSO 4 10 mg/l and MgSO 4 2.6 mg/l). Cultures were incubated at 30° C. ⁇ 2 at 150 rpm for 3 days. Again B. firmus living cells were separated from the supernatant by centrifugation (20 minutes at 4000x g).
  • the supernatant was filtered through a Millipore filter 0.45-0.22 pm to obtain the final bacterial biosurfactant product.
  • This final product was autoclaved twice for 30 minutes at 120° C. at 1 kg/cm 3 pressure to kill all the bacterium's living cells and inactivate its enzymes.
  • emulsifying activity of the cell-free supernatant was evaluated by mixing 0.5 ml with 0.5 ml of kerosene and 4 ml of distilled water to a disposable culture tube (borosilicate glass 16 ⁇ 150 mm).
  • the negative control consisted of distilled water and kerosene
  • the positive control consisted of distilled water, kerosene, and Triton X-100 (100 mg/ml).
  • Each tube was agitated in a vortex for 1 min and was left to stand for 24 hours. The height of the emulsification ring was then measured in millimeters and compared to that of the chemical emulsifier.
  • the third test consisted of an oil drop collapse in which one drop of the supernatant was placed on parafilm paper and a drop of oil was placed on top of it. If the drop of oil increased its diameter compared to the media control, the bacteria was considered to have produced a biosurfactant.
  • B. firmus GB-126 was grown aerobically on minimal salt medium containing (per liter) KH 2 PO 4 (2.0 g), K 2 HPO 4 (5.0 g), (NH 4 ) 2 SO 4 (3.0 g), NaNO 3 (2.0 g), NaCl (0.1 g), MgSO 4 H 2 O (0.2 g), 0 FeSO 4 7H 2 O (0.01 g), CaCl 2 (0.01 g), and 1 ml of a trace element solution.
  • the stock solution of trace elements contained (per liter) ZnSO 4 7H 2 O (2.32 g), MnSO 4 4H 2 O (1.78 g), H 3 BO 3 (0.56 g), CuSO 4 5H 2 O (1 g), Na 2 MoO 4 7H2O (0.39 g), CoCl 2 6H 2 O (0.42 g), EDTA (1 g), NiCl 2 6H 2 O (0.004g), and KI (0.66 g).
  • the medium was supplemented with 0.05% yeast extract (Vater, J. et al.) Glucose was added as a carbon source at a concentration of 2% (wt/vol).
  • the medium pH was 7.1 to 7.2.
  • the organism was grown at 37° C. for 48 h in 2-liter Erlenmeyer flasks containing 800 ml of medium and shaken at 200 rpm in a shaker incubator.
  • bacterial cells were removed from the surfactant-containing medium by centrifugation (13000x g for 15 min at 4° C.).
  • the biosurfactant was precipitated from the supernatant by adding 6 N HCl to obtain a final pH of 2.0.
  • the acid precipitates were recovered by centrifugation (13000x g for 15 min at 4° C.) and were extracted with dichloromethane or methanol (lipopeptide fraction). When methanol was used as the solvent, the extract was neutralized immediately to avoid formation of methyl esters.
  • Plants were planted in 960 cm 3 pots with each root half in a different cup. At 7 days after splitting the roots, a suspension of 50 mL of treatments was applied on the left side of the root. Five days later, the right side of the root was inoculated with 500 second stage juveniles of R. reniformis . The trial was harvested 45 DAP, and plant height, root fresh weight, and number of females and eggs per gram of root were measured. Each treatment had 6 replications and the entire trial was repeated twice.
  • the soil was a Decatur silty clay loam (sand-silt-clay: 17.5-51.3-31.2%; nitrogen: 0.16%; organic matter: 2.2; pH 7.24) from the Tennessee Valley Research and Extension Center (TVREC) near Belle Mina, Ala.
  • the soil was autoclaved using two 90-minute cycles at 130° C. at 15 psi with a 24 hour cool down between cycles.
  • Seed treatments were as follows: i) untreated seed with nematodes; ii) imidacloprid (500 g ai/100 kg) a standard insecticide; iii) B.
  • Rotlyenchulus reniformis vermiform life stages were extracted from the soil by modified gravity screening and sucrose centrifugation-flotation. Eggs were extracted from cotton roots by shaking the root system in a 1% NaOCl solution for four minutes at 120 rpm. The nematode suspension was and rinsed with water and collected on a 25 ⁇ m sieve. Females in roots were stained with acid fuschin to facilitate enumeration of the females invading the root. Vermiform life stages and eggs were counted under an inverted TS 100 Nikon microscope at 40 ⁇ magnification. Females embedded in the root systems were quantified at 5 ⁇ magnification utilizing the Nikon SMZ800 compound microscope.
  • Variables measured were plant height, shoot and root weight, females and eggs per gram of root, and the number of vermiform life stages in 500 cm 3 of soil. Greenhouse average temperature where plants were grown was 29° C. Soil moisture was maintained between 40-60% of the maximum water holding capacity. Data were analyzed in SAS 9.1 (SAS Institute Inc.). The distributional assumption was evaluated with the student panel graphs of the GLIMMIX procedure. Dunnett's option was used to assess the differences with the untreated control.
  • Biosurfactant production was confirmed by the emulsification of kerosene, oil drop collapse, and halo formation in blood agar ( FIG. 3 ).
  • the enzymatic profile of Bacillus firmus GB-126 indicated a high enzymatic activity for proteases, amylases, and cellulases forming a transparent halo in milk agar, starch agar, and CMC agar, respectively, within 24 hours. In contrast, no production of chitinases was observed under chitinase agar ( FIG. 7 ).
  • Bacillus firmus GB-126 inhibits the hatch of R. reniformis eggs and paralyzes second stage juveniles under in vitro conditions using secondary metabolites from this bacterium and also living cells at a concentration of 15 ⁇ 10 7 cfu/ml.
  • B. firmus GB-126 applied as a seed treatment at a rate of 7 ⁇ 10 6 cfu/seed reduced number of R. reniformis females in the root and juveniles in soil within the first 30 days of planting.
  • the effect of the insecticide imidacloprid which is used as a seed treatment and formulated with this bacterium, did not show any nematicidal activity.
  • B. firmus GB-126 the biocontrol activity of B. firmus GB-126 observed in previous trials under greenhouse and field conditions where eggs and juvenile stages were reduced can be explained because the bacterium is producing a biosurfactant that is toxic to the plant-parasitic nematode. No ISR was observed at rates tested. However, B. firmus GB-126 possibly has other mechanisms of action against R. reniformis due to the presence of proteases that can be deleterious to the nematode.
  • BioNemWP a unique tool for nematode control . http://agrogreen.coll/PDF/Bionem%20I0BC%202006.pdf.

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