<i>Bacillus thuringiensis </i>NOVONEST4 and applications thereof

Abstract

Strain of Bacillus thuringiensis novonest4 and applications thereof. The Bacillus thuringiensis is the Bacillus thuringiensis novonest4, with an Accession No. CCTCC NO: M2018443. The strain is firstly reported to have both the effect of preventing and controlling lepidoptera pests after foilar application and the effects of degrading carbendazim, promoting crop growth and increasing crop yield after soil application, and has wide application prospects in agricultural production.

Claims

1. A method for degrading carbendazim, comprising: incubating a strain of Bacillus thuringiensis to obtain a seed culture, wherein the strain of Bacillus thuringiensis is Bacillus thuringiensis novonest4 with CCTCC Accession No. M2018443, incubating the seed culture to obtain a fermentation stock solution, wherein the fermentation stock solution comprises spores and parasporal crystals of the strain of Bacillus thuringiensis, treating a system containing carbendazim with the fermentation stock solution and degrading the carbendazim in the system.

2. The method of claim 1, wherein the fermentation stock solution comprises the spores at a count of 5×10.sup.9 cfu/mL or more.

3. The method of claim 1, wherein at least 40% of the spores and the parasporal crystals are separated in the fermentation stock solution.

4. The method of claim 1, further comprising incubating the strain of Bacillus thuringiensis in a primary seed culture medium to obtain a primary seed culture, incubating the primary seed culture in a secondary seed culture medium to obtain a secondary seed culture, and incubating the secondary seed culture in a fermentation medium to obtain the fermentation stock solution.

5. The method of claim 1, wherein the system containing carbendazim is soil.

6. The method of claim 5, wherein the fermentation stock solution is at a concentration of 10.sup.8 spores per 1 g of soil or more for treating the soil containing carbendazim.

7. The method of claim 1, wherein the system containing carbendazim is treated with the fermentation stock solution at 30° C.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1 is a microphotograph (1000×) showing the Bacillus thuringiensis novonest4 of the present invention.

(2) FIGS. 2A and 2B show the effect of Bacillus thuringiensis novonest4 of the present invention on preventing and controlling tea geometrids, where FIG. 2A shows the effect of novonest 4; and FIG. 2B shows the effect of Fresh water.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention is described in details in the following examples, and the examples do not limit the scope of the inventoin. The reagents or materials are sourced from commercial channels and techniques are conventionally known, unless otherwise specified.

Example 1

Production and Identification of Bacillus thuringiensis Novonest4

(4) The Bacillus thuringiensis novonest4 was sourced from the Bacillus thuringiensis Resource Library of Hubei Biopesticide Engineering Research Center, and was simultaneously screened for the activity of killing lepidopteran pests and the activity of degrading carbendazim to obtain a dual-effect strain. The strain was further identified in its classification status by combining 16s rDNA and physiological and biochemical identification (Table 1, Table 2), and then was sent to the CCTCC for preservation, with a class designated as Bacillus thuringiensis novonest4 and the CCTCC Accession No. M2018443.

(5) In the invention, Bacillus thuringiensis novonest4 is alternatively abbreviated as novonest4.

(6) The colony of the Bacillus thuringiensis was milky white and grew well in the NA medium, and spores and parasporal crystals were observed under microscopic examination as shown in FIG. 1.

(7) TABLE-US-00001 TABLE 1 Physiological-biochemical characteristics of Bacillus thuringiensis novonest4: enzyme activity and carbon-source oxidation Test Substrates and Enzymes for Reaction Results ONPG o-nitrobenzene- β-galactosidase − galactoside ADH Arginine Arginine dihydrolase + LDC Lysine Lysine decarboxylase − ODC Ornithine Ornithine decarboxylase − CIT Sodium Citrate Citrate Utilization − H2S Sodium thiosulfate Generation of H2S + URE Urea Urease − TDA Tryptophan Tryptophan deaminase + IND Tryptophan Generation of Indole − VP Pyruvate 3-hydroxybutanone produced + acetylmethylcarbinol GEL Kohn gelatin Gelatinase + GLU Glucose Fermentation/Oxidation (4) + MAN Mannitol Fermentation/Oxidation (4) − INO Inositol Fermentation/Oxidation (4) − SOR Sorbitol Fermentation/Oxidation (4) − RHA Rhamnose Fermentation/Oxidation (4) − SAC Saccharose Fermentation/Oxidation (4) − MEL Melibiose Fermentation/Oxidation (4) − AMY Amygdalin Fermentation/Oxidation (4) − ARA Arabinose Fermentation/Oxidation (4) − +: positive reaction; +: negative reaction

(8) TABLE-US-00002 TABLE 2 Physiological-biochemical characteristics of Bacillus thuringiensis novonest4: acid production with a carbon source Tube/Substrate Tube/Substrate Corresponding Test Corresponding Test to Reagent Strip Result to Reagent Strip Results 0 Control − Esculin + 1 Glycerinum − 26 Salicin − 2 Erythritol − 27 Cellobiose + 3 D-Aarabinose − 28 Maltose + 4 L-Aarabinose − 29 Lactose − 5 Ribose + 30 Melibiose − 6 D-Xylose − 31 Sucrose + 7 L-Xylose − 32 Trehalose + 8 Adonitol − 33 Inulin − 9 β-methyl-D-xyloside − 34 Melezitose − 10 Galactose − 35 Raffinose − 11 Glucose + 36 Starch + 12 Fructose + 37 Glycogen + 13 Mannose + 38 Xylitol − 14 Sorbose − 39 Geraniol − 15 Rhamnose − 40 D-turanose − 16 Dulcitol − 41 D-lyxose − 17 Inositol − 42 D-tagatose − 18 mannitol − 43 D-fucose − 19 Sorbitol − 44 L-fucose − 20 α-methyl-D-mannoside − 45 D-arabitol − 21 α-methyl-D-glucoside − 46 L-arabitol − 22 N-acetyl-glucosamine + 47 Gluconate − 23 Amygdalin − 48 2-keto-gluconate − 24 Arbutin − 49 5-keto-gluconate − +: positive reaction; +: negative reaction;

Example 2

Fermentation of Bacillus thuringiensis Novonest4

(9) (1) Primary seed culture: 200 ml of primary seed medium was added to a 500 ml Erlenmeyer flask, and sterilized with moist heat at 120° C. for 15-30 min, and then, a freeze-dried tubed strain of live Bacillus thuringiensis was taken and inoculated into the primary seed medium, and then incubated at 35° C. for 10 h.

(10) The medium used for the primary seed culture included: 1% of glucose (g/L, the same below), 2% of beef extract, 1% of peptone, and 0.5% of sodium chloride, with a pH of 7.

(11) (2) Secondary seed culture: 150 L of secondary seed medium was added to a 400 L fermentor, and sterilized with moist heat at 120° C. for 30 min. 200 ml of primary seeds were inoculated into the secondary seed medium, and incubated at 35° C. for 10 h, with a controlled fermentor pressure of 0.05 Mpa, a stirring speed of 120 rpm, and a ventilation ratio of 1:1.0.

(12) The medium used for the secondary seed culture included: 2% of glucose, 1% of yeast extract, 2% of peptone, 0.05% of dipotassium hydrogen phosphate, 1% of sodium chloride, and 0.05% of magnesium sulfate. The pH of the medium was adjusted to 7.

(13) (3) Fermentation: 25 m3 of live Bacillus thuringiensis fermentation medium was added to a 40 m3 fermentor, and sterilized with moist heat at 120° C. for 30 minutes. 200 L of secondary seeds were inoculated into the live Bacillus thuringiensis fermentation medium, and incubated at 35° C. for 36 h, with a controlled fermentor pressure of 0.04 Mpa, a stirring speed of 100 rpm, and a ventilation ratio of 1:0.5.

(14) The raw materials and amount of the medium used for the fermentation included: 1% of corn starch, 2% of peanut cake powder, 1% of fish meal, 0.5% of yeast extract powder, 0.5% of peptone, 0.05% of potassium dihydrogen phosphate, and 0.05% of magnesium sulfate, with a pH of pH7.

(15) (4) The fermentation was ended when 40% of the spores and crystals were separated. After counting, the spores had a count of 5×10.sup.9 cfu/mL, and were used as a fermentation stock solution for the following embodiments.

Example 3

Insecticidal Activity of Bacillus thuringiensis Novonest4 Against Tea Geometrids

(16) The fermentation stock solution of Bacillus thuringiensis novonest4 prepared in Embodiment 2 was tested by a leaf soaking and feeding method. That is, 100-fold and 200-fold Bt solutions were prepared from sterile water, fresh water was used as a control, and the test was conducted in triplicate. Tea leaves were soaked for 10 min, removed, air-dried, and then fed to newly-hatched larvae of the tea geometrid. Laboratory tests shown that the Bacillus thuringiensis novonest4 diluted to 100 fold and 200 fold exhibited good prevention and control effect on the tea geometrids (FIGS. 2A and 2B). FIG. 2A shows diseased blades after 5 days after a 100-fold diluted solution of the microbial preparation of the invention is added, and FIG. 2B shows diseased blades after water is added as a control. As can be seen from FIGS. 2A and 2B and Table 3, the 100-fold diluted solution shows 100% of a protecting force for the blades. Statistics were conducted after 2d, 3d, 4d, and 5d. Each treatment was conducted in an artificial incubator at 25° C. with a relative humidity of 80% and a luminance of 2000 lx. Based on regular examinations every day, the number of deaths of test larvae were noted.

(17) TABLE-US-00003 TABLE 3 Control effect of Bacillus thuringiensis novonest4 against tea geometrids 2D after 3D after 4D after 5D after Application Application Application Application Control Control Control Control Treatment Effect (%) effect (%) effect (%) effect (%) 100-fold 89.30 94.43 100 100 diluted solution of Bacillus thuringiensis 200-fold 48.60 51.67 75.54 70.64 diluted solution of Bacillus thuringiensis Fresh water — — — —

(18) Due to space limitations, the insecticidal activity data of the Bacillus thuringiensis novonest4 on the tea geometrids were only recorded in the text of this application. During actual testing, the strain also shown good effect on killing tea tussock moths, corn borers, rice stem borers, diamond back moths, tea budworms, or Spodoptera litura.

Example 4

Degradation Effect of Bacillus thuringiensis Novonest4 Against Carbendazim

(19) (1) A basic medium having a volume of 180 ml (500 ml Erlenmeyer flask) was added with 80 mg of 50% carbendazim wettable powder, and then added with 20 ml of the BT fermentation stock solution prepared in Embodiment 2, with BT sterile water as a control. Both were incubated on a shaker (170 rpm/min) at 30° C. After 7 days, samples was taken for measurement. The control and 5 ml of a treated basic medium suspension were transferred into a 50 ml separating funnel, and extracted three times with 10 ml of dichloromethane. A lower organic phase passed through anhydrous sodium sulfate and was combined in a 250 ml round-bottom flask. The round-bottom flask was then placed on a rotary evaporator for reduced-pressure concentration to near-dryness, and then a resulting product was blown with nitrogen to dryness. Methanol was added to reach 10 ml, and HPLC was performed for testing.

(20) The formula of the basic medium was: 1.0 g of NH4NO3, 1.5 go of K2HPO4, 0.5 g of KH.sub.2PO.sub.4, 0.2 g of MgSO.sub.4, 0.5 g of NaCl, and the balance of water, which was added to reach 1 L.

(21) (2) 1000 g (dry weight) of soil to which carbendazim was not applied was weighed, added with carbendazim to allow the carbendazim to reach a concentration of 50 mg/kg, and then added with the novonest4 fermentation solution to allow the novonest4 to reach a concentration of 10.sup.8 spores per 1 g of soil. They were placed in an incubator at 30° C. and incubated in a dark condition at constant temperature. After 10 days, sampling was performed to determine the residual amount of the carbendazim in the soil. The results are shown in Table 4, where the degradation rate against the carbendazim was 53.45% in a shake flask, and reached 35.40% in soil.

(22) TABLE-US-00004 TABLE 4 Degradation rate of Bacillus thuringiensis novonest4 against Carbendazim Inorganic Medium salt medium Soil Degradation rate 53.45% 35.40%

Example 5

Growth-Promoting Effect of Bacillus thuringiensis novonest4 on Tea

(23) The tests were carried out in August 2018 in the tea garden of Yingshan County, Huanggang, Hubei, at an altitude of 107 meters. After the tea harvesting in summer, the novonest4 fermentation solution at a level of 5×10.sup.9 cfu/mL was sprayed on the roots of the tea trees at the use level of 20 L/mu. Those without the spraying of the BT fermentation solution were taken as a control. In October 2018, the budding density and the weight of one bud and two leaves were counted. With the application of the novonest4, an obvious growth-promoting effect was achieved on the tea, with the budding density increased by 26.34%, and the weight of one-hundred buds increased by 22.86% as shown in Table 5.

(24) TABLE-US-00005 TABLE 5 Growth-promoting Effect of Bacillus thuringiensis novonest4 on Tea Budding density Hundred-bud Treatment (0.1 m.sup.2) Weight (g) Bacillus thuringiensis novonest4 101.7 38.48 CK 80.5 31.32

(25) Due to space limitations, the growth-promoting effect data of the Bacillus thuringiensis novonest4 on the tea were only recorded in the text of this application. During actual testing, this strain also has a growth-promoting effect on tea, rice, corn, wheat, soybean, tomato, cabbage, pepper, cucumber and traditional herbal medicines such as Codonopsis pilosula, Magnolia officinalis, Atractylodes macrocephala and Coptis chinensis.