A multifunctional Bacillus thuringiensis LT2016 and its application

Abstract

The present invention provides a multifunctional Bacillus thuringiensis LT2016 and its application, belonging to the technical field of biocontrol bacteria. The Bacillus thuringiensis LT2016 of the present invention is deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee with a deposit number of CGMCC No. 28725 and a deposit date of October 24, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The Bacillus thuringiensis LT2016 of the present invention has three functions: preventing and controlling lepidopteran pests such as diamondback moth, inhibiting fungal growth, and promoting corn growth and increasing yield. The microbial agent prepared using the biocontrol bacteria can reduce the amount of other chemical pesticides, reduce the cost of corn disease prevention and control, promote corn growth, and has good promotion value.

Description

Technical Field

[0001] The present invention relates to the technical field of biocontrol bacteria, and in particular to a multifunctional Bacillus thuringiensis LT2016 and applications thereof. Background Art

[0002] As we all know, traditional chemical pesticides have problems such as low safety, high residual yields, and difficulty in degrading toxins. Irrational use often leads to water, soil, and air pollution, as well as toxic residues in agricultural products, seriously endangering ecosystems and human health. These factors have become a significant constraint on environmental governance, healthy living, steady agricultural growth, and sustainable development. Biological control is gaining increasing favor due to its high specificity, lack of toxicity to humans and animals, and the lack of resistance and environmental pollution.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium isolated from soil or dead insects. During spore formation, it produces one or more insecticidal crystal proteins (ICPs) with insecticidal activity against a wide range of pests, including Lepidoptera, Coleoptera, Diptera, Homoptera, Hymenoptera, Orthoptera, Isoptera, Trichoptera, and Thysanoptera. These insecticidal crystal proteins are harmless to humans and animals, environmentally friendly, and highly effective and specific against target pests. Consequently, B. thuringiensis has been widely used in biological pest control. For example, "Isolation and Screening of Bacillus thuringiensis Strains with High Toxicity to Lepidoptera Pests in Some Areas of Liaoning Province" (Ni Hejia et al. Journal of Agricultural Biotechnology. 2019, 27(8), 1513-1520) discloses multiple strains of Bacillus thuringiensis that have the activity of preventing and controlling lepidoptera pests; "The Effect of Bacillus thuringiensis LTS290 Strain on Inhibition of Fusarium" (Zhou Guowang et al. Biotechnology Bulletin. 2015, 31(8), 153-158) discloses a strain of Bacillus thuringiensis that has the effect of inhibiting Fusarium. However, the existing Bacillus thuringiensis has relatively single effects and has great limitations in practical field applications. So far, there has been no report on a strain of Bacillus thuringiensis that has the three effects of preventing and controlling lepidoptera pests such as diamondback moth, inhibiting fungal growth, and promoting corn root development. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a multifunctional Bacillus thuringiensis LT2016 and applications thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a Bacillus thuringiensis LT2016, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 28725, a deposit date of October 24, 2023, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention provides a method for preparing a bacterial liquid of the Bacillus thuringiensis LT2016, wherein the Bacillus thuringiensis LT2016 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

[0008] Preferably, the fermentation temperature is 28-32° C., the fermentation speed is 200-240 rpm, and the fermentation time is 45-50 h.

[0009] The present invention also provides the use of the Bacillus thuringiensis LT2016 in all or part of the following (1) to (8):

[0010] (1) Application in inhibiting pathogenic fungi;

[0011] (2) Application in the prevention and control of corn diseases;

[0012] (3) Application in the control of Lepidoptera insects;

[0013] (4) Application in promoting corn growth;

[0014] (5) Application in the preparation of fungal agents for inhibiting pathogenic fungi;

[0015] (6) Application in the preparation of microbial agents for preventing and controlling corn diseases;

[0016] (7) Application in the preparation of bacterial agents for controlling lepidopteran pests;

[0017] (8) Application in the preparation of seed coating agents.

[0018] Preferably, the pathogenic fungi include Bipolaris maydis, Fusarium graminearum, Fusarium pseudoverticillium and Fusarium fasciatum.

[0019] Preferably, the corn diseases include leaf spot, ear rot and stalk rot.

[0020] Preferably, the lepidopteran insects include corn borer, black cutworm, diamondback moth and Spodoptera litura of the Noctuidae family.

[0021] The present invention also provides a liquid microbial agent, wherein the microbial agent comprises a bacterial solution of the Bacillus thuringiensis LT2016, and the spore concentration in the bacterial solution is 1 to 9×10 8 CFU / mL.

[0022] The invention also provides a method for promoting corn growth, which comprises using the liquid bacterial agent to perform root irrigation treatment on the corn.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: the Bacillus thuringiensis LT2016 of the present invention has three functions: preventing and controlling lepidopteran pests such as diamondback moth, inhibiting fungal growth, and promoting corn growth and increasing yield. The microbial agent prepared using this biocontrol bacterium can reduce the amount of other chemical pesticides, reduce the cost of preventing and controlling corn diseases, promote corn growth, and has good promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a microscope photo of LT2016 spore crystals.

[0025] Figure 2 This is the growth curve of Bacillus thuringiensis LT2016.

[0026] Figure 3 Qualitative determination of IAA production by Bacillus thuringiensis LT2016.

[0027] Figure 4 is the IAA standard curve.

[0028] Figure 5 To qualitatively determine the siderophore production ability of Bacillus thuringiensis LT2016.

[0029] Figure 6 This is the antibacterial result of Bacillus thuringiensis LT2016 against corn leaf blight, Fusarium graminearum, Fusarium pseudoverticillium sclerotiorum and Fusarium fasciatum.

[0030] Figure 7 This figure shows the promoting effect of root irrigation with Bacillus thuringiensis LT2016 bacterial suspension on the corn root system.

[0031] Biological Deposit Description

[0032] The Bacillus thuringiensis LT2016 provided by the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 28725, a deposit date of October 24, 2023, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: Preparation of JQ23 strain microbial agent

[0035] Proceed as follows:

[0036] (1) Activation of strains: The preserved Bacillus thuringiensis strain LT2016 was activated on LB plate medium (its components and proportions are: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1 L water, pH 7.2). A single colony was picked and cultured on LB slant medium at 30°C for 3 days to obtain the activated strain.

[0037] (2) Preparation of seed solution: Use a sterile inoculating loop to scrape a small loop of the strain activated in step (1) and inoculate it into 50 mL of LB liquid culture medium (its components and proportions are: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1 L of water, pH 7.2). Cultivate at 30°C and a shaking speed of 220 rpm / min for 10 to 14 h to obtain seed solution.

[0038] (3) Fermentation culture: The seed liquid of step (2) was inoculated into a liquid fermentation medium (its components and weight percentages are: soybean cake powder 2.4%, cottonseed cake powder 2.3%, corn starch 1.6%, yeast powder 0.3%, KH2PO4 0.02%, MgSO4 0.03%) at a volume ratio of 1%, mixed and dissolved with water, adjusted to pH 7.2, sterilized at 121°C for 30 minutes, and then cooled to 30°C for standby use), cultured at 30±1°C, 200-220 rpm / min on a shaking table for 48 hours, and the number of bacteria and spores in the fermentation liquid was detected. When the separation of spores in the fermentation liquid reached 95%, the fermentation culture was stopped to obtain the fermentation liquid; the number of viable bacteria in the fermentation liquid was counted by the dilution plate count method and was greater than 1×10 9 cfu / mL.

[0039] (4) The pH value of the fermentation liquid obtained in step (3) is adjusted to 4.6-4.8, and 0.02%-0.1% sodium benzoate is added to obtain a microbial insecticide; or the fermentation liquid is centrifuged at 4°C and 5000 rpm / min for 30 min, the supernatant is discarded, and the resulting precipitate is the bacterial cell, which is diluted with water to 1×10 7 cfu / mL concentration, adjust the pH value to 4.6-4.8, and add 0.02%-0.1% sodium benzoate to obtain a liquid bacterial agent.

[0040] Example 2 Drawing of the Growth Curve of Bacillus thuringiensis LT2016

[0041] Seed solution was prepared by referring to the method of Example 1. The seed solution was inoculated into LB liquid medium at a ratio of 1%, and cultured in a shaking incubator at 37°C and 220 rpm. Samples were taken every 2 hours, repeated three times, and the OD was measured.600 The culture time of the fermentation broth was used as the horizontal axis, and the OD 600 The value is used as the vertical axis to draw the growth curve.

[0042] The growth curve of the strain to be tested is as follows Figure 2 As shown, the logarithmic growth period of Bacillus thuringiensis LT2016 is 6 to 14 hours. During this period, the strain multiplies rapidly and reaches a stable growth period at 14 hours.

[0043] Example 3 Determination of insecticidal activity of Bacillus thuringiensis LT2016 against lepidopteran pests

[0044] Pick a single Bt colony in 100 mL 1 / 2LB liquid medium and culture it at 30°C and 220 r / min until the spores separate. The bacterial solution concentration is determined by plate colony counting method, and then the bacterial solution is diluted to 1×10 8 , 3×10 7 , 1×10 7 , 3×10 6 , 1×10 6 , 3×10 5 , 1×10 5 , 1×10 4 CFU / mL, cabbage leaves were immersed in the gradient diluted bacterial solution for 1 minute, taken out and dried, and placed in a breeding box as the test feed. Cabbage leaves soaked in water were used as the blank control feed. 20 second-instar larvae were seeded into each dish, repeated three times, and raised in a light incubator at a temperature of 24±1°C, a humidity of 75-85%, and a photoperiod of light:dark = 14:10. The number of dead and live larvae was observed regularly. Based on the 72-h survey results, SPSS24.0 software was used to analyze and calculate the LC 50 , the results are shown in Table 1.

[0045] Table 1 Evaluation results of the insecticidal activity of LT2016 bacterial solution against 2nd instar larvae of various Lepidoptera

[0046]

[0047]

[0048] The results showed that Bacillus thuringiensis LT2016 had good insecticidal activity against the second-instar larvae of Lepidoptera, including cutworms, Asian corn borers, oriental armyworms, Spodoptera litura, and diamondback moths. 50 4.65×10 6 CFU / mL, 2.04×10 6 CFU / mL, 19.8×10 6 CFU / mL, 3.1×10 5CFU / mL, 3.4×10 5 CFU / mL.

[0049] Example 4 Determination of Secondary Metabolites of Bacillus thuringiensis LT2016

[0050] 1. Determination of IAA Production Capacity

[0051] (1) Qualitative determination

[0052] The fermentation broth prepared in Example 1 was inoculated into 20 mL of kingB liquid culture medium at a ratio of 1%. After shaking culture in a shaker at 28°C and 180 rpm for 5 days, it was centrifuged at 10,000 rpm for 10 minutes, the precipitate was discarded, and the supernatant was taken and stored in a refrigerator at 4°C for later use. Take 1 mL of supernatant in a test tube, add an equal volume of Salkowski colorimetric solution, and mix it thoroughly. Using kingB sterile culture medium as a control, place it in the dark at room temperature for 30 minutes and observe the color changes of each treatment. If the color turns red, it is a positive reaction, indicating that the strain can secrete IAA. The darker the color, the stronger the ability to produce IAA; if there is no obvious change in color, the reaction is negative, indicating that it does not have the ability to produce IAA. The results are as follows: Figure 3 As shown in the figure, the reaction of Bacillus thuringiensis LT2016 was positive, indicating that Bacillus thuringiensis LT2016 has the ability to secrete IAA.

[0053] (2) Quantitative determination

[0054] Weigh 10 mg of IAA powder, add 20 mL of ethanol to dissolve it, and obtain an IAA standard solution with a concentration of 500 mg / L. Pipette the above standard solutions into 100 mL volumetric flasks respectively, dilute to the scale with deionized water, and prepare a series of IAA standard solutions with concentrations of 0, 10, 20, 30, and 50 mg / L, and store them at room temperature away from light. Take 1 mL of each concentration standard solution respectively, add an equal volume of Salkowski colorimetric solution, and after dark treatment for 30 minutes, adjust to zero with a 0 mg / L standard solution, and use a spectrophotometer to detect the absorbance of each solution at a wavelength of 530 nm. Use the IAA solution concentration as the horizontal axis and the absorbance value as the vertical axis to draw the IAA standard curve. The results are as shown below. Figure 4 The standard curve regression equation y = 0.0017x-0.0001 was obtained and used for subsequent content detection.

[0055] Prepare fermentation broth according to Example 1. Centrifuge the fermentation broth at 10,000 rpm for 10 minutes, draw 1 mL of supernatant into a test tube, add an equal volume of Salkowski colorimetric solution, shake well, and let stand in the dark for 30 minutes. Measure the OD of each bacterial broth with blank LB medium as a control. 530The concentrations of IAA in the supernatants were calculated based on the standard curve. The results are shown in Table 2.

[0056] Table 2 Quantitative determination of IAA production by Bacillus thuringiensis LT2016

[0057]

[0058] The IAA quantitative detection test showed that the concentration of IAA produced by Bacillus thuringiensis LT2016 was 10.84 mg / mL, which had certain growth-promoting potential.

[0059] 2. Determination of Siderophore Production Capacity

[0060] (1) Qualitative determination

[0061] CAS solid culture medium was prepared according to the instructions of the siderophore detection kit (Shanghai Zeye Biotechnology Co., Ltd.). 2 μL of the fermentation broth of Bacillus thuringiensis LT2016 prepared in Example 1 was spotted on a CAS plate. After standing in a 30°C incubator for 48 hours, the colonies were observed to see if a yellow halo appeared around them. If so, it was determined that the strain was capable of secreting siderophores. The results were as follows: Figure 5 shown.

[0062] The results of the qualitative determination test of siderophore production showed that Bacillus thuringiensis LT2016 strain could grow normally on the CAS detection plate, and a yellow halo appeared around the colony, indicating that the bacteria could produce siderophores to chelate iron in the culture medium.

[0063] (2) Quantitative determination

[0064] The bacterial solution to be tested was inoculated into MKB liquid medium, cultured at 30°C and 180 rpm for 24 h, and then centrifuged at 5000 rpm for 10 min. 1 mL of the fermentation supernatant of the strain was mixed evenly with an equal volume of CAS detection solution preheated at 60°C. After standing in the dark for 60 min, the OD of each bacterial solution was measured using a microplate reader. 630 The absorbance value (As) was measured, and sterile water was used as a control to adjust the value to zero. Separately, 1 mL of sterile MKB medium was thoroughly mixed with an equal volume of CAS assay solution. The absorbance was also measured as a reference value (Ar). The siderophore synthesis rate was calculated to compare the siderophore secretion abilities of different strains. The calculation formula is: Siderophore synthesis rate (%) = (Ar - As) / Ar. The solubility index of Bacillus thuringiensis LT2016 was calculated to be 65.37%.

[0065] Example 5 Determination of antibacterial ability of Bacillus thuringiensis LT2016

[0066] The antibacterial ability of Bacillus thuringiensis LT2016 was preliminarily determined, and several representative pathogens were selected for determination: corn leaf blight, Fusarium graminearum, Fusarium pseudoverticularis, and Fusarium thuringiensis. First, the pathogens were activated. The activation method is: from the original pathogenic fungus culture plate, a certain number of bacterial cakes were punched at the outermost edge of the colony with a sterile puncher with a diameter of about 0.5 cm, and then the bacterial cakes (the side containing mycelium) were transferred to the center of a new PDA culture plate, and cultured in an incubator at a suitable temperature for 10-18 hours. When new mycelium grows around the bacterial cakes, they can be used for later use. After activation, they were inoculated in the center of the PDA plate, and 10 μL of the strain and the fermentation liquid prepared in Example 1 were inoculated at symmetrical positions 3 cm away from the center. The plate without the fermentation liquid of Bacillus thuringiensis LT2016 was used as a control. Each treatment was repeated three times, and the control plate was cultured at a constant temperature of 28°C until the mycelium on the control plate covered the control plate, and the antibacterial effect was observed ( Figure 6 ) and calculated the inhibition rate. The calculation formula is: Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - cake diameter) × 100%. Calculations show that Bacillus thuringiensis LT2016 has broad-spectrum antibacterial activity, with the greatest antagonism against southern corn leaf blight, achieving an inhibition rate of 65.83%. Its inhibition rate against most fungal pathogens is above 50%, demonstrating its potential for biocontrol.

[0067] Example 6 Growth-promoting effect of Bacillus thuringiensis LT2016 on corn

[0068] Indoor pot experiments were conducted using the "Zhengdan 958" corn variety as the research object. Healthy and uniform corn seeds were selected, disinfected with 10% sodium hypochlorite and 75% alcohol for 1 minute each, rinsed with sterile water several times, and then germinated in the dark for 2 days. When the corn sprouts grew to 1 cm, they were transplanted into square pots, with 4 plants planted per pot. Water was applied every 2 days to ensure that each treatment was watered with the same amount of water. When the corn reached the three-leaf and one-heart stage, 20 mL of a 1×10 8 CFU / mL Bacillus thuringiensis LT2016 suspension was used for root irrigation and the same treatment was repeated 7 days later. After 15 days, 4 plants were randomly selected from each group to measure various agronomic indicators such as plant height, leaf length, leaf width, root length, stem diameter, chlorophyll content, dry weight, and fresh weight. The results are shown in Tables 3, 4, and Figure 7 shown.

[0069] Table 3 Indoor growth promotion index measurement results 1

[0070]

[0071] Table 4 Indoor growth promotion index measurement results 2

[0072]

[0073] As shown in Tables 5 and 6, the agronomic indicators of corn treated with root irrigation of Bacillus thuringiensis LT2016 suspension, including plant height, leaf length, leaf width, root length, stem diameter, chlorophyll content, dry weight, and fresh weight, were all better than those of the CK group, indicating that Bacillus thuringiensis LT2016 has a growth-promoting effect on corn.

[0074] Example 8: Effect of Bacillus thuringiensis LT2016 on the prevention and control of corn diseases

[0075] Preparation of seed coating agent: LT2016 fermentation liquid and finished film-forming agent Gieli Film (Beinong Haili Zhuozhou Seed Coating Agent Co., Ltd.) were mixed in a ratio of 1:5, and the final concentration of the bacterial liquid was adjusted to 10 8 CFU / mL. Select several healthy corn seeds of similar size and sterilize them. Then add them to the above solution and stir quickly to evenly coat the seed coating agent on the seed surface. Then dry them in a cool place.

[0076] The field trial was conducted from June to September 2023 in Qingyuan District and Xushui District of Baoding City, and "Zhengdan 958" was selected as the planting variety.

[0077] The experimental plot was divided into several 100m 2 The plots were surrounded by 0.5-meter-wide protective rows. During manual sowing, the plant and row spacings were adjusted to 30 cm and 60 cm, respectively. The experiment was divided into three groups: white Zhengdan 958 seeds served as a blank control, Zhengdan 958 finished agent-coated seeds (Syngenta Group Co., Ltd.) served as treatment 1, and seeds treated with Bacillus thuringiensis LT2016 served as treatment 2. Each group had three replicates.

[0078] At the waxy stage of corn, 10 adjacent corn plants were randomly selected from each treatment plot for investigation. This method was repeated three times. According to the disease grading standard of GB / T 1.1-2009, the incidence of ear rot and large leaf spot was counted, and the disease index and control efficacy were calculated. The calculation formula is:

[0079]

[0080] The statistical results are shown in Tables 5 and 6:

[0081] Table 5 Control effects of different treatments on corn ear rot

[0082]

[0083] Table 6 Control effects of different treatments on corn leaf spot

[0084]

[0085] As can be seen from the above examples, the present invention provides a strain of Bacillus thuringiensis LT2016 and its applications. The present invention's Bacillus thuringiensis LT2016 simultaneously controls Lepidoptera pests such as diamondback moth, inhibits fungal growth, and promotes corn growth and yield. The biocontrol agent prepared using this biocontrol bacterium can reduce the use of other chemical pesticides, lower the cost of corn disease prevention and control, and promote corn growth, thus having excellent promotional value.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A strain of Bacillus thuringiensis ( Bacillus thuringiensis )LT2016, characterized in that, It is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 28725, the deposit date is October 24, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for preparing the bacterial solution of Bacillus thuringiensis LT2016 according to claim 1, characterized in that: Inoculating the Bacillus thuringiensis LT2016 into a fermentation medium for fermentation culture to obtain the bacterial solution; The fermentation temperature is 28-32° C., the fermentation speed is 200-240 rpm, and the fermentation time is 45-50 h.

3. Use of the Bacillus thuringiensis LT2016 according to claim 1 in any one of the following (1) to (8): (1) Application in inhibiting pathogenic fungi; (2) Application in the prevention and control of corn diseases; (3) Application in the control of Lepidoptera insects; (4) Application in promoting corn growth; (5) Application in the preparation of fungal agents for inhibiting pathogenic fungi; (6) Application in the preparation of microbial agents for preventing and controlling corn diseases; (7) Application in the preparation of bacterial agents for controlling lepidopteran insects; (8) Application in the preparation of seed coating agents; The pathogenic fungi are corn leaf blight fungus, Fusarium graminearum, Fusarium pseudoverticillium and Fusarium spp. The corn diseases are large leaf spot and ear rot; The lepidopteran insects are Asian corn borer, black cutworm, diamondback moth and Spodoptera litura.

4. A liquid bacterial agent, characterized in that: The liquid bacterial agent comprises the bacterial solution of Bacillus thuringiensis LT2016 according to claim 1, and the spore concentration in the bacterial solution is 1 to 9×10 8 CFU / mL.

5. A method for promoting corn growth, characterized in that: The liquid microbial agent according to claim 4 is used to perform root irrigation treatment on corn.

Images