Multifunctional bacillus thuringiensis LT2016 and application thereof

Abstract

The invention provides multifunctional bacillus thuringiensis LT2016 and application thereof, and belongs to the technical field of biocontrol bacteria. The bacillus thuringiensis LT2016 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.28725, the preservation date is October 24, 2023, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacillus thuringiensis LT2016 disclosed by the invention has three effects of preventing and treating lepidoptera pests such as plutella xylostella, inhibiting the growth of fungi, promoting the growth of corn and increasing the yield, and a fungicide prepared by adopting the biocontrol bacterium can reduce the dosage of other chemical pesticides, reduce the prevention and control cost of corn diseases and promote the growth of the corn, and has a good popularization value.

Description

Technical Field

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

[0002] As we all know, traditional chemical pesticides have problems such as low safety, large residues, and difficulty in degrading toxins. Irrational use often causes water, soil, and air pollution and toxic residues in agricultural products, which seriously endangers the ecosystem and human health. It has become an important factor restricting human environmental governance, healthy survival, steady agricultural growth, and sustainable development. Biological control is increasingly favored by people because of its strong specificity, non-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 the spore formation period, this type of bacteria can produce one or more insecticidal crystal proteins (ICPs) with insecticidal activity against a variety of pests such as Lepidoptera, Coleoptera, Diptera, Homoptera, Hymenoptera, Orthoptera, Isoptera, Trichoptera, Thysanoptera, etc. This insecticidal crystal protein is harmless to humans and animals, does not pollute the environment, and is highly effective and specific to target pests. Therefore, Bacillus thuringiensis has been most widely used in biological control of pests. 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, which have the activity of preventing and controlling lepidopteran 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, which has the effect of inhibiting Fusarium. However, the existing Bacillus thuringiensis has a relatively single effect and is relatively limited in practical application in the field. So far, there has been no report on a strain of Bacillus thuringiensis that has the three effects of preventing and controlling lepidopteran 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 strain of Bacillus thuringiensis LT2016, which is deposited in the General Microbiology Center of China Microorganism 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 No. 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 lepidopteran insects;

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

[0014] (5) Application in the preparation of bacterial 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 spp.

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

[0020] Preferably, the lepidopteran insects include Ostrinia furnacalis, cutworms, 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, wherein the spore concentration in the bacterial solution is 1 to 9×10 8 CFU / mL.

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

[0023] By adopting the above technical scheme, the present invention has the following beneficial effects: the Bacillus thuringiensis LT2016 of the present invention has three functions of preventing and controlling lepidopteran pests such as diamondback moth, inhibiting fungal growth and promoting corn growth and yield increase. The bacterial agent prepared by the biocontrol bacteria can reduce the dosage of other chemical pesticides, reduce the cost of corn disease prevention and control, 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 a graph showing the antibacterial effects of Bacillus thuringiensis LT2016 on Bacillus maydis, Fusarium graminearum, Fusarium pseudoverticillium sclerotium and Fusarium fasciatum.

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

[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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope 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 (whose components and proportions were: 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 an activated strain.

[0037] (2) Preparation of seed solution: Use a sterile inoculation loop to scrape a small loop of the strain activated in step (1) and inoculate it into 50 mL of LB liquid culture medium (whose 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), and culture 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) is inoculated into a liquid fermentation medium (whose components and weight percentages are: 2.4% soybean cake powder, 2.3% cottonseed cake powder, 1.6% corn starch, 0.3% yeast powder, 0.02% KH2PO4, 0.03% MgSO4) at a volume ratio of 1%, and water is added to mix and dissolve, the pH is adjusted to 7.2, sterilized at 121°C for 30 minutes, and then cooled to 30°C for standby use), and cultured at 30±1°C, 200-220rpm / min on a shaking table for 48 hours, and the number of bacteria and spores in the fermentation liquid is detected. When the separation of spores in the fermentation liquid reaches 95%, the fermentation culture is stopped to obtain a fermentation liquid; the number of viable bacteria in the fermentation liquid is greater than 1×10 by the dilution plate count method. 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 according to the method of Example 1, and 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 h, 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 are separated. The bacterial solution concentration is determined by the 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, the cabbage leaves were immersed in the gradient diluted bacterial solution for 1 min, taken out and dried, and placed in the feeding box as the test feed, and the cabbage leaves soaked in water were used as the blank control feed. Each dish was inoculated with 20 second-instar heads, and repeated three times. The temperature was 24±1℃, the humidity was 75-85%, and the photoperiod was light: dark = 14:10. The number of dead and live worms was observed regularly. According to the 72h 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 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 cutworm, Asian corn borer, oriental armyworm, Spodoptera litura, and diamondback moth. 50 They are 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 and culturing in a shaker at 28°C and 180 rpm for 5 days, the culture was centrifuged at 10,000 rpm for 10 min, the precipitate was discarded, and the supernatant was stored in a refrigerator at 4°C for later use. Take 1 mL of the 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 at room temperature away from light 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, and 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, it can be seen that the reaction of Bacillus thuringiensis LT2016 is positive, indicating that Bacillus thuringiensis LT2016 has the ability to secrete IAA.

[0053] (2) Quantitative determination

[0054] Weigh 10 mg of IAA powder and add 20 mL of ethanol to dissolve it to 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 protecting from light for 30 minutes, adjust to zero with a 0 mg / L standard solution. 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 follows: Figure 4 The standard curve regression equation y=0.0017x-0.0001 was obtained and used for subsequent content detection.

[0055] Prepare fermentation broth with reference to Example 1, centrifuge the fermentation broth at 10000 rpm for 10 min, draw 1 mL of supernatant into a test tube, add an equal volume of Salkowski colorimetric solution, shake well and keep in dark for 30 min. Use blank LB medium as control and measure the OD of each bacterial broth. 530The concentration of IAA in the supernatant was calculated according to 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 a 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 iron carrier detection kit (Shanghai Zeye Biotechnology Co., Ltd.). 2 μL of the fermentation broth of Bacillus thuringiensis LT2016 prepared in Example 1 was inoculated on the CAS plate, and then placed in a 30°C incubator for 48 hours to observe whether a yellow halo appeared around the colony. If so, it was determined that the strain was able to secrete iron carriers. 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 culture medium, cultured at 30°C and 180 rpm for 24 h, centrifuged at 5000 rpm for 10 min, 1 mL of the fermentation supernatant of the strain was taken and mixed with an equal volume of CAS detection solution preheated at 60°C, and allowed to stand in the dark for 60 min. The OD of each bacterial solution was measured with an ELISA instrument. 630 Value (As), take sterile water as a control to zero. Take another 1mL sterile MKB medium and mix it with an equal volume of CAS detection solution. Measure its absorbance value as the reference value (Ar) in the same way. Compare the ability of different strains to secrete iron carriers by calculating the iron carrier synthesis rate. The calculation formula is: Iron carrier synthesis rate (%) = (Ar-As) / Ar. After calculation, the solubility index of Bacillus thuringiensis LT2016 is 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 fasciatum. First, the pathogens were activated. The activation method is: from the original pathogenic fungus culture plate, a certain number of bacterial cakes are 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) are 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, it can be used for 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 on all sides. 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 of 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 group colony diameter - treatment group colony diameter) / (control group colony diameter - bacterial cake diameter) × 100%. According to calculations, Bacillus thuringiensis LT2016 has a broad-spectrum antibacterial property and has the best antagonistic effect on corn leaf blight, with an inhibition rate of 65.83%. The inhibition rate of most pathogenic fungi is above 50%, and all have biocontrol potential.

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

[0068] Indoor pot experiments were all conducted with the "Zhengdan 958" corn variety as the research object. Healthy and uniform-sized corn seeds were selected, disinfected with 10% sodium hypochlorite and 75% alcohol for 1 min respectively, rinsed with sterile water several times, and 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 in each pot. Water was applied every 2 days to ensure that the same amount of water was applied to each treatment. When the corn grew to the three-leaf and one-heart stage, 20 mL of 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 determination results 1

[0070]

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

[0072]

[0073] It can be seen from Tables 5 and 6 that the agronomic parameters of corn treated with root irrigation with bacterial suspension of Bacillus thuringiensis LT2016, such as plant height, leaf length, leaf width, root length, stem diameter, chlorophyll content, dry weight and fresh weight, were better than those of the CK group, indicating that Bacillus thuringiensis LT2016 has a growth-promoting effect on corn.

[0074] Example 8 Control effect of Bacillus thuringiensis LT2016 on corn diseases

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

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

[0077] The experimental plot was divided into several 100m 2 The plot was surrounded by a 0.5m wide protective row. When sowing manually, the plant spacing and row spacing were adjusted to 30cm and 60cm respectively. The experiment was divided into three groups, with "Zhengdan 958" white seeds as blank control, Zhengdan 958 finished agent coated seeds (Syngenta Group Co., Ltd.) as treatment 1, and seeds treated with "Bacillus thuringiensis LT2016" as treatment 2, with 3 replicates in each group.

[0078] At the waxy stage of corn, 10 adjacent corn plants were randomly selected from each treatment plot for investigation. This method was repeated 3 times. According to the disease grading standard of GB / T 1.1-2009, the occurrence of ear rot and large leaf spot were counted, and the disease index and control effect were calculated. The calculation formula is:

[0079]

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

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

[0082]

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

[0084]

[0085] It can be seen from the above embodiments that the present invention provides a strain of Bacillus thuringiensis LT2016 and its application. The Bacillus thuringiensis LT2016 of the present invention has three functions of preventing and controlling Lepidoptera pests such as diamondback moth, inhibiting fungal growth, and promoting corn growth and yield increase. The microbial agent prepared by the biocontrol bacteria can reduce the dosage of other chemical pesticides, reduce the cost of corn disease prevention and control, and promote corn growth, and has good promotion 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus thuringiensis LT2016, characterized in that: It is deposited in the General Microbiology Center of China Microorganism 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: The Bacillus thuringiensis LT2016 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

3. The preparation method according to claim 2, characterized in that: The temperature of the fermentation culture is 28-32° C., the rotation speed of the fermentation culture is 200-240 rpm, and the time of the fermentation culture is 45-50 h.

4. Use of the Bacillus thuringiensis LT2016 of claim 1 in all or part 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 lepidopteran insects; (4) Application in promoting corn growth; (5) Application in the preparation of bacterial 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 pests; (8) Application in the preparation of seed coating agents.

5. The use according to claim 4, characterized in that: The pathogenic fungi include Microsporum zeae, Fusarium graminearum, Fusarium pseudoverticillium and Fusarium spp.

6. The use according to claim 4, characterized in that: The corn diseases include leaf spot, ear rot and stalk rot.

7. The use according to claim 4, characterized in that: The lepidopteran insects include corn borer, cutworm, diamondback moth and Spodoptera litura of the Noctuidae family.

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

9. A method for promoting corn growth, characterized in that: The liquid bacterial agent according to claim 8 is used to perform root irrigation treatment on corn.

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