Butanol-producing bacillus strain, bacterial agent and application thereof

Abstract

This invention provides a butanol-producing Bacillus strain, inoculant, and its application. The butanol-producing Bacillus is a butanol-producing Bacillus (… Peribacillus butanolivorans CSQK4, deposited at the China Center for Type Culture Collection (CCTCC) NO: M 2025043, dated January 6, 2025. The inoculant is obtained from *Bacillus butyricum* through fermentation. This *Bacillus butyricum* of the present invention has a dual function as both a pesticide and fertilizer, promoting tomato growth and increasing yield while enhancing its resistance, exhibiting advantages such as high efficiency, non-toxicity, and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to a butanol-based Bacillus strain, its inoculant, and its application. Background Technology

[0002] The extensive use of agricultural chemicals, especially chemical pesticides and fertilizers, has played a crucial role in ensuring high and stable crop yields and promoting the development of modern agriculture. However, long-term irrational input has severely damaged the diversity and stability of farmland ecosystems, constraining the sustainability of agricultural production and posing a potential threat to sustained high and stable crop yields. For example, excessive fertilizer application reduces soil biodiversity, causes soil acidification and compaction, leading to a decline in arable land quality, which in turn affects crop yield and quality. Simultaneously, fertilizer runoff increases the concentration of nutrients in farmland water bodies, damaging water quality and threatening aquatic biodiversity. The irrational use of chemical pesticides easily leads to a rapid increase in pesticide resistance in target pathogens and pests, resulting in a continuous decline in control effectiveness and creating a vicious cycle of "increased resistance - increased pesticide use." Pesticide residues can also cause agricultural products to exceed standards and accumulate in the ecosystem through the food chain, harming non-target organisms and even human health. Therefore, the scientific application of chemical pesticides and the safety of agricultural product quality are receiving increasing attention from society.

[0003] Microbial preparations, possessing both growth-promoting and disease-preventing functions similar to fertilizers, represent an important green alternative for alleviating the aforementioned problems. However, the industrial application of microbial preparations currently faces numerous bottlenecks, such as a scarcity of highly active and environmentally adaptable superior strains, the potential for environmental pollution during fermentation processes, insufficient product stability, and a need to improve market acceptance. To date, research on butanol-containing Bacillus subtilis in promoting tomato growth, inducing plant resistance, and increasing yield has not been systematically reported, and related products have not yet been registered for application. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a *Bacillus butyricum* strain, its inoculant, and its application. *Bacillus butyricum* has a dual function as both a pesticide and fertilizer, promoting tomato growth and increasing yield while enhancing its resistance. The preparation process of its inoculant is environmentally friendly, simple, and has low production costs. It can be used in tomato field production, providing a dual function as both a pesticide and fertilizer, and can partially replace agricultural chemicals.

[0005] To solve the above-mentioned technical problems, the present invention provides a butanol-eating Bacillus strain, wherein the butanol-eating Bacillus is a butanol-eating Bacillus ( Peribacillus butanolivorans CSQK4, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025043, deposited on January 6, 2025.

[0006] Based on a general technical concept, the present invention provides a microbial agent obtained by fermentation of the aforementioned butyrol-type Bacillus.

[0007] The fermentation process of the above-mentioned microbial agent is further described as follows: S1. Activation: Culture the butyrate-producing Bacillus CSQK4 strain until a single colony appears; S2. Seed culture: The single colony is inoculated into a seed culture medium and cultured until the logarithmic growth phase to obtain a seed solution; S3. Production culture: Inoculate the seed liquid into the production culture medium at an inoculation rate of 1% of the total volume of the production culture medium, and culture until the logarithmic growth phase; S4. The bacterial agent is prepared by dispensing and packaging.

[0008] Furthermore, the culture conditions described in S1, S2 and S3 of the above-mentioned bacterial agent are as follows: culture is carried out at 30℃~35℃ with shaking at a shaking speed of 300 rpm.

[0009] Furthermore, in the above-mentioned microbial agent, the seed culture medium in S2 is TCA liquid culture medium with a pH value of 7.0 to 7.2.

[0010] Furthermore, in the above-mentioned microbial agent, the production culture medium in S3 is TCA liquid culture medium with a pH value of 7.0 to 7.2. The aforementioned microbial agent, further, has a concentration of 2 × 10⁻⁶. 9 cfu / mL ~5×10 9 cfu / mL.

[0011] Based on a general technical concept, the present invention provides an application of the aforementioned microbial agent in improving the germination rate of tomato seeds and promoting root growth.

[0012] Based on a general technical concept, the present invention provides an application of the aforementioned microbial agent in enhancing the resistance of tomatoes to viral diseases.

[0013] Based on a general technical concept, the present invention provides an application of the aforementioned microbial agent in increasing tomato yield.

[0014] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides a butanol-containing Bacillus CSQK4 strain, which has a dual function of "medicine and fertilizer", which can promote tomato growth, increase yield, and enhance its resistance.

[0015] (2) This invention provides a butanol-based Bacillus agent with environmentally friendly and simple production process, low production cost, and fast strain growth rate. The bacterial agent can be prepared by a simple and quick operation method. The bacterial agent prepared using the strain of this invention has the advantages of environmentally friendly production process and low cost.

[0016] (3) This invention provides an application of a butanol-based Bacillus inoculant. Butanol-based Bacillus inoculants have advantages such as good growth-promoting effects on tomatoes, improved resistance, and increased yield, enabling high and stable tomato production. The strains of this invention can be used in tomato field production, possessing a dual function of "pesticide and fertilizer," and can partially replace agricultural chemicals.

[0017] A butanol-eating Bacillus, wherein the butanol-eating Bacillus is a butanol-eating Bacillus ( Peribacillus butanolivorans CSQK4, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025043, deposited on January 6, 2025. Attached Figure Description

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0019] Figure 1 This is a colony diagram of the strain from Example 1 of the present invention.

[0020] Figure 2 This is a bar chart showing the effect of different concentrations of microbial agents on the germination rate of tomatoes in Example 3 of the present invention.

[0021] Figure 3 This is a phenotypic diagram showing the effect of different concentrations of microbial agents on the growth of tomato radicles in Example 3 of the present invention.

[0022] Figure 4 This is a bar chart showing the effect of different concentrations of microbial agents on the growth of tomato radicles in Example 3 of the present invention.

[0023] Figure 5 The bar chart shows the effect of different concentrations of bacterial agents on the resistance of tomatoes to tomato brown wrinkled fruit virus in Example 4 of the present invention.

[0024] Figure 6 This is a bar chart showing the effect of different concentrations of microbial agents on tomato yield increase in Example 5 of the present invention. Detailed Implementation

[0025] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0026] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0027] TCA solid culture medium composition: 15 g / L tryptone, 5 g / L soybean papain hydrolysate, 5 g / L NaCl, 15 g / L agar, pH 7.0–7.2.

[0028] Example 1 A type of butyrol-containing Bacillus ( Peribacillus butanolivorans CSQK4, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025043, deposited on January 6, 2025.

[0029] The butyrol-containing Bacillus CSQK4 of this invention was obtained by screening using the following method: Five g of tomato rhizosphere soil samples collected from tomato-growing areas in Changsha, Hunan Province, were added to 5 mL of sterile phosphate buffer, sealed, and vortexed for 10 min, followed by serial dilution. 10 -1 ~10 -3 1 mL of diluent was added to 9 mL of TCA liquid medium and incubated at 35°C for 24 h. The bacterial suspension was then diluted and spread onto TCA solid medium. The culture was incubated until colonies appeared. Bacteria with different colony morphologies were picked and inoculated separately into TCA liquid medium to screen for butanol-containing Bacillus. Peribacillus butanolivorans CSQK4 is deposited at the China Center for Type Culture Collection.

[0030] Figure 1 This is a colony diagram of the butanol-producing Bacillus CSQK4 strain of this invention on TCA medium. As can be seen from the diagram, the colonies are milky white, round, and have smooth edges.

[0031] The 16S rDNA sequence is 1118 bp in length and has accession number C_AA165263.1 in the National Genome Science Data Center (Beijing) GenBase.

[0032] Example 2 A microbial agent, composed of the butyrate-type Bacillus (Bacillus) from Example 1. Peribacillus butanolivorans CSQK4 is obtained through fermentation. The specific fermentation process includes: (1) Activation: The cryopreserved strain of Bacillus CSQK4 was cultured by plate culture until a single colony appeared. The plate culture method is preferably carried out using TCA solid medium with a pH of 7.0 to 7.2 and a culture temperature of 30℃ to 35℃.

[0033] (2) Seed culture: Inoculate the single colony cultured in step (1) into an Erlenmeyer flask and culture it in seed culture medium until the logarithmic growth phase; the seed culture medium is preferably TCA liquid culture medium with a pH of 7.0 to 7.2, a culture temperature of 30 to 35°C, and a shaking speed of 300 rpm.

[0034] (3) Production culture: Inoculate 1% of the total volume of the bacterial culture medium obtained after step (2) into the production bottle, and culture with TCA liquid. The culture medium components and culture conditions are the same as in step (2). Culture until the logarithmic growth phase.

[0035] (4) Dispense the bacterial agent into bottles. The entire process of this embodiment involves a culture time of 3-5 days. After the culture is completed, the number of viable bacteria reaches 3×10⁻⁶. 9 cfu / mL or higher.

[0036] Example 3 The application of the microbial agent of Example 2 in improving the germination rate of tomato seeds and promoting root growth. Its application method includes: (1) The butanol-containing Bacillus strain CSQK4 prepared in Example 2 was diluted 200 times and 100 times with phosphate buffer (PBS) respectively, and added to filter paper to moisten the filter paper.

[0037] (2) Sow 100 tomato seeds evenly on filter paper and culture at 25±2℃ for 7 days.

[0038] Figure 2 The bar chart shows the effect of different concentrations of the inoculant on the germination rate of tomatoes. The chart shows that when the inoculant was diluted 100-fold and 200-fold, the tomato germination rates were 93.25% and 96.50%, respectively, both significantly higher than the PBS-based control (tomato germination rate 85.00%). The tomatoes were then cultured at 25±2℃ for another 9 days.

[0039] Figure 3 Phenotypic diagram showing the effects of different concentrations of microbial agents on the growth of tomato radicles.

[0040] Figure 4 The bar chart shows the effect of different concentrations of the inoculant on the growth of tomato radicles. As can be seen from the figure, when the inoculant was diluted 100 times and 200 times, the length of the tomato radicle was 8.17 cm and 8.66 cm, respectively, both of which were significantly higher than the PBS-based control (tomato radicle length was 3.5 cm).

[0041] Example 4 The application of the microbial agent of Example 2 in enhancing the resistance of tomatoes to viral diseases. Its application method includes: (1) Preparation of bacterial culture: The bacterial culture of Bacillus CSQK4, a butanol-containing Bacillus strain, was diluted 200 times and 100 times with phosphate buffer (PBS).

[0042] (2) Inoculate the tomato seedlings with the virus 7 to 10 days after transplanting and recovery. Start spraying the bacterial solution 24 hours later. It is best to apply the bacterial solution on a cloudy or sunny day and there should be no rain within 24 hours after application.

[0043] (3) After 14 days, the viral load was detected by specific RT-qPCR. The gene sequence of primer F2 is shown in SEQ ID NO.1, specifically: 5'-CTT CCA AAC GTG TAC GCA C-3'; the gene sequence of primer R2 is shown in SEQ ID NO.2, specifically: 5'-GTC GAG ATA TGT CGA ATA GA-3'.

[0044] Figure 5 The bar chart shows the effect of different concentrations of the inoculant on the relative genmic RNA level of TobRFV resistance in tomatoes. As can be seen from the figure, at 100-fold and 200-fold dilutions of the inoculant, the relative content of ToBRFV genomic RNA in tomato leaves was 5.64 and 5.48, respectively, both significantly lower than the PBS-based control (relative content of ToBRFV genomic RNA in tomatoes was 10.84).

[0045] Example 5 Application of the microbial agent of Example 2 in increasing tomato yield.

[0046] The test site was located in Gaoqiao Town, Changsha County, Changsha City, Hunan Province, covering an area of ​​approximately 667 m². 2 The previous crop was tomatoes, with a soil pH of 6.75 and an organic matter content of 1.65%. No other microbial agents were used throughout the experiment, and other field management practices followed local conventions.

[0047] The experiment included four treatments: a 100X dilution of the inoculant, a 200X dilution, a phosphate-buffered saline (PBS) group, and a water control (ddH2O) group. The application method was as follows: the first application of the inoculant solution was given 7 days after the tomato seedlings had established themselves; subsequent applications were given at the initial flowering and fruiting stages, for a total of three applications. All three applications were conducted on cloudy or sunny days, and there was no rainfall within 24 hours of application. The average daily temperature during the field trial was 25.5℃. Yields in each plot were measured at harvest, and the converted yield and yield increase rate were calculated.

[0048] Figure 6This is a bar chart showing the effect of different concentrations of the microbial agent on tomato yield (Yield of field plots). The chart shows that dilutions of the microbial agent 100-fold and 200-fold resulted in tomato yield increases of 9.19% and 10.56%, respectively, both higher than the PBS control (tomato yield increase of 1.03%). The tomato yield increase rate was calculated as follows: the tomato yield obtained from spraying tomatoes with the microbial agent diluted 100-fold and 200-fold, minus the tomato yield obtained from spraying with PBS, divided by the PBS-sprayed tomato yield, multiplied by 100%.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A butanol-producing Bacillus strain, characterized in that, The butyrol-containing Bacillus is a butyrol-containing Bacillus ( Peribacillus butanolivorans CSQK4, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2025043, deposited on January 6, 2025.

2. A microbial agent, characterized in that, The bacterial agent is obtained by fermentation from the butanol-containing Bacillus strain described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The fermentation process is as follows: S1. Activation: Culture the butyrate-producing Bacillus CSQK4 strain until a single colony appears; S2. Seed culture: The single colony is inoculated into a seed culture medium and cultured until the logarithmic growth phase to obtain a seed solution; S3. Production culture: Inoculate the seed liquid into the production culture medium at an inoculation rate of 1% of the total volume of the production culture medium, and culture until the logarithmic growth phase; S4. The bacterial agent is prepared by dispensing and packaging.

4. The microbial agent according to claim 3, characterized in that, The cultivation conditions described in S1, S2 and S3 are as follows: oscillation cultivation at 30℃~35℃ and an oscillation speed of 300 rpm.

5. The microbial agent according to claim 3, characterized in that, The seed culture medium in S2 is TCA liquid culture medium with a pH value of 7.0 to 7.

2.

6. The microbial agent according to claim 3, characterized in that, The production culture medium in S3 is TCA liquid culture medium with a pH value of 7.0 to 7.

2.

7. The microbial agent according to any one of claims 2 to 6, characterized in that, The concentration of the bacterial agent is 2×10⁻⁶. 9 cfu / mL ~5×10 9 cfu / mL.

8. The application of the microbial agent according to any one of claims 2 to 7 in improving the germination rate of tomato seeds and promoting root growth.

9. The microbial agent according to any one of claims 2 to 7, in enhancing the resistance of tomatoes to Tomato Brown Ruffle Virus (TRUV). Tomato brown rugose fruit virus Applications in resistance.

10. The application of the microbial agent according to any one of claims 2 to 7 in increasing tomato yield.

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