Bacillus cereus bc24 and application thereof

Abstract

This invention relates to a rhizosphere probiotic of the genus Bacillus cereus ( Bacillus cereus This invention relates to the field of microbial technology, specifically to BC24 and its applications. The strain is a microorganism isolated from tomato soil subjected to continuous cropping for many years. This strain exhibits a strong ability to degrade coumaric acid, an autotoxic substance in tomatoes. It can utilize ferulic acid and caffeic acid as the sole carbon source for growth and reproduction, achieving degradation rates exceeding 80% for both autotoxic substances. Cultivation with a solution containing the BC24 bacterium effectively mitigates the stress of phenolic acids on tomato plants, promoting normal growth and development. The rhizosphere probiotic BC24 of this invention possesses excellent biocontrol potential and can be used to develop biocontrol agents that alleviate continuous cropping obstacles.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a rhizosphere probiotic Bacillus cereus BC24 and its applications. Background Technology

[0002] Tomatoes are an important economic crop in my country, widely cultivated due to their rich nutritional value and mature cultivation techniques. In recent years, with the increasing demands of modern people, economic crops have developed rapidly in agriculture. However, the increasing multiple cropping index and intensification of tomato greenhouse cultivation have led to rapid soil degradation and increasingly serious problems with continuous cropping obstacles, directly resulting in weakened plant growth, decreased quality and yield, and an increase in soil-borne diseases and pests.

[0003] The causes of continuous cropping obstacles are complex, involving factors such as deterioration of soil physical and chemical properties, accumulation of autotoxic substances, and imbalance of soil microecology. For tomatoes, coumaric acid is a type of phenolic acid allelochemical autotoxic substance that significantly impacts tomato growth. It can induce autotoxicity, affecting tomato seed germination and plant growth, thus restricting high-quality and high-yield tomatoes. Currently, continuous cropping obstacles seriously affect the sustainable development of the tomato planting industry and have become a pressing production problem that needs to be solved.

[0004] Therefore, the inventors utilized functional bacterial strains to degrade phenolic acids in the soil environment used for tomato cultivation to prevent autotoxicity. Some indigenous beneficial microorganisms can use phenolic acids as their sole carbon and energy source for growth, thus achieving biodegradation. Simultaneously, phenolic acids are difficult to eliminate through physical or chemical methods; therefore, microbial methods are used to address the accumulation of phenolic acid autotoxic substances in the soil. For economic crops such as tomatoes, the *Bacillus cereus* BC24 isolated by the inventors has a significant effect on the degradation of coumaric acid, ferulic acid, and caffeic acid, effectively mitigating the stress of phenolic acids on tomato plants, promoting normal growth and development. Furthermore, this process is effective and safe, and is of great significance for preventing continuous cropping obstacles caused by phenolic acid autotoxicity and protecting the agricultural ecological environment. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly efficient rhizosphere probiotic for degrading tomato autotoxins and its applications. This strain belongs to the genus *Bacillus*, and its preservation name is *Bacillus cereus* (…). Bacillus cereus BC24 can degrade autotoxic substances secreted by tomato roots, thereby slowing down the occurrence of continuous cropping obstacles in tomatoes.

[0006] As a first aspect of the present invention, a rhizosphere probiotic of the genus Bacillus cereus is provided. Bacillus cereusBC24, the genus mentioned, is a rhizosphere probiotic Bacillus cereus BC24 isolated from soil of tomatoes that has been continuously cropped for many years and is capable of degrading the toxin paracoumaric acid in tomatoes. This genus was deposited on August 31, 2023 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO.28308.

[0007] The rhizosphere probiotics provided by this invention have been identified as Bacillus genus. They are characterized by: Gram-positive bacterial cells, short and thick rod-shaped, without spores or flagella, and nearly circular colonies with neat edges, glossy, opaque, milky white, and sticky surfaces when grown on LB agar plates.

[0008] As a second aspect of the present invention, the application of the rhizosphere probiotic Bacillus cereus BC24 in the efficient degradation of phenolic acid autotoxic substances is provided.

[0009] This bacterium exhibits a strong ability to degrade phenolic acids. After 24 hours of liquid shake-flask culture, the degradation rates of coumaric acid at substrate concentrations of 300 mg / L and 700 mg / L were 100% and 89.6%, respectively. After 48 hours, the degradation rates of ferulic acid and caffeic acid at substrate concentrations of 300 mg / L were 100% and 66.2%, respectively. After 72 hours, the degradation rate of caffeic acid was 82.3%.

[0010] As a third aspect of the invention, the application of the rhizosphere probiotic Bacillus cereus BC24 is provided to alleviate the stress of eucoumaric acid on the growth and development of tomato seedlings. In the embodiments, it was verified that Bacillus cereus BC24 can reduce the physiological stress of eucoumaric acid on tomatoes and promote seedling growth.

[0011] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention provides a rhizosphere probiotic, Bacillus cereus BC24, which has the ability to degrade tomato autotoxic substances, including coumaric acid, ferulic acid, and caffeic acid. 2. The rhizosphere probiotic Bacillus cereus BC24 provided by this invention can alleviate the physiological stress of coumarin on tomatoes and promote seedling growth. Attached Figure Description

[0012] Figure 1 These are morphological photos of strain BC24 on LB medium; Figure 2 This is a phylogenetic tree diagram of strain BC24; Figure 3 This is the HPLC standard curve for coumaric acid; Figure 4 This is a bar chart showing the effect of strain BC24 on the degradation of p-coumaric acid; Figure 5 These are chromatographic separation diagrams of six phenolic acids; Figure 6 This is a bar chart showing the degradation effect of strain BC24 on ferulic acid and caffeic acid; Figure 7 This is a line graph showing the optimal growth curve of strain BC24. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings.

[0014] Example 1: 1. Isolation and identification of functional bacteria that degrade tomato autotoxins The isolation of functional bacteria that degrade tomato autotoxins mainly includes four parts: enrichment, selective culture, purification, and domestication.

[0015] (1) Enrichment. Rhizosphere soil samples from tomatoes grown in continuous cropping for several years were collected. 10.00 g of soil sample was passed through a 2 mm sieve and cultured in 50 mL of 100 mg / L p-coumaric acid inorganic salt medium on a shaker (30℃, 180 r / min) for 3 days. Every 3 days thereafter, 5 mL of the soil enrichment solution was transferred from the shake flask to 45 mL of fresh p-coumaric acid inorganic salt medium (inoculation rate 2%), with the p-coumaric acid concentration increased accordingly. The acclimatization concentration gradient was 100, 300, 500, 700, and 1000 mg / L. The inorganic salt medium used in the experiment consisted of (NH4)2SO4 2 g / L, KH2PO4 2 g / L, Na2HPO4 1.3 g / L, p-coumaric acid 100–1000 mg / L, NaCl 5 g / L, with the remainder being water.

[0016] (2) Selective culture. Dilute the last enrichment culture medium and spread it (10). -4 10 -5 10 -6 The samples were cultured on LB solid medium, with each dilution repeated three times, and incubated at 30°C for 4 days. Lysogeny broth (LB) medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L, natural pH.

[0017] (3) Purification. After a clear colony has grown, pick a single colony and streak it on LB solid medium. Once the colony has grown, observe its culture characteristics, including colony color, surface smoothness (smooth, rough, wrinkled, concentric rings, radial), whether the substrate produces water-soluble pigments, size, shape, edges (smooth, serrated, wavy, fibrous, etc.), ridges (convex, concave, flat), presence or absence of fluorescence, transparency, and the color and viscosity of the medium. If the culture characteristics are completely consistent, they can be considered as the same bacteria. If no single colony is found, repeated streaking and purification are necessary.

[0018] (4) Domestication. After isolation and purification, the strains were domesticated. Inorganic salt culture medium was prepared in the same way. The selected strains were then inoculated back into inorganic salt culture medium containing p-coumaric acid, and the carbon source concentration was increased to 700 mg / L. Some microorganisms that could adapt or produce mutations remained and became the dominant bacterial group.

[0019] 2. Identification of BC24 strain (1) Characteristics of bacterial cell morphology BC24 strain is Gram-positive. Figure 1 It is a short, thick rod-shaped organism, without spores or flagella. When it grows on LB agar plates, the colonies are nearly round with neat edges, a glossy surface, are opaque, milky white, and sticky.

[0020] (2) 16S rDNA sequence analysis The 16S rDNA sequence of strain BC24 obtained from the secondary screening was determined. The 16S rDNA sequencing was performed by Shanghai Biopharmaceutical Co., Ltd., and the 16S rDNA sequence length was 1457 bp, as shown in SEQ ID NO: 1. This sequence fragment was subjected to sequence homology analysis using the NCBI database to obtain the 16S rDNA of similar strains. The obtained 16S rDNA of similar strains was then subjected to multiple sequence homology analysis using MEGA 5.0 software, and a phylogenetic tree was constructed (…). Figure 2 ).

[0021] Based on morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain BC24 was identified as belonging to the genus *Bacillus cereus*. Bacillus cereus ).

[0022] This strain was deposited on August 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO.28308.

[0023] Example 2: Determination of the degradation rate of coumaric acid by tomato autotoxins Chromatographic conditions were established: the standard sample was of chromatographic purity; the column temperature was 25 ℃; the UV detection wavelength was 280 nm; mobile phase B was 0.25% aqueous acetic acid solution; and mobile phase C was methanol. Separation of the samples was achieved using gradient elution with the following conditions: mobile phase B: 70% (0 min) → 50% (15 min) → 30% (16 min) → 0 (30 min) → 70% (35 min); flow rate 1.0 mL / min; and injection volume 20 µL. Standard curve preparation: standard solutions of 0, 100, 300, 500, 700, and 1000 mg / L p-coumaric acid were prepared. Analysis was performed under the above chromatographic conditions, and a standard curve was constructed with standard concentration as the coordinate axis and peak area as the ordinate. Figure 3 ).

[0024] Determination of coumaric acid degradation capacity: The isolated and screened Bacillus cereus BC24 was placed in LB medium and shaken for 24 hours. The cells were then collected by centrifugation at 5000 r / min, and the OD value was adjusted. 600 =0.2, 4 ml were transferred to 200 ml of sterile inorganic salt liquid culture medium with p-coumaric acid substrate concentrations of 0, 300, and 700 mg / L (inoculum rate 2%), and cultured at 37℃ with shaking at 180 r / min for 7 days. 4 ml of sterile water was added as a control. Each treatment was replicated twice, and samples were taken at 0, 12, 24, 36, 48, 72, 96, and 168 h. The residual p-coumaric acid was calculated based on the standard curve described above. Degradation status is shown in […]. Figure 4 .

[0025] At 24 hours, strain BC24 achieved degradation rates of 100% and 89.6% for p-coumaric acid at substrate concentrations of 300 mg / L and 700 mg / L, respectively. This case demonstrates that the isolated Bacillus cereus strain BC24 can grow and reproduce using p-coumaric acid as the sole carbon source and possesses a highly efficient ability to degrade p-coumaric acid.

[0026] Example 3: Determination of the broad-spectrum substrate activity of strain BC24 One ml of BC24 strain culture broth was inoculated into 50 ml of inorganic salt liquid culture medium containing protocatechuic acid, syringic acid, caffeic acid, coumarin, ferulic acid, and salicylic acid as substrates, respectively, at a substrate concentration of 300 mg / L. The culture was incubated at 37℃ with shaking at 180 rpm. Samples were filtered through a 0.22 µm microporous membrane at 0, 48, and 72 h, and the substrate amount was determined by HPLC. The substrate degradation efficiency was calculated as [(substrate amount at zero time - substrate amount at the end time) / substrate amount at zero time] × 100%, yielding the substrate degradation spectrum of the phenolic acid degrading bacteria. Qualitative analysis was performed using standard retention time analysis, determining the retention times of the six phenolic acids as follows: protocatechuic acid 5.183 min, syringic acid 8.321 min, caffeic acid 9.538 min, coumarin 11.708 min, ferulic acid 12.892 min, and salicylic acid 16.813 min. The separation chromatograms are shown below. Figure 5 .

[0027] Strain BC24 exhibits a certain ability to degrade both ferulic acid and caffeic acid. After 48 hours, the degradation rates for ferulic acid and caffeic acid were 100% and 66.2%, respectively, and after 72 hours, the degradation rate for caffeic acid was 82.3%. Experimental results are shown below. Figure 6 .

[0028] Example 4: Determination of optimal growth conditions for strain BC24 Prepare 50 mL LB medium solutions with pH values ​​of 4, 5, 6, 7, 8, and 9, and NaCl concentrations of 0.5%, 1%, 2%, and 4%. Sterilize the LB medium solutions with different pH and NaCl concentrations at 121°C for 20 min. After cooling to room temperature, inoculate with 2% of the isolated single bacterial strain suspension (OD). 600 =0.2). Add 0.2 mL of culture medium to each well, with 5 replicates per group. Place the sample plate in a Bioscreen C (FP-1100-c, Bioscreen C, Finland) incubator and incubate at medium speed and 37°C for 72 h. Determine the optimal growth pH and salt concentration based on the growth curve of the strain.

[0029] The optimal growth conditions for strain BC24 are a salt concentration of 0.5-1% and a pH of 5.0-7.0. Experimental results are shown below. Figure 7 .

[0030] Example 5: Experiment on functional bacteria alleviating tomato seedling stress caused by coumarin acid stress Tomato seedlings were cultured in a substrate. Once they reached the four-leaf stage, uniformly growing seedlings were transplanted into 50mL centrifuge tubes, one seedling per tube. 40mL of a 1 / 2MS culture medium containing 0 mg / L and 100 mg / L coumaric acid was added, along with inoculation with 4% of the isolated single-strain bacterial suspension (OD200). 600=0.2), the control treatment was inoculated with an equal volume of sterile water. There were four treatments: ① 0 mg / L p-coumaric acid; ② 100 mg / L p-coumaric acid; ③ 0 mg / L p-coumaric acid + BC24; ④ 100 mg / L p-coumaric acid + BC24. Each treatment was replicated three times, with each replicate including five tomato seedlings. The seedlings were then placed in a light incubator (33℃ / 16 h photoperiod, 28℃ / 8 h dark period, light intensity 400 μmol·m⁻¹). -2 ·s -1 The seedlings were cultured in [a specific environment] for 7 days. After the experiment, the plant height and fresh weight of the tomato seedlings were recorded. Fusarium wilt was classified into five levels: 0-4. Level 0: healthy plants with no yellowing leaves; Level 1: no wilting symptoms, but less than 50% of leaves are yellow; Level 2: no wilting symptoms, but more than 50% of leaves are yellow; Level 3: wilting leaves and lesions at the base of the stem; Level 4: plant death. The disease index (DI) was calculated using the following formula: Disease index = Σ (number of infected plants at each level × representative value for that level) / (total number of plants surveyed × highest representative value for the disease level) × 100.

[0031] The experimental group supplemented with strain BC24 showed better growth than the control group supplemented with sterile water. The experimental results are shown in Table 1.

[0032] Table 1

[0033] It was verified that Bacillus BC24 can alleviate the physiological stress of coumarin on tomatoes and promote seedling growth.

[0034] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rhizosphere probiotic, Bacillus cereus BC24, characterized by: The strain was deposited on August 31, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO. 28308.

2. The application of the rhizosphere probiotic Bacillus cereus BC24 as described in claim 1, characterized in that: The rhizosphere probiotic Bacillus cereus BC24 is used to degrade the tomato rhizosphere phenolic acid autotoxic substances, which include p-coumaric acid, ferulic acid, and caffeic acid.

3. The application of the rhizosphere probiotic Bacillus cereus BC24 according to claim 2, characterized in that, The application is to alleviate the problem of continuous cropping of tomatoes.

4. The application of the rhizosphere probiotic Bacillus cereus BC24 according to claim 3, characterized in that: The purpose of alleviating the continuous cropping obstacle of tomatoes is to alleviate the stress effect of the phenolic acid on tomato plants, which is beneficial to the normal growth and development of tomato plants.

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