Saline-alkali tolerant bacillus cereus and application thereof

Abstract

The invention discloses saline-alkaline tolerant bacillus cereus and application thereof, and belongs to the technical field of microorganisms. The bacillus cereus X2-1-4 is separated from a soil sample, integrates multiple functions of high salt resistance, alkali resistance, alkali reduction, soil improvement, disease control and the like, has a relatively strong bacteriostatic effect on the sclerotinia sclerotiorum, can be used for preventing and treating the sclerotinia rot of soybeans or sunflower caused by the sclerotinia sclerotiorum, and has a good application prospect in prevention and treatment of the sclerotinia rot of the soybeans or the sunflower caused by the sclerotinia sclerotiorum. The method can be widely applied to soybean and sunflower planting in large saline-alkali soil in China.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a salt-tolerant Bacillus cereus strain and its applications. Background Technology

[0002] Soybeans and sunflowers are important dual-purpose crops for grain and oil in northern my country, and pioneer crops for the improvement and utilization of saline-alkali land. Soybeans can grow normally in slightly saline-alkali land (soil pH 7.5-8.5, total salt content ≤0.3%), while sunflowers are even more salt-tolerant and can withstand moderately saline-alkali land (soil pH 8.0-9.0, total salt content ≤0.6%). Soybeans, as a core source of high-quality plant protein, account for more than 40% of the plant protein intake of Chinese residents; sunflowers are a characteristic oilseed crop in the main saline-alkali producing areas of northern China, with sunflower seeds containing 45%-55% oil. Both play an irreplaceable role in ensuring national food security and improving agricultural economic benefits, and also play a key role in the development of agriculture in saline-alkali land.

[0003] Sclerotinia stem rot in soybeans and sunflowers are devastating diseases that restrict the growth of these two major crops. The damage is even more severe in saline-alkali environments. Salt and alkali stress reduces the crop's own disease resistance and accelerates the germination and infection of sclerotia, resulting in a disease incidence rate that is 15%-25% higher than in non-saline-alkali areas. In severe years, the incidence of soybean sclerotinia stem rot in major producing areas (including saline-alkali land) can reach 40%-60%, resulting in a yield reduction of 30%-50%, and some continuously cropped saline-alkali plots suffer total crop failure due to the disease (Zhang Li, Wang Ying, Li Juan, et al. Screening and genetic mechanism of soybean sclerotinia stem rot resistance resources [J]. Chinese Journal of Oil Crops, 2021, 43 (2): 312-320); In sunflower, the incidence of sunflower sclerotinia stem rot in major saline-alkali producing areas such as Inner Mongolia and Xinjiang is generally 25%-45%, which not only causes a yield loss of 20%-40%, but also leads to a mold rate of more than 25% in infected flower heads, a decrease in sunflower seed oil content of 8%-12%, and a serious reduction in commercial quality (Liu Yang, Zhang Wei, Chen Ming, et al. Evaluation and screening of sunflower sclerotinia stem rot resistance germplasm resources [J]. Crop Journal, 2022, (3): 89-95).

[0004] Both diseases are caused by Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorumWhile the pathogens are caused by saline-alkali soil, significant physiological race differentiation exists, and the pathogenicity of dominant races is exacerbated by saline-alkali soil environments. Zhao Kai et al. identified at least three dominant physiological races (races I, II, and III) in 186 strains of *Sclerotinia sclerotiorum* from 15 major soybean and sunflower producing areas (including 6 saline-alkali plots) in northern China: Race I is a soybean-specific pathogenic race, with a 20%-30% increase in pathogenicity in saline-alkali soils. It can rapidly invade the stems through soybean petals, leading to a stem rot rate of over 80%. Race III is a sunflower-specific pathogenic race, with a 40% increase in infection efficiency under moderate saline-alkali conditions. It can directly infect the flower heads and spread rapidly, with a flower head rot rate of over 75%. Race II has the ability to infect both crops, but its pathogenicity is weak (contributing less than 10% to disease incidence in the field). Further investigation revealed that the segregation rate of race I in soybean sclerotinia rot diseased plants reached 68%, while the segregation rate of race III in sunflower sclerotinia rot diseased plants reached 72% (Zhao Kai, Wang Feijuan, Pan Xuebo, et al. Study on physiological race differentiation and pathogenic characteristics of sclerotinia rot in soybean and sunflower [J]. Acta Microbiologica Sinica, 2020, 60 (7): 1423-1435). This indicates that the host specificity of different physiological races and the synergistic effect of saline-alkali environment are key factors that make it difficult to control the two diseases simultaneously. In addition, Sclerotinia sclerotiorum can form black sclerotia to overwinter in the soil. The survival period of sclerotia in saline-alkali soil is extended to 4-6 years, which is 20%-40% higher than that in non-saline-alkali soil. Under continuous cropping conditions, the accumulation of sclerotia in the soil increases by an average of 15%-20% per year, which further aggravates the occurrence of diseases (Wang Hao, Li Na, Zhao Qiang, et al. Effects of chemical fungicides on the resistance of Sclerotinia sclerotiorum and soil microbial diversity [J]. Journal of Agricultural Environmental Science, 2019, 38 (8): 1856-1863).

[0005] Currently, the main methods for controlling sclerotinia stem rot in soybeans and sunflowers are chemical control. Commonly used chemical agents include iprodione, sclerotinia sclerotinia var. malignant, and isopyrazosulfuron, while biological agents mainly include jinggangmycin and kasugamycin. However, in saline-alkali soil environments, the shortcomings of these control methods are more prominent. The field efficacy of chemical agents against soybean sclerotinia stem rot can reach 55%-70% in non-saline-alkali soils, but in saline-alkali soils, due to decreased agent stability (high pH leading to partial agent decomposition), the efficacy drops to 40%-55%. The efficacy against sunflower sclerotinia stem rot drops from 50%-65% in non-saline-alkali soils to 35%-50%. The efficacy of bio-based agents is even lower, with only 35%-50% efficacy against soybean sclerotinia stem rot, further decreasing to 25%-40% in saline-alkali soils (Li Zhe, Wang Li, Liu Min, et al. Monitoring and analysis of resistance of Sclerotinia stem rot to iprodione in major soybean producing areas [J]. Journal of Plant Protection, 2020, 47 (5): 1089-1096; Wang Hao, Li Na, Zhao Qiang, et al. Effects of chemical fungicides on resistance of Sclerotinia stem rot and soil microbial diversity [J]. Journal of Agricultural Environmental Science, 2019, 38 (8): 1856-1863).

[0006] In addition, due to limited arable land resources, the continuous cropping cycle in major soybean and sunflower producing areas has generally been shortened to 2-3 years, and the accumulation of Sclerotinia sclerotiorum in the soil has increased by 15%-20% annually. This has resulted in the control efficacy of chemical agents against Sclerotinia sclerotiorum in the soil being less than 30%, and the disease has become increasingly severe year by year (Wang Hao, Li Na, Zhao Qiang, et al. Effects of chemical fungicides on Sclerotinia sclerotiorum resistance and soil microbial diversity [J]. Journal of Agricultural Environmental Science, 2019, 38 (8): 1856-1863). Moreover, long-term use of chemical agents has led to serious resistance. Li Zhe et al. found that the resistance rate to iprodione in 128 strains of Sclerotinia sclerotiorum in major soybean producing areas such as Heilongjiang and Jilin reached 35.2%, of which highly resistant strains accounted for 12.5%, resulting in the field control efficacy dropping from the initial 70% to below 40% (Li Zhe, Wang Li, Liu Min, et al. Monitoring and analysis of Sclerotinia sclerotiorum resistance to iprodione in major soybean producing areas [J]. Journal of Plant Protection, 2020, 47(5): 1089-1096); Meanwhile, the acute toxicity (LD50) of sclerotinia sulfadiazine to sunflower pollinating bees was also investigated. 50 =0.03 mg / bee) limited the application of the pesticide during the flowering period (Huang Wei, Chen Tao, Zhou Lin, et al. Toxicity evaluation of the pesticide for controlling sunflower sclerotinia stem rot to pollinating bees [J]. Acta Phytopathologica Sinica, 2018, 48 (4): 521-528), while the flowering period is the key infection period of sunflower sclerotinia stem rot, which further reduced the control effect.

[0007] The main technology for achieving zero growth in chemical pesticide use is the substitution of chemical pesticides with biopesticides and biological agents. In saline-alkali land agriculture, salt-tolerant biocontrol agents are the core requirement for overcoming the bottleneck in pest control. However, in current soybean and sunflower production, there is a severe shortage of highly effective biological agents targeting sclerotinia rot. The only known agents, Bacillus subtilis and Trichoderma viride, not only have a narrow spectrum of control but also lack salt tolerance. When the soil pH is ≥8.0, the survival rate of Bacillus subtilis drops below 50%, and the spore germination rate of Trichoderma viride is less than 30%, making them completely unsuitable for saline-alkali land environments. Zhao Kai et al. isolated Bacillus subtilis B1, which showed an indoor inhibition rate of 81.2% and a field control efficacy of 62.3% against soybean sclerotinia stem rot (race I). However, it showed an inhibition rate of only 45.8% and a control efficacy of only 31.5% against sunflower sclerotinia stem rot (race III). Moreover, its colonization in saline-alkali soil with a pH of 8.5 was less than 10% of that in non-saline-alkali soil (Zhao Kai, Wang Feijuan, Pan Xuebo, et al. Study on physiological race differentiation and pathogenic characteristics of Sclerotinia sclerotiorum in soybean and sunflower [J]. Acta Microbiologica Sinica, 2020, 60 (7): 1423-1435). Chen Xi et al. reported that Trichoderma viride T2 showed a control efficacy of 50.1% against sunflower sclerotinia stem rot (race III) and less than 30% against soybean sclerotinia stem rot (race I). It was almost completely inactivated in soil with a total salt content of 0.4% (Chen Xi, Liu Jie, Zhang Meng, et al. Study on the control effect and colonization characteristics of Trichoderma viride against sunflower sclerotinia stem rot [J]. Journal of Biological Control, 2021, 37 (2): 345-353. Furthermore, the existing biocontrol bacteria suffer from weak colonization and short-lasting effects, which are more pronounced in saline-alkali soils. For example, the population of *Trichoderma viride* T2 in the soybean rhizosphere peaked at 1.2 × 10⁻⁶ after 7 days. 6 CFU / g (soil) decreased to 1.5×10 after 21 days. 5 CFU / g soil (only a peak of 12.5%) cannot cover the entire growth period of soybeans and sunflowers (about 120-150 days), resulting in a recurrence rate of more than 40% in the later stage of the disease (Chen Xi, Liu Jie, Zhang Meng, et al. Study on the control effect and colonization characteristics of Trichoderma viride on sunflower sclerotinia disease [J]. Journal of Biological Control, 2021, 37 (2): 345-353).

[0008] Bacillus cereus ( Bacillus cereusSalt-tolerant Bacillus cereus is a type of Gram-positive bacteria widely found in soil. Some strains naturally possess a certain degree of stress resistance, while salt-tolerant strains, which can remain active in high-salt, high-pH environments, have become ideal resources for biological control in saline-alkali lands. These strains are characterized by spore production, strong stress resistance (tolerant to high temperatures of 40-50℃, drought, and salt-tolerant strains can tolerate pH 4.0-10.0 and total salt content ≤1.0%), and rapid reproduction (logarithmic growth phase of only 2-3 hours). Their spores can survive for 3-5 years in saline-alkali soils, demonstrating the potential for long-term control of soil-borne diseases. Salt-tolerant Bacillus cereus can inhibit pathogen growth by producing antimicrobial peptides (such as Bacillus cereus extract), chitinase, and cellulase, or by forming a biological barrier through competition for nutrients and spatial sites to prevent pathogen infection. Simultaneously, its metabolites can slightly improve soil salinity (lowering pH by 0.2-0.5), creating a synergistic effect of "biological control + soil improvement". Domestic studies have confirmed that *Bacillus cereus* is effective against gray mold in vegetables. Botrytis cinerea ), wheat scab ( Fusarium graminearum It has good antagonistic effects against diseases such as sclerotinia stem rot in soybeans and sunflowers, and the field control efficacy of some strains can reach 60%-70% (Sun Tao, Li Ming, Zhao Jing, et al. Optimization of liquid fermentation process and cost analysis of formulation of biocontrol bacteria [J]. Transactions of the Chinese Society of Agricultural Engineering, 2022, 38 (11): 256-263). However, there are no reports on salt-tolerant Bacillus cereus against sclerotinia stem rot in soybeans and sunflowers, showing great potential for biocontrol applications.

[0009] The patent, with patent number CN 112322134 A and titled "A strain of Bacillus cereus and its application in the control of vegetable diseases," tested the control effects of the described Bacillus cereus on diseases such as tomato gray mold, cucumber downy mildew, and eggplant verticillium wilt, but did not test its control effects on soybean sclerotinia stem rot and sunflower sclerotinia stem rot, nor did it mention the strain's salt and alkali tolerance characteristics (Shandong Agricultural University. A strain of Bacillus cereus and its application in the control of vegetable diseases: CN112322134 A [P]. 2021-02-02); *Sclerotinia sclerotiorum*, which causes soybean sclerotinia stem rot and sunflower sclerotinia stem rot (… Sclerotinia sclerotiorum ) and the tomato gray mold in the patent ( Botrytis cinerea Botrytis cinerea, a fungus belonging to the Deuteromycetes, and the cucumber downy mildew fungus ( Pseudoperonospora cubensis The genus *Pseudomonas* (Oomycetes) belongs to a different group of microorganisms, and its pathogenic mechanisms (such as *Sclerotinia sclerotiorum* overwintering by producing sclerotia and secreting oxalic acid toxins to destroy host cells) are significantly different from the diseases mentioned above. Furthermore, the physiological race differentiation characteristics of *Sclerotinia sclerotiorum* and its synergistic effect in saline-alkali soil environments are not mentioned in this patent. Therefore, it is impossible to deduce its ability to control sclerotinia sclerotiorum in soybeans and sunflowers through this patented technology, and it is also not suitable for saline-alkali soil planting scenarios. This results in a huge technological gap in the application of salt-tolerant *Bacillus cereus* in the control of sclerotinia sclerotiorum in the two major crops. Summary of the Invention

[0010] This invention overcomes the shortcomings of the prior art and proposes a salt- and alkali-resistant Bacillus cereus strain and its application; This invention is achieved through the following technical solution: A salt-tolerant Bacillus cereus strain, specifically Bacillus cereus ( Bacillus cereus X2-1-4, this strain was deposited on December 30, 2025 at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological preservation number: CGMCC No. 37243.

[0011] The *Bacillus cereus* strain provided by this invention was isolated from soil samples in Xuekulu Township, Shanyin County, Shuozhou City, Shanxi Province. This salt- and alkali-tolerant *Bacillus cereus* X2-1-4 strain integrates multiple functions, including high salt tolerance, alkali tolerance, alkali reduction, soil improvement, and disease control. It exhibits a strong inhibitory effect on *Sclerotinia sclerotinia*, and can control *Sclerotinia sclerotinia* disease in soybeans or sunflowers caused by *Sclerotinia sclerotinia*.

[0012] Inoculants containing the aforementioned Bacillus cereus are also within the scope of protection of this invention.

[0013] In the bacterial agent, Bacillus cereus can exist in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

[0014] Preferably, the formulation of the microbial agent can be an emulsion, suspension, granules, wettable powder, or water dispersible agent.

[0015] This invention provides the above-mentioned Bacillus cereus or an inoculant containing Bacillus cereus for the prevention and control of crops infected with Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Sclerotinia sclerotiorum Applications in diseases caused by ).

[0016] The disease in question is either soybean sclerotinia stem rot or sunflower sclerotinia stem rot.

[0017] The method for controlling sclerotinia stem rot in soybeans or sunflowers is to inoculate Bacillus cereus X2-1-4 into NB medium to prepare OD. 600 Soak soybean leaves in a Bacillus suspension with a concentration of 0.5 g / L, and then inoculate the leaves with the Bacillus cereus suspension using a pathogenic fungal cake.

[0018] The beneficial effects of this invention compared to the prior art are as follows: 1. The Bacillus cereus X2-1-4 described in this invention has extremely strong salt and alkali resistance, making it suitable for extreme salt and alkali environments. High salt tolerance: It can grow normally in culture medium with a concentration of 50~150 g / L NaCl and can tolerate an extreme high salt environment of 200 g / L (belonging to extreme halophilic bacteria). Even under high salt stress, it can still survive and maintain growth activity, breaking through the salt tolerance limit of ordinary microorganisms. Dual benefits of alkali resistance and alkali reduction: The Bacillus cereus X2-1-4 described in this invention has outstanding alkali resistance and strong alkali reduction ability. After 24 hours of fermentation and culture, the alkali reduction rate can reach up to 19.1% (all ≥15.0%), which can effectively reduce the pH value of the environment (such as soil) and alleviate the harm of salt and alkali stress to plants.

[0019] 2. Excellent EPS production capacity, contributing to soil ecological improvement. High extracellular polysaccharide (EPS) synthesis efficiency: the extracellular polysaccharide synthesis efficiency per unit biomass reaches 46.2%, and EPS production is sufficient; it indirectly improves soil salinization, creates suitable conditions for microbial and plant growth, and increases the biodiversity of the soil ecosystem.

[0020] 3. It exhibits strong antagonistic activity against pathogens and has significant potential for controlling crop diseases. Broad-spectrum antibacterial effect: It has a significant inhibitory effect on pathogens such as Sclerotinia sclerotiorum of soybean and Sclerotinia sclerotiorum of sunflower, with an antagonism coefficient of about 2.1 and an average inhibition rate of 62.9% (Sclerotinia sclerotiorum of soybean) and 58.7% (Sclerotinia sclerotiorum of sunflower), respectively. Moreover, the antagonistic effect is long-lasting, the edges of the inhibited pathogen hyphae are even, and the antibacterial effect is stable. Highly effective disease control: In practical applications, after treating soybean leaves with a bacterial suspension (OD600=0.5), the bacteria (Sclerotinia sclerotiorum)... Sclerotinia sclerotiorum The incidence rate of diseases caused by this fungicide was only 6.4%, far lower than that of the control group (76.5%), and the control effect increased with the increase of the concentration of the fungal suspension, showing significant control effect on related crop diseases.

[0021] In summary, the salt-tolerant Bacillus cereus X2-1-4 strain of this invention integrates multiple functions such as high salt tolerance, alkali tolerance, alkali reduction, soil improvement, and disease control. It exhibits a strong inhibitory effect on Sclerotinia sclerotinia, the causal agent of soybean sclerotinia, and can control soybean sclerotinia or sunflower sclerotinia caused by this pathogen. The results of this invention demonstrate that this strain has a strong inhibitory ability against Sclerotinia sclerotinia, providing a good biocontrol resource for controlling soybean or sunflower sclerotinia diseases. This strain can be directly applied to the development and utilization of saline-alkali land and soil ecological restoration, while effectively controlling crop diseases such as sclerotinia sclerotinia. It provides core microbial resource support for improving the quality and efficiency of agricultural production in saline-alkali areas and reducing the damage caused by salinity and alkali and disease losses. Its application scenarios are wide-ranging and highly practical. Attached Figure Description

[0022] Figure 1The colony morphology of Bacillus cereus strain X2-1-4 described in this invention; A: Colony morphology on the front side of the plate; B: Colony morphology on the back side of the plate; Figure 2 Gram-stained microscopic (40X) photograph of the Bacillus cereus strain X2-1-4 described in this invention; Figure 3 This is a Neighbor-Joining phylogenetic tree constructed based on the 16S rRNA gene nucleic acid sequence of Bacillus cereus strain X2-1-4 described in this invention (using the neighbor-joining method, bootstrap 1000). Figure 4 This is the amplification result of the gyrB and XRE gene fragments of Bacillus cereus strain X2-1-4 described in this invention; In the figure: M: marker; 1: gyrB water control; 2: gyrB gene amplification of strain X2-1-4; 3: XRE water control; 4: XRE gene amplification of strain X2-1-4; Figure 5 This describes the growth of Bacillus cereus X2-1-4 described in this invention in liquid culture media with different salt concentrations; Figure 6 This describes the growth of Bacillus cereus X2-1-4 as described in this invention on solid culture media with different salinity and alkalinity. Figure 7 The plate confrontation method is used to test Bacillus cereus X2-1-4 against Sclerotinia sclerotiorum var. soybeanus (…). Sclerotinia sclerotiorum The antagonistic effect of Bacillus cereus X2-1-4 and Bacillus cereus in the figure; A: Sclerotinia sclerotiorum var. soybeanis; B: culture of Bacillus cereus X2-1-4 and Sclerotinia sclerotiorum var. soybeanis. Figure 8 The plate confrontation method is used to test Bacillus cereus X2-1-4 against Sclerotinia sclerotiorum var. sunfloweris (…). Sclerotinia sclerotiorum The antagonistic effect of ) in the figure; A: Sclerotinia sclerotiorum var. sunfloweris; B: Bacillus cereus X2-1-4 and Sclerotinia sclerotiorum var. sunfloweris in culture.

[0023] Figure 9 This invention describes the control effect of Bacillus cereus X2-1-4 against soybean sclerotinia stem rot; in the figure, A: inoculation with soybean sclerotinia stem rot fungus; B: via OD... 600 =0.5 Bacillus cereus X2-1-4 was treated with soybean sclerotinia rot fungus. Detailed Implementation

[0024] To make the technical problems to be solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The technical solutions of this invention are described in detail below with reference to the embodiments and accompanying drawings, but the scope of protection is not limited thereto.

[0025] This invention proposes a salt- and alkali-tolerant Bacillus cereus strain X2-1-4, which is classified and named Bacillus cereus. Bacillus cereus X2-1-4, the *Bacillus cereus* X2-1-4, was deposited on December 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC NO. 37243. The *Bacillus cereus* provided by this invention was isolated from soil samples in Xuekulun Township, Shanyin County, Shuozhou City, Shanxi Province. Example

[0026] This embodiment describes the isolation, purification process, and identification results of the strain.

[0027] 1.1 Isolation and purification of Bacillus Five samples were taken from both the topsoil and deep soil layers. 2 g of each sample was placed in a sterilized Erlenmeyer flask, 100 mL of sterile water was added, and the flask was incubated at 28°C and 150 rpm for 1 hour. After incubation, the sample was diluted to a 10⁻⁶ ppm. -1 and 10 -2 bacterial solutions of varying concentrations were prepared in both stock solution and 10... -1 and 10 -2 Take 100 μL of the bacterial suspension at a specific concentration and spread it onto beef extract peptone plates, with three replicates. Incubate upside down in a 25°C incubator for 2–3 days. Remove the plates and observe the growth of the bacterial strain, colony morphology, and color.

[0028] Single colonies from the culture were streaked onto beef extract peptone medium for purification. Single colonies with different morphologies were picked and incubated in beef extract peptone liquid medium at 28°C in a shaker for 24 h. Each bacterial culture was then preserved with glycerol and stored at -80°C for later use.

[0029] 1.2 Morphological observation The purified strain X2-1-4 was streaked and Gram-stained for observation. We found that X2-1-4 is a Bacillus species characterized by a grayish-white color, a rough, waxy surface, irregular edges, and stringy texture (e.g., ...). Figure 1 Under an optical microscope, it appears as a short rod and shows a positive Gram reaction (e.g.) Figure 2 ).

[0030] 1.3 Molecular biological identification Single colonies were picked from the streaked plate and diluted in 50 μL of sterile water to serve as DNA templates. Using primers 27f: 5'-GTAAAACGACGGCCAGT-3' and 1492r: 5'-CAGGAAACAGCTATGAC-3', a 30 μL PCR reaction system was established to amplify the 16S rDNA sequence fragment. The reaction system is as follows: ; The reaction conditions are as follows: ; After the polymerase chain reaction (PCR) was completed, 5 μL of gel electrophoresis was performed to preliminarily determine that the extended fragment was approximately 1500 bp. The remaining PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for 16S sequencing. The sequencing results were compared with the 16S rDNA gene sequences of some registered strains using the BLAST tool in NCBI. A Neighbor-Joining phylogenetic tree was constructed using the 16S rRNA gene nucleic acid sequence (using the neighbor-joining method, bootstrap 1000) (e.g.) Figure 3 The results showed that it clustered with Bacillus cereus and Bacillus thuringiensis.

[0031] Following the method of Chelliah R et al. (Chelliah R, Wei S, Park BJ, et al. Wholegenome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers[J]. Microbial pathogenesis, 2019, 129: 284-297.), primers for the bacterial gyrB gene (approximately 220 bp) were selected. gyr BF 5'-GCCCTGGTATGTATATTGGATCTAC-3' and gyrPrimers XRE-F5'-AAGATATTGCAAGCGGTAAGAT-3' and XRE-R 5'-GTTTTGTTTCAGCATTCCAGTAA-3' for the XRE gene (approximately 250 bp) were used to amplify the gyrB and XRE sequence fragments in a 30 μL PCR reaction system using genomic DNA of strain X2-1-4 as a template for further identification of strain X2-1-4. The reaction system is as follows: ; The reaction conditions are as follows: ; After the reaction is complete, take 5 μL of gel for electrophoresis and analyze the results (e.g., ...). Figure 4 The results show that X2-1-4 genomic DNA was used as a template. gyr BF, gyr BR primer PCR amplification yielded a sample approximately 220 bp in size. gyr The target band was not found when PCR amplified with XRE-F and XRE-R primers, which is consistent with the characteristics of Bacillus cereus. Therefore, strain X2-1-4 was further identified as Bacillus cereus.

[0032] Based on the above individual morphology, colony morphology characteristics, Gram staining observation results, and molecular identification, and referring to the common bacterial systematic identification manual, the taxonomic position of the isolated and purified strain X2-1-4 can be preliminarily determined to be Bacillus cereus. B. cereus ). Example

[0033] This embodiment describes the salt and alkali resistance activity test of Bacillus cereus X2-1-4; 2.1 Salt tolerance test Bacillus cereus X2-1-4 with good growth was inoculated at a volume fraction of 5% into LB liquid medium with NaCl concentration gradients of 50, 100, 150, and 200 g / L, and cultured for 36 h. OD was then measured. 600 Values. The culture conditions were 28℃ and 180 rpm, with three replicates for each treatment, using LB liquid medium with a NaCl concentration of 10 g / L as a control.

[0034] The test results are as follows Figure 5 OD of strain X2-1-4 600 The OD value decreased with increasing NaCl concentration, and the growth inhibition was relatively low at a NaCl concentration of 50 g / L; above 50 g / L, the growth of the strain was limited and reproduction slowed down. At a NaCl concentration of 100 g / L, the OD value...600 >0.8; OD at NaCl concentration of 150 g / L 600 The concentration dropped below 0.8; it could still survive and grow slowly at salt concentrations up to 200 g / L. In summary, *Bacillus cereus* X2-1-4 can grow normally in 50–150 g / L NaCl medium and can tolerate high salt environments up to 200 g / L, classifying it as an extreme halophile.

[0035] 2.2 Alkalinity Reduction Capacity Test Plate validation of alkalinity tolerance: Bacillus cereus X2-1-4 was streaked onto LB medium at pH 8.0 and 9.0, with sodium chloride concentrations of 10 g / L and 50 g / L, respectively. The culture was carried out at 28°C for 5 days, and growth was observed and recorded periodically to determine the strain's salt and alkali tolerance. All other conditions remained unchanged, with three replicates for each treatment. LB medium at pH 7.0, with sodium chloride concentrations of 10 g / L and 50 g / L, served as controls.

[0036] Verification of alkalinity-reducing ability through shake culture: *Bacillus cereus* X2-1-4 strains with good plate growth were inoculated into LB liquid medium at pH 9.0 and fermented for 7 h, 24 h, and 31 h, respectively. The growth and pH changes were observed after each fermentation. The inoculum size was 5% of the strain. The alkalinity-reducing rate of the strain was calculated using the following formula: ; In the formula: pH before fermentation represents the pH value of the liquid culture medium before fermentation; pH after fermentation represents the pH value of the liquid culture medium after fermentation; η is the alkalinity reduction capacity, expressed as %.

[0037] Plate validation of the strain's alkali-reducing ability, such as Figure 6 As shown, the results indicate that strain X2-1-4 possesses a certain degree of salt and alkali tolerance. Except for failing to grow in LB solid medium with 50 g / L NaCl and pH=9 for 5 days, it can grow under other salt concentrations and pH conditions. Specifically, single colonies can rapidly grow in LB medium with 10 g / L NaCl and pH 7-9 within 1 day. Furthermore, Bacillus cereus X2-1-4 can also grow normally on solid medium with normal salt concentration and pH=9, indicating it is an alkali-tolerant microorganism. The highest alkali reduction rate (19.1%) after 24 hours of cultivation for X2-1-4 is observed, demonstrating a strong alkali-reducing ability. It can effectively lower soil pH, indirectly increasing soil ecosystem biodiversity and providing support for the development and utilization of saline-alkali land and mitigating the harm caused by saline-alkali soil.

[0038] 2.3 Testing of EPS (extracellular polymers) production capacity Bacillus cereus X2-1-4 strain was inoculated at a 5% inoculum into an EPS-producing fermentation medium (3 g / L yeast extract, 20 g / L sucrose, 0.2 g / L K₂HPO₄, 0.5 g / L KH₂PO₄, 100 g / L NaCl, 0.5 g / L MgSO₄·7H₂O). The culture was incubated at 28℃ and 180 rpm for 3 days. After incubation, 10 mL of the fermentation broth was centrifuged at 6000 rpm for 15 minutes, and the cells were dried and weighed. 95% ethanol solution was added to the supernatant at a 1:3 volume ratio, and the mixture was allowed to stand at low temperature (4℃) for 12 hours to promote polysaccharide precipitation. Then, the mixture was centrifuged at 10000 rpm for 30 minutes, the supernatant was discarded, the precipitate was collected, dried, and weighed. Finally, the extracellular polysaccharide synthesis efficiency per unit biomass was calculated by comparing the ratio of polysaccharide precipitate to cell weight.

[0039] Halophytic adaptive secretory products (EPS) utilize their rich molecular structure containing active groups such as carboxyl, thiol, and hydroxyl groups to biofix heavy metal ions in the environment. This extracellular polymer can specifically complex sodium and potassium cations in the soil, effectively alleviating the osmotic stress effect caused by cation accumulation in high-salt environments. Furthermore, through intermolecular interactions, it forms stable complexes with soil colloidal substances, promoting matrix structure improvement and enhancing plant salt tolerance. Results showed that the extracellular polysaccharide synthesis efficiency per unit biomass of Bacillus cereus X2-1-4 strain reached 46.2%. Example

[0040] This embodiment describes the determination of the antagonistic properties and control effects of Bacillus cereus X2-1-4; 3.1 In-plate antagonistic effect determination The plate confrontation culture method was used. A 5 mm diameter soybean sclerotium cake (or sunflower sclerotium cake) was placed in the middle left side of a PDA plate. Purified inoculum was picked up with an inoculation needle and inoculated 2.5 cm from the cake. Soybean and sunflower sclerotium cakes were used as controls. Each treatment was replicated in triplicate. The plates were incubated at 25°C for 2 days until the plant pathogen reached 4 / 5 of the plate. The radius of the inhibition zone (from the center of the bacterial colony to the edge of the pathogen hyphae) and the radius of the antagonistic bacterial colony were measured. The antagonism coefficient and inhibition rate were calculated using the following formulas: ; ; Where R1: colony radius of antagonistic bacteria in the treatment group, R2: colony radius of pathogenic bacteria in the treatment group, CK: colony radius of pathogenic bacteria in the control group, and T: colony radius of pathogenic bacteria in the treatment group, all in cm.

[0041] The results showed that Bacillus cereus X2-1-4 inhibited the growth of pathogens, and the inhibited pathogen hyphae had even edges and a long-lasting antagonistic effect. The antagonism coefficient against the pathogens was approximately 2.1. The average inhibition rate against Sclerotinia sclerotinia of soybean was 62.9%, and the average inhibition rate against Sclerotinia sclerotinia of sunflower was 58.7% (e.g., Figure 7 , Figure 8 ).

[0042] 3.2 Prevention and control effect detection Single colonies were picked from the streaked plate of Bacillus cereus X2-1-4 and inoculated into beef extract peptone liquid medium. The culture was incubated overnight at 28°C and 180 rpm to prepare OD. 600 The bacterial suspensions were prepared at concentrations of 0.5, 0.1, and 0.02. Healthy soybean leaves at the 6-8 leaf stage and the same growth stage were selected. The soybean leaves were soaked in bacterial suspensions of different concentrations for 2-3 minutes. Fresh soybean sclerotium cakes with a diameter of 5 mm were dipped into bacterial suspensions of different concentrations and inoculated into the center of the dried leaves (avoiding the veins). Water was used as a control. Each treatment was repeated 3 times. The leaves were placed in a 25℃ incubator for humidification and the disease incidence was observed regularly.

[0043] 72 hours later (e.g.) Figure 9 The control group suffered extensive leaf infection by the pathogen, with severe disease affecting approximately 76.5% of the entire leaf. The severity of the disease was reduced in all treatment groups. Specifically, the control effect increased with increasing bacterial suspension concentration, and OD... 600 The optimal concentration was 0.5, with a soybean leaf disease incidence rate of only 6.4%, indicating that this strain was effective against *Sclerotinia sclerotiorum* (…). Sclerotinia sclerotiorum It has significant potential for controlling crop diseases caused by ).

[0044] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0045] The detailed descriptions listed above are merely specific descriptions of feasible implementation methods of this application and are not intended to limit the scope of protection of this application. All equivalent implementation methods or modifications made without departing from the spirit of the art of this application should be included within the scope of protection of this invention.

Claims

1. A salt- and alkali-tolerant Bacillus cereus strain, characterized in that, Bacillus cereus ( Bacillus cereus X2-1-4, with accession number CGMCC No. 37243.

2. An inoculum containing the salt-tolerant Bacillus cereus as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the bacterial agent, the Bacillus cereus exists in the form of cultured live bacteria, fermentation broth, or bacterial suspension.

4. The microbial agent according to claim 3, characterized in that, The formulation of the microbial agent is an emulsion, suspension, granules, wettable powder, or water dispersible agent.

5. The Bacillus cereus strain according to claim 1 or the inoculant according to any one of claims 2-4 in controlling crops infected with Sclerotinia sclerotiorum (… Sclerotinia sclerotiorum Applications in diseases caused by ).

6. The application according to claim 5, characterized in that, The disease in question is either soybean sclerotinia stem rot or sunflower sclerotinia stem rot.

7. The application according to claim 6, characterized in that, The method for controlling sclerotinia stem rot in soybeans or sunflowers is to inoculate Bacillus cereus X2-1-4 into NB medium to prepare OD. 600 A Bacillus suspension with a concentration of 0.5 was used to inoculate soybean leaves onto the surface of the leaves using a bacterial cake dipped in the Bacillus cereus suspension.

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