Bacillus cereus strain and application thereof
By inoculating the roots of chili peppers with Bacillus cereus B2, the problem of crop yield reduction in saline soils was solved, and the growth performance and salt tolerance of chili peppers in high-salt environments were improved.
Patent Information
- Application Number
- CN202511073499.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-21
AI Technical Summary
Salinization leads to soil structure degradation and crop yield reduction. Existing technologies are insufficient to effectively improve crop growth performance and salt tolerance in saline soils.
The Bacillus cereus strain B2 was used to inoculate pepper roots, promoting root growth, regulating ion transport, and improving the adaptability of peppers to high-salt environments.
It significantly increases the root length and number of lateral roots of chili peppers, enhances root vitality, improves K+ absorption capacity, reduces the Na+/K+ ratio, promotes the growth and development of chili peppers in high-salt environments, and enhances their salt tolerance.
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Figure CN120988884A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microorganisms, and particularly relates to a bacillus cereus strain and application thereof. BACKGROUND
[0002] Salinization not only causes soil structure degradation, but also causes salt accumulation, damages the normal growth and development mechanism of crops, and ultimately leads to crop yield reduction and land productivity decline. In facility planting, due to high temperature and humidity, long-term high-fertilization and irrigation, the phenomenon of secondary salinization of soil is particularly prominent. Secondary salinization of soil can increase the content of soil salt, inhibit the absorption of water by crops and the development of root system, and ultimately lead to yield reduction and quality decline. Related studies have pointed out that salinization can hinder the growth of plant roots, aggravate the accumulation of active oxygen, thereby interfering with photosynthesis and inhibiting plant growth. In recent years, the use of plant growth-promoting rhizobacteria (PGPR) to enhance the tolerance of crops to abiotic stress has become a promising biological solution due to its environmental friendliness, cost-effectiveness and sustainability. SUMMARY
[0003] The purpose of the present application is to provide a bacillus cereus strain and application thereof.
[0004] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:
[0005] The bacillus cereus B2 of the present application was preserved in the China General Microbiological Culture Collection Center on July 08, 2025, and the preservation number is CGMCC No.34800. After bacterial strain identification, it was found that the strain is bacillus cereus. The 16S rRNA gene sequence of the strain is shown as SEQ ID NO.1.
[0006] The separation method of the above-mentioned bacillus cereus strain is as follows: in a facility pepper shed harmed by secondary salinization, the surface layer 10-20cm soil is preserved in a 4℃ refrigerator, 10g of soil sample is weighed, 90mL of sterile water is added and mixed thoroughly to prepare a 10% soil suspension. The suspension is placed in a 30℃ shaking table for 30min to mix the sample thoroughly. Then the suspension is diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5Isometric gradient dilution, respectively, coated on LB solid medium, 28 DEG C culture 4d, form colonies, pick single colony, expand culture, repeated purification 3 times or more, select B2 strain, according to whole genome sequencing and construction of housekeeping gene phylogenetic tree, determine it is Bacillus cereus.
[0007] The preparation method of the LB solid medium is as follows: 1g of trypsin peptone, 0.5g of yeast extract, 1g of sodium chloride and 1.5g of agar are dissolved in 100ml of distilled water, then sterilized at 121 DEG C for 20min, naturally cooled to about 55 DEG C, then the medium is poured into a culture dish, and used after cooling and solidification.
[0008] The preservation method of the Bacillus cereus strain is also disclosed, which comprises the following steps: inoculating the single colony of the Bacillus cereus B2 into LB liquid medium, culturing at 37 DEG C for 10h on a 200r / min shaking table, obtaining bacterial culture solution with an OD600 value greater than 1, uniformly mixing 1ml of the bacterial culture solution with 420ml of 50% glycerol, rapidly cooling with liquid nitrogen, and storing at-80 DEG C.
[0009] The preparation method of the LB solid medium is as follows: 1g of trypsin peptone, 0.5g of yeast extract, 1g of sodium chloride and 1.5g of agar are dissolved in 100ml of distilled water, then sterilized at 121 DEG C for 20min, and used after natural cooling. In addition, the preparation method of the Bacillus cereus strain suspension is also disclosed, which comprises the following steps: transferring the purified strain B2 into a sterilized conical flask containing 100ml of LB liquid medium, and culturing at 37 DEG C with oscillation at 200r / min. 8 The concentration of the suspension is adjusted to 10
[0010] The most important purpose of the present application is to disclose the application of the Bacillus cereus strain in improving the high-salt environment adaptability of pepper plants.
[0011] The salt tolerance of the pepper can be significantly improved by treating the plant roots with the bacterial agent containing the Bacillus cereus bacteria.
[0012] Specifically, under salt stress, compared with the control, the following performances are shown:
[0013] A: the total root length and the number of lateral roots of the pepper plants inoculated with the B2 strain are significantly increased, and the root activity is enhanced; + the absorption capacity is enhanced, and the Na + / K + ratio is significantly reduced.
[0014] B: The expression amount of ion transport related genes in the leaves of pepper plants inoculated with B2 strain is significantly up-regulated;
[0015] C: The height and stem diameter of pepper plants inoculated with B2 strain are higher than those of the control.
[0016] The ion transport related genes include K + Transporter CaHAK1, cation transporter CaHKT2-1 and Na + / H + Antiporter CaNHX6.
[0017] Advantages of the present application:
[0018] The Bacillus cereus B2 of the present application is collected from a facility pepper shed in Zhongmou County and Fugou County of Henan Province which is damaged by secondary salinization, and is isolated and identified for its function, and it is found that it can secrete auxin, promote the growth and development of pepper root system in a high salt environment, regulate the sodium and potassium ion transport in the pepper plant, and improve the salt environment adaptability of the pepper plant. The present application has important application value for the planting of pepper in salinized soil.
[0019] The Bacillus cereus strain B2 of the present application is a kind of efficient microbial resource, which can be used for developing microbial inoculants or biological fertilizers to promote the growth of pepper and other crops in salinized soil, improve the salt tolerance, and guarantee and improve the sustainable production capacity of pepper in salinized soil. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The phylogenetic tree of strain B2 is constructed.
[0021] Figure 2 The IAA production ability detection of Bacillus cereus B2 and the influence on the root system morphology of pepper seedlings under normal and salt stress conditions are shown in the figure, (A) Salkowski method is used to detect the IAA secretion ability of strain B2, (B) the content of IAA secreted by strain B2, and (C) the root system phenotype.
[0022] Figure 3 The influence of Bacillus cereus B2 on the root system structure of pepper seedlings under normal and salt stress conditions is shown in the figure, (A) root activity, (B) total root length, (C) total root volume, (D) total root surface area, (E) bifurcation number, and (F) root tip number.
[0023] Figure 4 The influence of Bacillus cereus B2 on the Na + and K + content of pepper seedlings under normal and salt stress conditions is shown in the figure, (A) sodium ion content, (B) potassium ion content, and (C) Na+ Ratio. + Ratio.
[0024] Figure 5 Relative expression levels of ion transport genes (A) CaHKT2-1, (B) CaNHX6, (C) CaHAK1 in leaves of pepper plants inoculated or not with Bacillus cereus B2 under normal and salt stress conditions.
[0025] Figure 6 Effects of Bacillus cereus B2 on the growth of pepper seedlings under normal and salt stress conditions; in the figures, (A) pepper phenotype; (B) plant height; (C) stem diameter. DETAILED DESCRIPTION
[0026] The present application will be further described below in connection with specific embodiments, and its advantages and characteristics will become more apparent with the description. However, the specific test methods involved in the following examples are conventional methods or are carried out according to the suggested conditions of the manufacturer's instructions, unless otherwise specified.
[0027] If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods, unless otherwise specified. The reagents and materials used, unless otherwise specified, can be obtained commercially. All quantitative tests were performed in triplicate biological replicates, and the results were averaged.
[0028] I. Test materials and preparation of culture media
[0029] Pepper (Capsicum annuum L.) material: variety named "203".
[0030] LB liquid medium: 1 g of tryptone, 0.5 g of yeast extract and 1 g of sodium chloride were dissolved in 100 ml of distilled water, which was then sterilized at 121 °C for 20 min, and used after cooling.
[0031] LB solid medium: 1.5 g / mL of agar was added to the LB liquid medium, which was then sterilized at 121 °C for 20 min. The medium, cooled to about 55 °C, was poured into Petri dishes and allowed to cool naturally.
[0032] II. Isolation of Bacillus cereus B2
[0033] 1、Soil samples were collected from the greenhouse of pepper in Zhongmou and Fugou county of Henan province, which were damaged by secondary salinization. The topsoil of 10-20cm was stored in 4℃ refrigerator. 10g soil sample was added with 90ml sterilized water, and mixed well to prepare 10%(w / v) soil suspension. The suspension was shaken in 30℃ shaker for 30min to mix well. Then the suspension was diluted by 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 gradient. The dilutions were spread on LB medium, and incubated at 28℃ for 4d to form colonies.
[0034] 2、After step 1, single colony was picked up for amplification. The bacteria was purified for more than 3 times. The screened bacteria was named as B2.
[0035] III. Identification and preservation of the strain
[0036] Strain identification: After 16S rRNA sequencing of bacteria B2 (the sequence is shown as SEQ ID NO. 1), alignment was performed in the NCBI database, and the results showed that the 16S rRNA homology of bacteria B2 reached more than 99% with Bacillus cereus, Bacillus tropicus and Bacillus proteolyticus and other multiple species. In view of the high similarity of 16S rRNA of bacteria of different species of Bacillus, in order to further classify the B2 strain, the whole genome of bacteria B2 was sequenced. According to the UBCG V2 (http: / / leb.snu.ac.kr / ubcg2) process, 81 core housekeeping genes (alaS, cgtA, dnaG, dnaX, engA, era, ffh, fmt, frr, ftsY, gmk, hisS, ileS, infB, infC, ksgA, lepA, leuS, nusA, nusG, pheS, pheT, prfA, recA, rplA, rplB, rplC, rplD, rplE, rplF, rplI, rplJ, rplK, rplL, rplM, rplN, rplO, rplP, rplQ, rplR, rplS, rplT, rplU, rplV, rplW, rplX, rpmA, rpmC, rpmI, rpoA, rpoB, rpsB, rpsC, rpsD, rpsE, rpsF, rpsG, rpsH, rpsI, rpsJ, rpsL, rpsM, rpsO, rpsP, rpsQ, rpsR, rpsS, rpsT, rsmH, ruvB, secA, secY, serS, smpB, tilS, trmD, truB, tsaD, tsf, ybeY, ychF) of bacteria of different species of Bacillus were selected by FastTree software to construct a phylogenetic tree by the neighbor-joining method. As shown in FIG. 1, bacteria B2 is most closely related to Bacillus cereus. Therefore, it is determined to belong to Bacillus cereus. Figure 1
[0037] The strain has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 34800, and the preservation date is July 8, 2025.
[0038] Strain preservation: Bacillus cereus B2 single colony was inoculated into LB liquid medium, and cultured at 37°C, 200r / min for 10h, to obtain bacterial culture solution with OD600 value greater than 1. 1ml bacterial culture solution and 420ml 50%(v / v) glycerol were mixed uniformly, rapidly cooled with liquid nitrogen, and stored at -80°C.
[0039] IV. Preparation of Bacillus cereus B2 suspension
[0040] The purified strain B2 was transferred to a sterilized conical flask containing 100 mL of LB liquid medium and cultured at 37°C with 200 r / min shaking. Until the OD600 (optical density) reached 1.0-1.5. Then, the cultured bacterial solution was centrifuged at 8000 rpm for 10 min at 4°C, the precipitate was retained and the liquid phase was removed. Finally, the precipitated bacterial bodies were resuspended with an appropriate amount of sterile distilled water, and the concentration of the bacterial suspension was adjusted to 10 8 cfu / mL.
[0041] V. Effects of Bacillus cereus B2 bacterial agent on peppers
[0042] (I) Test method
[0043] 1. Take pepper seeds and perform the following disinfection treatments in sequence: 75% (v / v) ethanol solution immersion for 30 seconds; sterile water rinsing for 3 times; 2.6% (v / v) sodium hypochlorite (NaClO) aqueous solution immersion for 6 min; sterile water rinsing for 3 times, and then place the disinfected seeds in 55°C warm water for seed soaking. After seed soaking, germinate the seeds in a 28°C constant temperature incubator. After the seeds germinate, sow them in a seedling plug tray.
[0044] 2. When the pepper seedlings in the plug tray grow to the two-leaf stage, transplant them into nutrient pots for further cultivation. When the seedlings grow to the three-leaf stage, select healthy and uniform plants for subsequent tests.
[0045] 3. Set up the following four treatment groups:
[0046] CK group (blank control): use distilled water to irrigate the plants.
[0047] CK+B2 group (bacterial agent control): use Bacillus cereus B2 bacterial suspension to irrigate the plants.
[0048] S group (salt stress control): use a 300 mM NaCl solution to irrigate the plants.
[0049] S+B2 group (salt stress + bacterial agent treatment): use a 300 mM NaCl solution and Bacillus cereus B2 bacterial suspension to irrigate the plants.
[0050] 4. Start the treatment when the pepper seedlings are at the three-leaf stage.
[0051] For the CK+B2 group and the S+B2 group of plants: irrigate each pepper plant with 10 mL of Bacillus cereus B2 bacterial suspension. Perform two irrigation treatments, with a 5-day interval. The CK group and the S group are irrigated with distilled water.
[0052] After the irrigation of the bacterial suspension was completed, the plants in groups S and S+B2 began to receive salt stress treatment: each plant was irrigated with 30 ml of a 300 mM NaCl solution (the subsequent irrigations maintained this salt concentration). At the same time, the plants in groups CK and CK+B2 were irrigated with distilled water.
[0053] The above salt stress treatment lasted for 2 weeks, and then after the salt stress treatment ended, plant phenotype observation and index determination were performed. All index determinations were set up with three biological replicates.
[0054] 1. Qualitative and quantitative detection of the level of auxin IAA secreted by the B2 strain, and observation of root morphology under each treatment. It can be seen from Figure 2 that the strain B2 can secrete indole-3-acetic acid (IAA), which as a plant auxin helps to stimulate plant root development, promote root growth, and thus enhance the plant's ability to absorb water and nutrients, and improve the plant's adaptability under stress environments (such as salt stress),
[0055] 2. By determining the root morphology-related indicators, the statistical results are shown in Figure 3 It can be seen that compared with the control CK, the total root length, the number of bifurcations, and the number of root tips of the CK+B2 group increased significantly.
[0056] Compared with the control S group, the total root length, the total volume, the total surface area of the root system, the number of bifurcations, and the number of root tips of the S+B2 group increased significantly, and the root system activity was enhanced.
[0057] It can be seen that whether under normal conditions or under salt stress conditions, the B2 strain can improve the growth of the pepper root system.
[0058] 3. Ion content determination: The Na + content and K + content in the leaves and roots of the pepper plants were determined respectively. The Na + / K + ratio was calculated, and the ion content and ratio statistical results are shown in Figure 4 It can be seen that the K + absorption ability was enhanced, and the Na + / K + ratio decreased significantly.
[0059] 4. Effect on ion transport: The effect on key ion transport protein genes in pepper was determined (as shown in Figure 5 Under salt stress, compared with the control, the ion transport-related genes (high-affinity K + transporter CaHAK1, cation transporter CaHKT2-1, and Na + / H +The expression level of the antiporter CaNHX6 was significantly up-regulated.
[0060] 5. The aboveground phenotype observation and growth index determination, such as Figure 6 As shown in the aboveground morphology observation, the wilting of the S group was more serious than that of the S+B2 group under salt stress, and the plant height and stem diameter of the S group were also lower than those of the S+B2 group, among which the stem diameter decreased significantly. It can be seen that inoculation of the B2 strain can effectively reduce the damage of salt stress to the pepper plants.
[0061] It can be seen that under salt stress conditions (S+B2 group vs. S group), Bacillus cereus B2 promotes the growth of pepper roots and the formation of lateral roots in a high-salt environment, maintains the root activity in a high-salt environment, regulates the expression of ion transport genes in pepper plants, promotes the absorption of K + in a high-salt environment, effectively reduces the salt ion toxicity, and significantly enhances the adaptability of pepper in a high-salt environment.
[0062] The sequence of SEQ ID NO. 1 is as follows:
[0063]
[0064]
Claims
1. A Bacillus cereus strain, characterized in that, The strain is Bacillus cereus strain B2 (Bacillus cereus), which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 34800.
2. The Bacillus cereus strain according to claim 1, wherein the 16S rRNA gene coding sequence of the strain is shown as SEQ ID NO.
1.
3. The isolation method of Bacillus cereus strain in claim 1, the method is: in the facility pepper greenhouse endangered by secondary salinization, the surface layer 10-20 cm soil is stored in the 4℃ refrigerator, 10 g soil sample is weighed, 90 mL sterile water is added and mixed thoroughly to prepare 10% soil suspension. The suspension is placed in a 30℃ shaking table for 30 min to mix the sample thoroughly. Then the suspension is diluted according to the proportion gradient of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 , respectively coated on LB solid medium, cultured at 28℃ for 4d to form colonies, single colonies are picked and expanded, purified repeatedly for more than 3 times, and B2 strain is screened out, which is determined as Bacillus cereus according to whole genome sequencing and construction of housekeeping gene phylogenetic tree.
4. The method of isolation of a Bacillus cereus strain according to claim 3, characterized in that, The preparation method of the LB solid culture medium is as follows: 1 g of tryptone, 0.5 g of yeast extract, 1 g of sodium chloride and 1.5 g of agar are dissolved in 100 ml of distilled water, and then sterilized at 121 ℃ for 20 min; after natural cooling to about 55 ℃, the culture medium is poured into a culture dish, and used after cooling and solidification.
5. The method for preserving the Bacillus cereus strain according to claim 1, characterized in that, The method is as follows: Bacillus cereus B2 single colony is inoculated into LB liquid culture medium, and cultured at 37 ℃ and 200 r / min for 10 h on a shaking table to obtain a bacterial culture solution with an OD600 value greater than 1; 1 ml of the bacterial culture solution is mixed with 420 ml of 50% glycerol, uniformly cooled by liquid nitrogen, and stored at -80 ℃.
6. The method for preserving the Bacillus cereus strain according to claim 5, characterized in that, The preparation method of the LB liquid culture medium is as follows: 1 g of tryptone, 0.5 g of yeast extract and 1 g of sodium chloride are dissolved in 100 ml of distilled water, and then sterilized at 121 ℃ for 20 min; after natural cooling, the culture medium is used.
7. The method of preparing a suspension of Bacillus cereus strain according to claim 1, characterized in that, The method is: after purification, the strain B2 is transferred to a sterilized conical flask containing 100 mL of LB liquid medium, and is cultured at 37°C with 200 r / min shaking until OD600 reaches 1.0-1.5, then the cultured bacterial liquid is centrifuged at 8000 rpm for 10 min at 4°C, the precipitate is reserved and the liquid phase is removed, finally, the precipitated bacterial body is resuspended with an appropriate amount of sterile distilled water, and the suspension concentration is adjusted to 10 8 cfu / mL.
8. The Bacillus cereus strain of claim 1 is used for improving the high-salt environment adaptability of pepper plants.
9. Use according to claim 8, characterized in that, Under salt stress, compared with the control, the expression levels of ion transport-related genes in the leaves of pepper plants inoculated with the B2 strain are significantly up-regulated; A: the total root length and lateral root number of pepper plants inoculated with B2 strain increased significantly, and the root activity was enhanced; K + absorption capacity was enhanced, and the Na + / K + ratio decreased significantly; B: the expression levels of ion transport-related genes in the leaves of pepper plants inoculated with the B2 strain are significantly up-regulated; C: the height and stem diameter of pepper plants inoculated with the B2 strain are higher than those of the control.
10. Use according to claim 9, characterized in that, The ion transport-related genes include K + Transporter CaHAK1, cation transporter CaHKT2-1 and Na + / H + Antiporter CaNHX6.