Bacillus velezensis y10 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bacillus velezensis Y10 and application thereof. The bacillus velezensis Y10 is preserved in the China General Microbiological Culture Collection Center on December 5, 2024, and has a preservation number of CGMCC No.32927. The bacillus velezensis Y10 can improve the resistance of cotton to salt stress, and provides a new microbial resource for improving the salt tolerance of cotton.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Bacillus belye Y10 and its applications. Background Technology

[0002] cotton( Gossypium spp. Cotton is an important economic crop and a significant source of feed and oil. Currently, cotton fiber, seeds, and straw are widely used in textiles, food and feed processing, and papermaking. Due to competition from grains and rising labor costs, major cotton-producing areas are gradually shifting to regions with higher soil salinization. However, severe secondary soil salinization significantly restricts cotton growth. Although cotton has a certain salt tolerance and is often used as a pioneer crop in the development of saline-alkali land, excessive salt ions significantly inhibit its growth. After salt stress treatment, seed biomass, superoxide dismutase, catalase, and peroxidase activities decreased significantly, while malondialdehyde content increased. Salt stress also significantly reduces cotton fiber length and elongation, severely impacting cotton yield and quality.

[0003] The plant microbiome, often referred to as the plant's second genome, plays a crucial role in regulating the rhizosphere environment, controlling growth and development, and enhancing plant stress resistance. Under abiotic stress, the complex dynamic interactions between microorganisms and plants can promote crop growth and mitigate salt stress damage without harming the environment. Bio-agents are also widely used in plant growth and development regulation and disease resistance research. As the role of microorganisms in plant adaptation is gradually revealed, many beneficial microorganisms that help plants cope with adversity have been isolated and reintroduced into the soil to increase crop yields.

[0004] Current research on cotton salt tolerance mainly focuses on genes, recombinant vectors, or proteins. To provide more ways to improve cotton salt tolerance, it is necessary to study plant microorganisms and provide a new microbial resource for improving cotton salt tolerance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Bacillus belyssus Y10 and its applications. The Bacillus belyssus Y10 of this invention can effectively improve the salt tolerance of cotton.

[0006] The first aspect of this invention provides a *Bacillus belyssus* Y10, which is classified as *Bacillus belyssus* at the China General Microbiological Culture Collection Center. Bacillus velezensis The collection date is December 5, 2024, and the collection number is CGMCC No. 32927.

[0007] The application isolates and screens a bacterial strain from the well-grown cotton root soil in a comprehensive demonstration base of saline-alkali land in the Yellow River Delta of Dongying City, Shandong Province, and finds through experiments that the strain can improve the resistance of cotton to salt stress, providing a new microbial resource for improving the salt tolerance of cotton.

[0008] The application provides application of the Bacillus velezensis Y10 in preparing a product for improving the resistance of cotton to salt stress.

[0009] In another preferred embodiment, the product is the Bacillus velezensis Y10 as the only effective component.

[0010] In another preferred embodiment, the plant is cotton.

[0011] The application provides application of the Bacillus velezensis Y10 in preparing a plant growth promoter.

[0012] In another preferred embodiment, the growth promoter is used for promoting the growth of plant height, stem diameter, leaf area, fresh weight and dry weight.

[0013] In another preferred embodiment, the plant is cotton.

[0014] In another preferred embodiment, the growth promoter is the bacterial body and / or fermentation liquor of the Bacillus velezensis Y10.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The application isolates and screens a bacterial strain from the well-grown cotton root soil in a comprehensive demonstration base of saline-alkali land in the Yellow River Delta of Dongying City, Shandong Province, and finds through experiments that the strain can improve the resistance of cotton to salt stress, providing a new microbial resource for improving the salt tolerance of cotton.

[0017] The application finds through experiments that the Bacillus velezensis Y10 can promote the growth of cotton seedlings, and the plant height of the cotton seedlings under salt stress is increased by 9.80%, the stem diameter is increased by 38.0%~39.2%, the leaf area is increased by 10.3%~25.3%, the fresh weight is increased by 41.8%~50.5%, and the dry weight is increased by 46.8%~54.6%. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The phylogenetic tree of Y10.

[0019] Figure 2 Morphology diagram of Y10 on LB solid medium in the application.

[0020] Figure 3 Figure of the influence of different treatment conditions on cotton seed germination under salt stress in the application; wherein, A is the germination diagram of seeds under CK treatment; B is the germination diagram of seeds under Salt treatment; C is the germination diagram of seeds under Y10 treatment.

[0021] Figure 4 Figure of the influence of different treatment conditions on cotton seed germination under salt stress in the application; wherein, A is the germination diagram of seeds under CK treatment; B is the germination diagram of seeds under Salt treatment; C is the germination diagram of seeds under Y10 treatment.

[0022] Figure 5 Figure of the influence of different treatment conditions on cotton seed germination under salt stress in the application; wherein, A is the germination diagram of seeds under CK treatment; B is the germination diagram of seeds under Salt treatment; C is the germination diagram of seeds under Y10 treatment.

[0023] Figure 6 Figure of the influence of different treatment conditions on cotton seed germination under salt stress in the application; wherein, A is the germination diagram of seeds under CK treatment; B is the germination diagram of seeds under Salt treatment; C is the germination diagram of seeds under Y10 treatment.

[0024] Figure 7 Figure of the influence of different treatment conditions on cotton seed germination under salt stress in the application; wherein, A is the germination diagram of seeds under CK treatment; B is the germination diagram of seeds under Salt treatment; C is the germination diagram of seeds under Y10 treatment.

[0025] Figure 8Fig. 1 is a diagram of the effects of different concentrations of Y10 on the growth of cotton seedlings under salt stress in the present application; wherein A is a diagram of the effects of different concentrations of Y10 on the plant height of cotton under salt stress, B is a diagram of the effects of different concentrations of Y10 on the stem diameter of cotton under salt stress, C is a diagram of the effects of different concentrations of Y10 on the root length of cotton under salt stress, D is a diagram of the effects of different concentrations of Y10 on the leaf area of cotton under salt stress, E is a diagram of the effects of different concentrations of Y10 on the fresh weight of cotton under salt stress, and F is a diagram of the effects of different concentrations of Y10 on the dry weight of cotton under salt stress; in the diagram, "*" indicates a significant difference, P < 0.05, and "**" indicates a very significant difference, P < 0.01.

[0026] Figure 9 Fig. 2 is a diagram of the determination results of the antioxidant enzyme activity of cotton seedlings, wherein A is the determination results of superoxide dismutase, B is the determination results of peroxidase, and C is the determination results of catalase. DETAILED DESCRIPTION

[0027] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0028] The total superoxide dismutase determination kit was purchased from A001-1 from Nanjing Jiancheng Bioengineering Institute; the peroxidase determination kit was purchased from A084-3-1 from Nanjing Jiancheng Bioengineering Institute; and the catalase determination kit was purchased from A007-1-1 from Nanjing Jiancheng Bioengineering Institute.

[0029] The superoxide dismutase is abbreviated as SOD, the peroxidase is abbreviated as POD, and the catalase is abbreviated as CAT.

[0030] The Bacillus velezensis Y10 in the present application was preserved in the China General Microbiological Culture Collection Center, and the classification name was Bacillus velezensis Bacillus velezensis , the preservation time was December 05, 2024, and the preservation number was CGMCC No. 32927. The following Bacillus velezensis Y10 is referred to as Y10.

[0031] The 16S rDNA sequence of the Y10 is shown as SEQ ID NO. 1.

[0032]

[0033] Isolation and identification of Y10.

[0034] 1. Isolation of Y10: Y10 was isolated from the rhizosphere soil of cotton growing well in the Yellow River Delta Saline-alkali Land Comprehensive Demonstration Base in Dongying City, Shandong Province. The strain was isolated by dilution plate method, and the specific operation method is as follows.

[0035] Take 10 g of soil sample and put it into 90 mL of sterile water and shake it evenly to get a 10 -1 g / mL concentration of bacterial solution, which is diluted to 10 -2 g / mL, 10 -3 g / mL and 10 -4 g / mL bacterial solution respectively and spread on PDA plates, dry and invert in a 25℃ incubator for 5d~7d. Pick single colonies with bacterial morphology and transfer to LB plates for purification culture. After preliminary identification by microscopy, number and transfer to LB slant for culture. After growing well, store in the refrigerator. Take strain Y10 for the following identification.

[0036] 2. Identification of Y10.

[0037] 1) Morphological observation of Y10: inoculate Y10 on PDA medium and observe the colony morphology at 28℃, as shown in Figure 2 . The results show that Y10 colony on PDA medium is milky white, round, smooth and slightly convex in texture.

[0038] Among them, the potato dextrose agar medium: peel the potato, 200g cut into small pieces, add water and boil for 30min, filter with 4 layers of gauze, add 20g glucose, agar 17g, distilled water to 1000mL, boil and mix evenly, sterilize at 121℃ for 20min, get PDA medium.

[0039] 2) Molecular biology identification of strain: pick Y10 and inoculate on PDA medium, cultivate at 28℃ for 10d, collect the bacterial slurry, extract the genomic DNA of Y10 using 16sDNA identification method, and amplify its gene sequence using universal primer 27f / 1492r.

[0040] The sequencing results are searched for similarity in NCBI using Blast software. The DNA sequence alignment results of Y10 sequencing are more than 99% similar to Bacillus velezensis and Bacillus subtilis subsp. subtilis, so it is classified as Bacillus velezensis Bacillus velezensis . The phylogenetic tree of Y10 is shown in Figure 1 .

[0041] Example 2, Y10 on cotton germination test under salt stress.

[0042] The well-grown strain Y10 on the plate was picked up with a sterilized toothpick and inoculated into a 500 mL flask containing 200 mL of LB medium, and cultured at 28°C, 180 r / min for 1 d to obtain Y10 fermentation broth.

[0043] LB liquid medium: 10 g of tryptone, 10 g of yeast extract, 10 g of NaCl, and distilled water to 1000 mL, pH adjusted to 7.0, sterilized at 121°C for 20 min.

[0044] Cotton seed treatment liquid in different groups: CK group only with water, Salt group using 150 mM NaCl, Y10 group using 150 mM NaCl solution to dilute Y10 fermentation broth 20 times.

[0045] Two filter papers were laid in a 9 cm sterile disposable culture dish, 8 mL of the above three treatment liquids were poured, 20 seeds were evenly placed on the filter paper, and another layer of filter paper was laid on top of the seeds, 4 mL of the above treatment liquid was poured, and the culture dish was covered. Label the culture dish, and each treatment was repeated 3 times. The culture dish was placed in a constant temperature incubator at 28°C and 80% relative humidity for dark culture.

[0046] The number of germinated seeds was recorded on the 4th and 7th days, and the germination potential and germination rate of the seeds were calculated. After the germination test, healthy germinated seedlings were taken from each culture dish and the hypocotyl and root length of each treatment were measured using a vernier caliper. The test results are shown in Tables 1 and 2, and the phenotypic results are shown in Figure 3 .

[0047] Table 1 Germination results

[0048]

[0049] As can be seen from Table 1, the highest germination potential of CK was 76.67%, the germination potential of salt treatment was 31.67%, which was 45.00% lower than that of CK, and the difference between the two groups was significant. Salt stress also inhibited the vitality of cotton seeds. The germination potential of cotton seeds treated with Y10 was 58.33%, which increased by 26.66% compared with salt stress.

[0050] Table 2 Germination rate results

[0051]

[0052] As can be seen from Table 2, the cotton seed germination rate under the condition of CK group is 86.67%, and the cotton seed germination rate under the condition of salt treatment group is 38.33%, which is significantly reduced by 48.34% compared with the control. The germination rate of Y10 treatment is 76.67%, which is increased by 38.34% compared with the control.

[0053] Example 3, Y10 promotes the growth of cotton seedlings.

[0054] The following fermentation broth is the Y10 fermentation broth in Example 2, and the specific process is as follows.

[0055] Select uniform and full cotton seeds, surface sterilize with 5wt% H2O2 solution for 10min, rinse the seeds repeatedly with deionized water for several times, soak the seeds in deionized water for 24h, then place them on a culture dish covered with wet gauze, and germinate at 28℃ for 2d. Select seeds with uniform germination and sow them in plastic pots, 4 seeds per pot.

[0056] Set up 4 treatments, namely blank group, treated with water, marked as CK; treatment 1 group, treated with 50 times diluted Y10 fermentation broth, marked as Y10-1; treatment 2 group, treated with 100 times diluted Y10 strain fermentation broth, marked as Y10-2; treatment 3 group, treated with 200 times diluted Y10 strain fermentation broth, marked as Y10-3. The treatment method of strain fermentation broth is root irrigation, 50mL of corresponding dilution multiple strain fermentation broth is poured around the rhizosphere each time, once when the cotyledon is flat, and once again when the first true leaf is flat, and normal water is poured at other times. Sample and determine the indicators after 10d of treatment. The whole process is cultured under the conditions of room temperature of 25℃~28℃, humidity of 30%, and light time not less than 8 hours.

[0057] Test test: slowly pull out the cotton seedlings treated by the above method from the nutrient pot, gently flush the soil attached to the root with running water, wipe off the excess water with toilet paper, place the cotton seedlings flat on the table, measure the plant height and root length with a scale; use a vernier caliper to measure the stem base of cotton; measure the length and width of the cotton leaf with a ruler, and calculate the leaf area using formula (1); absorb the surface water of the cotton seedlings with absorbent paper, and weigh the fresh weight of the whole cotton plant; the plant with recorded fresh weight is killed at 105℃ for 20min, and then dried at 80℃ until constant weight, and weighed. The test results are shown in Figure 4 , and the phenotype results are shown in Figure 5 .

[0058] Single leaf area = leaf length x leaf width x 0.73 formula (1).

[0059] From Figure 4 A and Figure 4B in FIG. 6 can be seen that the application of 100-fold diluted Y10 fermentation broth can significantly improve the plant height of cotton, and the 100-fold diluted fermentation broth treatment can significantly improve the stem diameter of cotton. The root length of cotton seedlings after Y10 fermentation broth treatment, as shown in C in FIG. 6, the leaf area, as shown in D in FIG. 6, the fresh weight, as shown in E in FIG. 6, and the dry weight, as shown in F in FIG. 6, do not change significantly. Figure 4 Figure 4 Figure 4 Figure 4

[0060] Example 4, salt tolerance test of Y10 on cotton seedlings.

[0061] The fermentation broth described below is the Y10 fermentation broth in Example 2, and the specific process is as follows.

[0062] First, the potting salt concentration screening is carried out, and different salt concentration gradient tests are carried out after the cotyledon of cotton seedlings is flat, and the treatment is as follows: 5 treatments are set, and 5 treatment combinations are respectively: CK group, i.e. water treatment; S1 group is 200 mmol / L NaCl treatment; S2 group is 250 mmol / L NaCl treatment; S3 group is 300 mmol / L NaCl treatment; S4 group is 350 mmol / L NaCl treatment. Each treatment is set with 3 repeats, and each treatment has 6 cotton seedlings. After 10 days of salt stress treatment, the plant height of cotton seedlings is observed to determine the optimum salt concentration. The culture conditions are the same as in Example 3, and the results are shown in Table 1. Figure 6 From Table 1, it can be seen that the S4 group is the optimum salt concentration, and the verification experiment of the strain for improving the salt tolerance of cotton is carried out after 350 mmol / L salt treatment. Figure 6

[0063] 5 treatments are set, which are CK1 group, i.e. water treatment; CK2 group is 350 mmol / L NaCl treatment; Y10salt-1 group is 50-fold diluted fermentation broth and 350 mmol / L NaCl treatment; Y10salt-2 group is 100-fold diluted fermentation broth and 350 mmol / L NaCl treatment; Y10salt-3 group is 200-fold diluted fermentation broth and 350 mmol / L NaCl treatment, and the culture conditions are the same as in Example 3. In the test, the treatment mode is root irrigation, and 50 mL of strain fermentation broth of corresponding dilution multiple is poured around the rhizosphere each time, and poured once when the cotyledon is flat and once again when the first true leaf is flat; the salt adding mode is to pour 350 mmol / L salt solution after one week of the last bacteria treatment. After 10 days of salt stress treatment, the biological traits of cotton seedlings are measured. The test method is consistent with the phenotype determination method of cotton seedlings in Example 3. The test results are shown in Table 2. Figure 8 The phenotype results are shown in Table 3. Figure 7

[0064] Figure 8 ​​​​​​As can be seen, compared with the CK group, 350 mmol / L salt treatment can significantly inhibit the plant height of cotton seedlings, and compared with the salt stress group, Y10 bacterial liquid diluted 100 times can significantly increase the plant height of cotton seedlings, as shown in A of Figure 8 ; the stem diameter of cotton seedlings treated with bacterial liquid of three concentrations can be increased, as shown in B of Figure 8 ; the effect on the root length of cotton seedlings is not significant, as shown in C of Figure 8 ; the leaf area of cotton seedlings treated with Y10 bacterial liquid diluted 50 times and 100 times is significantly increased, as shown in D of Figure 8 ; compared with salt stress, the fresh weight and dry weight of cotton seedlings treated with bacterial liquid of three concentrations are significantly increased, as shown in E and F of Figure 8 . Among them, the plant height of cotton seedlings treated with strain Y10 under salt stress is significantly increased by 9.80%, the stem diameter is significantly increased by 38.0%~39.2%, the leaf area is significantly increased by 10.3%~25.3%, the fresh weight is significantly increased by 41.8%~50.5%, and the dry weight is significantly increased by 46.8%~54.6%.

[0065] Cotton seedling antioxidant enzyme activity determination.

[0066] 1) Superoxide dismutase: determined using a total superoxide dismutase determination kit. After 3 days of salt stress, 0.15 g of sample was weighed, 1.35 mL of phosphate buffer with pH 7.2 was added, and it was ground under ice bath conditions, centrifuged at 3500 r / min for 10 min, the supernatant was taken, and the absorbance value was determined at a wavelength of 550 nm according to the kit instructions.

[0067] 2) Peroxidase: determined using a peroxidase determination kit. After 3 days of salt stress, 0.15 g of sample was weighed, 0.6 mL of phosphate buffer with pH 7.2 was added, and it was ground under ice bath conditions, centrifuged at 3500 r / min for 10 min, the supernatant was taken, and the absorbance value was determined at a wavelength of 420 nm according to the kit instructions.

[0068] 3) Catalase: determined using a catalase determination kit. After 3 days of salt stress, 0.15 g of sample was weighed, 0.6 mL of phosphate buffer with pH 7.2 was added, and it was ground under ice bath conditions, centrifuged at 2500 r / min for 10 min, the supernatant was taken, and the absorbance value was determined at a wavelength of 405 nm according to the kit instructions.

[0069] The determination results are shown in Figure 9 , from Figure 9It can be seen that under 350mmol / L salt stress, Y10 fermentation broth diluted 50 times and diluted 100 times can significantly improve the SOD activity, POD activity and CAT content of the root of cotton seedlings. Y10 fermentation broth diluted 50 times can improve the SOD activity, POD activity and CAT content of the root of cotton seedlings by 28.46%, 26.65% and 49.47% respectively; Y10 fermentation broth diluted 100 times can improve the SOD activity, POD activity and CAT content of the root of cotton seedlings by 40.57%, 40.83% and 41.74% respectively.

[0070] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that such additions and modifications be included within the ambit of the present application.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis The application of Y10 in the preparation of products that improve the salt stress resistance of plants is characterized by, The product is a growth promoter; The Bacillus velezensis Y10 is preserved in the China General Microbiological Culture Collection Center, and the preservation time is December 5, 2024, and the preservation number is CGMCC No. 32927. The plant is cotton.

2. Use according to claim 1, characterized in that, The growth promoter is used to promote the increase of plant height, stem diameter, leaf area, fresh weight and dry weight under salt stress.

3. Use according to claim 1, characterized in that, The growth promoter is the fermentation liquor of Bacillus velezensis Y10.

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