Bacillus pseudomycoides CB59-7 and application of bacillus pseudomycoides CB59-7 in improvement of saline-alkaline tolerance of plants
By applying Bacillus pseudomycetamol CB59-7 inoculant to saline-alkali land, the problem of castor bean growth inhibition in saline-alkali environment was solved, resulting in increased plant height and leaf quantity, and improved agricultural production efficiency in saline-alkali land.
Patent Information
- Application Number
- CN202511534613.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-01-13
AI Technical Summary
Castor beans suffer from stunted growth, poor root development, and yellowing leaves in saline-alkali environments, which severely impacts yield and quality. Current technologies lack effective microbial methods to improve their salt and alkali tolerance.
The Bacillus pseudomycosis CB59-7 inoculant was applied to the plant roots to enhance the plant's salt and alkali tolerance and promote its growth in saline-alkali environments.
It significantly increases castor plant height and leaf quantity, improves agricultural production in saline-alkali land, increases yield, and has high environmental and biological safety.
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Figure CN121320161A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a pseudofungus bacillus CB59-7 and its application in improving the salt and alkali tolerance of plants. Background Technology
[0002] Saline-alkali land is widespread worldwide. Improving and utilizing it can effectively expand arable land area, enhance food production, and is of great significance to sustainable agricultural development. Currently, the types of vegetation that can be planted on saline-alkali land are limited. Comprehensive research on the impact of soil microbial communities and salinity on plant alkali resistance, as well as how vegetation restoration affects soil microbial communities, organic matter, and other soil properties, is relatively lagging. There is an urgent need to develop key technologies for planting on saline-alkali land. Among these, microbial improvement technology has opened up new avenues for saline-alkali land management. Microorganisms restore saline-alkali land by improving crop salt tolerance and altering the geochemical properties of saline-alkali soil.
[0003] castor bean ( Ricinus communis Castor bean is an important economic crop, with its seeds containing over 50% oil, which can be widely used in industrial lubricants, biodiesel, pharmaceuticals, and cosmetics. However, castor beans have limited adaptability to saline-alkali environments. Under high salinity and alkalinity conditions, root development is hindered, leaves wither and turn yellow, and plant growth is slow, severely affecting yield and quality. Therefore, improving the tolerance of castor beans to saline-alkali stress has become an important research direction for improving agricultural production in saline-alkali land. Summary of the Invention
[0004] The purpose of this invention is to provide a pseudomycosis fungus CB59-7 and its application in improving the salt and alkali tolerance of plants. The pseudomycosis fungus CB59-7 can improve the salt and alkali tolerance of plants and promote plant growth in saline-alkali environments.
[0005] This invention provides a pseudomycosis fungus ( Bacillus pseudomycoides CB59-7, the aforementioned Bacillus pseudomycosis CB59-7 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.32784.
[0006] The present invention also provides a microbial agent, wherein the effective component of the microbial agent includes Bacillus pseudomycosis CB59-7 as described in the above-described scheme.
[0007] As a preferred embodiment, the effective viable count of *Bacillus pseudomycosiscinus* CB59-7 in the bacterial agent is ≥2 × 10⁻⁶. 6 CFU / mL or ≥2×10 6 CFU / g.
[0008] The present invention also provides the application of the above-described Bacillus pseudomycosis CB59-7 or the described inoculant in improving the salt and alkali tolerance of plants.
[0009] As a preferred embodiment, improving the plant's salt and alkali tolerance includes increasing at least one of plant height, number of leaves, and yield.
[0010] As a preferred option, the plant includes castor bean.
[0011] The present invention also provides a method for improving the salt and alkali tolerance of plants, comprising the following steps: applying the *Bacillus pseudomycosiscinus* CB59-7 or the bacterial agent described above into the soil in which plants are planted.
[0012] As a preferred embodiment, the application site includes the plant roots.
[0013] As a preferred option, the amount applied is 2~5L / mu or 2~5kg / mu.
[0014] As a preferred option, the application is performed during the late seedling stage of the plant.
[0015] This invention provides a pseudomycosis fungus ( Bacillus pseudomycoides CB59-7, the *Bacillus pseudomyophyte* CB59-7 described in this invention, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32784. This *Bacillus pseudomyophyte* CB59-7 can improve the salt and alkali tolerance of plants and promote the growth of castor beans under saline-alkali conditions, resulting in increased plant height and significantly greener leaves. Furthermore, CB59-7 is a natural microorganism with high safety for plants, the environment, and humans and animals, and does not produce toxic or harmful substances during fermentation and bacterial culture preparation. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0017] Figure 1 The images show the colony morphology of some of the purified bacteria; where a is the colony morphology of strain CB59-7; and b-d are the colony morphology of other strains screened out. Figure 2 The images show the colony morphology of some of the purified bacteria under 100 mM NaHCO3; where a is the growth of CB59-7 under 100 mM NaHCO3; and b to d are the colony morphology of other purified strains. Figure 3 Phylogenetic tree of 16S rRNA sequence of strain CB59-7; Figure 4 The graph shows the effect of strain CB59-7 on castor bean seed germination. Figure 5 The effect of strain CB59-7 on the salt and alkali tolerance of castor beans is shown in the figure. This indicates that the data show significant differences; Figure 6 The effect of strain CB59-7 inoculant on salt and alkali tolerance of castor beans in the field was investigated.
[0018] Biological Preservation Instructions Strain CB59-7, classified as *Bacillus pseudomycosis* ( Bacillus pseudomycoides The specimen was deposited on November 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32784. Detailed Implementation
[0019] This invention provides a pseudomycosis fungus ( Bacillus pseudomycoides CB59-7, the aforementioned Bacillus pseudomycosis CB59-7 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.32784.
[0020] The *Bacillus pseudomyophyte* CB59-7 described in this invention was screened from biogas slurry from a biogas digester. The colonies are off-white in color, irregularly round or nearly round, with irregular wrinkles on the surface. Gram staining, mannitol, glucose, nitrate reduction reaction, catalase reaction, and methyl red test results were positive; starch hydrolysis, VP reaction, citrate, gelatin liquefaction, indole test, and urease test results were negative. It also exhibits some salt tolerance, growing in <9% (w / v) NaCl medium. Based on morphological, physiological, biochemical, and molecular biological analysis, CB59-7 is identified as *Bacillus pseudomyophyte*. Bacillus pseudomycoides .
[0021] The *Bacillus pseudomyophyte* CB59-7 described in this invention exhibits strong stress resistance and environmental adaptability, enhancing the plant's resilience. Furthermore, CB59-7 is a natural microorganism, possessing high safety for plants, the environment, and humans and animals, and does not produce toxic or harmful substances during fermentation and broth preparation. Example results show that under saline-alkali conditions, CB59-7 can promote plant growth, increase plant height, and promote an increase in the number and greening of leaves.
[0022] The present invention provides a microbial agent, wherein the effective component of the microbial agent includes *Bacillus pseudomycosiscinus* CB59-7 as described in the above-mentioned scheme.
[0023] As one embodiment, the effective viable count of *Bacillus pseudomycosiscinus* CB59-7 in the bacterial agent is ≥2 × 10⁻⁶. 6 CFU / mL; as another embodiment, the effective viable count of Bacillus pseudomycosis CB59-7 in the bacterial agent is ≥2×10⁻⁶. 6 CFU / g.
[0024] The present invention also provides the application of the above-described Bacillus pseudomycosis CB59-7 or the described inoculant in improving the salt and alkali tolerance of plants.
[0025] As one implementation method, improving the salt and alkali tolerance of plants includes increasing at least one of plant height, number of leaves, and yield.
[0026] In one embodiment, the plant includes castor bean.
[0027] The present invention also provides a method for improving the salt and alkali tolerance of plants, comprising the following steps: applying the *Bacillus pseudomycosiscinus* CB59-7 or the bacterial agent described above into the soil in which plants are planted.
[0028] In one embodiment, the application site includes the plant roots. Applying the *Bacillus pseudomycosiscinus* CB59-7 inoculant to the plant roots according to this invention is beneficial for improving the plant's salt and alkali tolerance.
[0029] In one embodiment, when the microbial agent is a liquid formulation, the application amount is 2-5 L / mu. In a specific embodiment of the present invention, the application amount can be any value within the range of 2-5 L / mu, such as 2, 2.5, 3, 3.5, 4, 4.6, or 5 L / mu. In another embodiment, when the microbial agent is a solid formulation, the application amount is 2-5 kg / mu. In a specific embodiment of the present invention, the application amount can be any value within the range of 2-5 kg / mu, such as 2, 2.5, 3, 3.5, 4, 4.6, or 5 kg / mu.
[0030] In one implementation method, the application is performed during the late seedling stage of the plant. This invention selects the late seedling stage for application of the microbial agent, as the root tips of castor bean seedlings are in an elongated state and root hairs are abundant, allowing for sufficient contact with the microbial agent.
[0031] To further illustrate the present invention, the technology provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1: Screening of salt-tolerant bacteria The method for screening Bacillus pseudomycosis CB59-7 from biogas digester slurry according to the present invention is as follows: Take 5 mL of biogas fermentation broth sample and mix thoroughly on a vortex mixer. Add 500 μL of sample to 4.5 mL of sterile water and perform a 10-minute incubation. -1 ~10 -4 Serial dilutions were performed. The diluted solution was evenly spread onto LB agar plates using the plate spread method and incubated at 37°C with three replicates. Colonies were observed after 24 hours. Colonies of different morphology, color, and size were picked and streaked for purification. The LB agar consisted of: 6 g yeast extract, 10 g tryptone, 10 g sodium chloride, 17 g agar, and deionized water to a final volume of 1 L, adjusted to pH 7.
[0033] After the above steps, 24 bacteria of varying colors, shapes, and sizes were selected. Some of these bacteria, such as... Figure 1 As shown, where Figure 1 In the middle, 'a' is CB59-7, for future use.
[0034] Twenty-four bacterial strains selected above were streaked onto LB agar plates containing 100 mM NaHCO3 at pH 8 and cultured at 37°C in triplicate. Salt and alkali tolerance of the bacteria was observed after 24 h. CB59-7 showed the best growth on LB agar containing 100 mM NaHCO3. (See [link to relevant documentation]). Figure 2 In the 'a', some bacteria, such as Figure 2 As shown in b~d in the diagram.
[0035] Example 2: Identification of salt-tolerant bacteria CB59-7 (1) Morphological, physiological and biochemical identification The CB59-7 cells are off-white, nearly round, with an uneven surface and slight ridges. Physiological and biochemical identification was performed by referring to the "Handbook of Systematic Identification of Common Bacteria" (edited by Dong Xiuzhu and Cai Miaoying, Science Press, 2001). The specific results are shown in Table 1.
[0036] Table 1. Results of Physiological and Biochemical Tests on CB59-7
[0037] Note: "+" indicates a positive test result, and "-" indicates a negative test result; Preliminary morphological and physiological biochemical tests have determined that CB59-7 belongs to Bacillus.
[0038] (2) Molecular identification Genomic DNA was extracted from CB59-7 using a bacterial genomic DNA rapid extraction kit. The primer sequences required for gyr B sequencing were: UP-1S (SEQ ID NO.1): 5′-GAAGTCATCATGACCGTTCTGCA-3′; UP-2Sr (SEQ ID NO.2): 5′-AGCAGGGTACGGATGTGCGAGCC-3′, as shown in SEQ ID NO.2. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 60℃ annealing for 2 min, 72℃ extension for 2 min, 30 cycles; 72℃ reaction for 10 min.
[0039] The 16S rRNA and gyr B sequences obtained from sequencing were compared with nucleic acid data in GenBank using BLAST for homology analysis. When constructing a phylogenetic tree using the combined 16S rRNA and gyr B sequences, *E. coli* K-12 (J01695) was used as an outgroup, and the Neighbour-joining method was employed. Figure 3 As shown.
[0040] Homology comparison of CB59-7 using 16S rRNA and gyr B sequences revealed that CB59-7 shared 100% homology with *Bacillus pseudomycosiscinus* NBRC 101232, and the phylogenetic tree results were consistent. Figure 3 Based on a comprehensive analysis of morphology, physiology, biochemistry, and molecular biology, *Bacillus pseudomycosis* CB59-7 was identified. Bacillus pseudomycoides The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 25, 2024, with accession number CGMCC No. 32784.
[0041] Example 3: Effect of CB59-7 on castor bean germination Castor seeds under different salinity and alkalinity conditions were treated with CB59-7 bacterial solution to detect the effect of Bacillus mycoides CB59-7 on castor germination.
[0042] 1. Castor bean cultivation treatment (1) Select healthy and plump castor seeds "Bilu No. 1" (castor seeds were donated by Zibo Academy of Agricultural Sciences), soak the seeds in a 0.5% (v / v) sodium hypochlorite solution for 10 min for disinfection, rinse with sterile water at least 3 times, wipe off excess water on the seed surface, and place the seeds in a ventilated place to dry for later use.
[0043] (2) Filter the nutrient soil using a 5 mm sieve, and sterilize the sieved soil. High-pressure steam sterilization (121℃, 30 min) can be used to eliminate soil microorganisms that may affect the experimental results. Evenly fill the sterilized soil into glass containers, ensuring that the amount of soil in each container is consistent.
[0044] (3) To avoid mutual interference between seedlings, sow 8 castor bean seeds in each container, cover with a thin layer of soil after sowing, and gently compact. Place the containers in a greenhouse with an average temperature of 25°C and a light exposure time of 12 h.
[0045] 2. The *Bacillus pseudomycosis* CB59-7 screened in Example 1 was fermented and cultured, as follows: (1) Activation of bacterial strain: CB59-7 was activated using LB solid medium. The activation method was streak plate method. A small amount of CB59-7 bacterial strain was dipped into a sterile bacterial inoculation loop and streaked evenly in a Z-shape on an LB plate. The culture was incubated in the dark at 35°C for 24 h. The formula of the LB solid medium was: 6 g yeast extract, 10 g tryptone, 10 g sodium chloride, 17 g agar, and deionized water was added to a final volume of 1 L and the pH was adjusted to 7. (2) Preparation of CB59-7 seed culture: Pick a single colony of activated CB59-7 and transfer it to LB liquid medium and culture at 30℃ and 150rpm for 24 h; The LB solid medium formula is: 6 g yeast extract, 10 g tryptone, 10 g sodium chloride, deionized water to 1 L, and pH adjusted to 7. (3) Preparation of fermentation broth: CB59-7 seed culture was added to the modified LB medium at a volume ratio of 1:200 between the seed culture and the fermentation broth. The culture was carried out at 35℃ and 180 rpm for 120 h. The modified LB medium formula is as follows: 6 g yeast extract, 10 g tryptone, 10 g sodium chloride, 5 g potassium dihydrogen phosphate, and deionized water to a final volume of 1 L, with the pH adjusted to 7.5.
[0046] Take 5 mL of CB59-7 fermentation broth (concentration 1×10⁻⁶). 8 The CFU / mL bacterial solution was diluted 50 times with 0.5% (w / v) NaHCO3 solution to obtain the diluted bacterial solution (50-fold diluted bacterial solution) for later use.
[0047] 3. Group processing (1) The castor seeds treated in step 1 were randomly divided into 3 groups, which were named salt-alkali treatment group, bacterial solution treatment group and blank control group respectively.
[0048] (2) The procedures for sowing castor beans in different treatment groups were as follows: Salt-alkali treatment group (0.5% NaHCO3 treatment): At the time of sowing, 30 mL of 0.5% (w / v) NaHCO3 solution was applied. Seven days later, another 30 mL of 0.5% (w / v) NaHCO3 solution was applied.
[0049] Bacterial solution treatment group (0.5% NaHCO3+ bacterial solution treatment): At the time of sowing, 30 mL of the diluted bacterial solution from step 2 was applied. After 7 days, another 30 mL of the diluted bacterial solution was applied.
[0050] Blank control group (water treatment): When sowing, water with 30 mL of water, and 7 days later, water with another 30 mL of water.
[0051] Take photos to observe the germination of castor beans on the 14th day after sowing. The results are as follows: Figure 4 As shown, the germination rate of castor beans under saline-alkali treatment was significantly improved after adding a bacterial solution diluted 50 times.
[0052] Example 4: Effect of CB59-7 on the salt and alkali tolerance of castor bean To investigate the effect of Bacillus pseudomycetes CB59-7 on the salt and alkali tolerance of castor beans grown under different saline-alkali treatments, CB59-7 bacterial solution was applied to the roots.
[0053] Castor bean seedlings in the late seedling stage and with uniform growth were selected from the water group and saline-alkali treatment group in Example 3. The plant height and number of leaves of the castor bean seedlings were measured. Salt-alkali treatment + bacterial agent application group: Every 3 days, the bacterial solution prepared in step 2 of Example 3 was diluted 50 times and applied to the roots. Each pot was irrigated with 50 mL each time. After 2 treatments, photos were taken on the 7th day and the plant height and number of leaves were counted.
[0054] Salt-alkali treatment + no bacterial agent application group: The treatment is the same as the "salt-alkali treatment + bacterial agent application group", the difference is that 50mL of clean water is applied.
[0055] Clean water group: The treatment is the same as the "salt-alkali treatment + application of bacterial agent group", the difference is that 50 mL of clean water is poured in.
[0056] The percentage increase in leaf area before treatment is set as 1. The formula for calculating the percentage increase in leaf area after treatment is as follows: Leaf increase rate = average leaf area of the treatment with fungicide / average leaf area of the control.
[0057] Photographs and statistical results are as follows Figure 5 As shown in Table 2, the results indicate that in the saline-alkali treatment group, the application of microbial agents can increase plant height and significantly increase the proportion of leaf growth.
[0058] Table 2. Increase in plant height and leaf area in different groups
[0059] Example 5: Field effect of CB59-7 on salt and alkali tolerance of castor bean Experimental location: Castor bean planting fields in Heilongjiang Province, and the tested variety was "Bilv No. 1".
[0060] Test strain: CB59-7.
[0061] Experimental treatments: The experiment included two treatments, each lasting 30 m. 2 Three replicates were set. Normal sowing time was used as the standard, with normal fertilization and field management; no fungicides were applied throughout the entire growth period.
[0062] Treatment 1: During the later stages of castor bean growth, use CB59-7 fermentation stock diluted 50 times (concentration 1×10⁻⁶). 7 The spray treatment was carried out using CFU / mL; the dosage for both spray treatments was 4 L / acre. Treatment 2: Water control. Spray with 4 L / acre of water; no fungicides were used throughout the entire growth period. The later stages of castor bean growth are as follows: Figure 6 As shown, the growth of castor beans was significantly improved after treatment with a 50-fold dilution of CB59-7 fermentation stock solution. After the castor beans were harvested, the yield was calculated: castor beans treated with CB59-7 yielded 300 catties / mu, while those not treated with CB59-7 yielded 220 catties / mu.
[0063] In summary, CB59-7 can improve the growth status of castor beans in saline-alkali land and increase yield.
[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A type of pseudofungi (Bacillus pseudofungus) Bacillus pseudomycoides CB59-7, characterized in that, The *Bacillus pseudomycosis* CB59-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32784.
2. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes Bacillus pseudomycosis CB59-7 as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The effective viable count of *Bacillus pseudomycosiscinus* CB59-7 in the bacterial agent is ≥2 × 10⁻⁶. 6 CFU / mL or ≥2×10 6 CFU / g.
4. The application of Bacillus pseudomycosis CB59-7 as described in claim 1 or the inoculant as described in claim 2 or 3 in improving the salt and alkali tolerance of plants.
5. The application according to claim 4, characterized in that, The improvement of plant salt and alkali tolerance includes increasing at least one of plant height, number of leaves, and yield.
6. The application according to claim 4 or 5, characterized in that, The plant mentioned includes castor bean.
7. A method for improving the salt and alkali tolerance of plants, characterized in that, The method includes the following steps: applying the Bacillus pseudomycosis CB59-7 of claim 1 or the inoculum of claim 2 or 3 into the soil where plants are established.
8. The method according to claim 7, characterized in that, The application site includes the plant roots.
9. The method according to claim 7, characterized in that, The application rate is 2-5 L / mu or 2-5 kg / mu.
10. The method according to claim 7, characterized in that, The application period is during the late seedling stage of the plant.