Bacillus pumilus 21-14 and uses thereof

Abstract

The present application belongs to the technical field of bioengineering. The present application discloses a bacillus pumilus 21-14, the strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 6, 2024, the center is abbreviated as CGMCC, the strain preservation center registration number is CGMCC NO.31892, the strain preservation date is September 6, 2024, and the address of the preservation unit is No.3, Beichen West Road, Chaoyang District, Beijing. The bacillus pumilus strain 21-14 provided by the present application is simple to culture, short in cycle, free of ecological toxicity, rich in types of available carbon sources, strong in salt and alkali tolerance, capable of promoting the growth and development of potatoes through phosphorus solubilization and IAA production, beneficial to promoting the improvement of the biomass of crops in saline-alkali soil, and capable of effectively inhibiting the growth of streptomyces prolixus.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. Background Technology

[0002] Bacillus produces antibacterial substances that can control various plant diseases. Bacillus possesses biological activities such as phosphorus solubilization, potassium solubilization, and nitrogen fixation, which are beneficial for increasing crop yields. Furthermore, Bacillus exhibits good stress resistance. The antibacterial substances produced by Bacillus generally have a broad spectrum of inhibition, exhibiting broad-spectrum bactericidal activity against a wide range of Gram-negative and Gram-positive bacteria, and generally possess good thermal stability. They also have high environmental compatibility and heavy metal ion adsorption capacity, and can improve soil physicochemical properties, playing an important role in plant disease control and soil remediation.

[0003] Soil-borne diseases of potatoes are one of the limiting factors affecting potato quality. Controlling these diseases through microbial agents has become a new research direction. Therefore, seeking green and environmentally friendly microbial agents to improve fertilizer utilization, reduce the occurrence of soil-borne diseases, and improve the soil ecological environment is one of the urgent problems to be solved in the current agricultural fertilizer utilization process. Summary of the Invention

[0004] In view of this, the first objective of the present invention is to provide a strain of Bacillus pumilus 21-14, with accession number CGMCC NO.31892.

[0005] The Bacillus pumilus 21-14 provided by this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.31892 and deposit date of September 6, 2024. The center is abbreviated as CGMCC and its address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] A second objective of this invention is to provide the application of Bacillus pumilus 21-14 in the preparation of inoculants that inhibit the growth of potato scab pathogens, in the preparation of microbial saline-alkali soil conditioners, in the preparation of inoculants that convert ineffective phosphorus into plant-absorbable effective phosphorus, in the preparation of formulations that promote plant root growth and development, and in the preparation of inoculants for the prevention and control of potato scab.

[0007] The Bacillus pumilus 21-14 provided by this invention is simple to cultivate, non-ecotoxic, salt-tolerant, and has a rich variety of available carbon sources. It can decompose insoluble phosphorus in the soil and produce cellulase, siderophores, etc. At the same time, it can effectively inhibit the growth of potato scab pathogens. It has very broad application prospects in the biological control and growth promotion of potato scab. Attached Figure Description

[0008] Figure 1 It is a phylogenetic tree of Bacillus pumilus 21-14 16S.

[0009] Figure 2 This is a single colony image of Bacillus pumilus 21-14.

[0010] Figure 3 This is an electron microscope image of Bacillus pumilus 21-14.

[0011] Figure 4 This is a Gram staining image of Bacillus pumilus 21-14.

[0012] Figure 5 This is an antagonistic diagram of Bacillus pumilus 21-14.

[0013] Figure 6 This is a graph showing the results of phosphate solubilization assay for Bacillus pumilus 21-14.

[0014] Figure 7 This is a graph showing the results of the assay for siderophores produced by Bacillus pumilus 21-14.

[0015] Figure 8 This is a graph showing the results of cellulase production by Bacillus pumilus 21-14.

[0016] Figure 9 This is a potted plant image showing the effect of Bacillus pumilus 21-14 in controlling potato scab. Detailed Implementation

[0017] Unless otherwise specified, the strains tested in the following examples all refer to Bacillus pumilus 21-14.

[0018] Unless otherwise specified, activation in the following examples refers to the three-stage expansion culture of Bacillus pumilus 21-14 on PDA solid medium.

[0019] Example 1

[0020] Isolation and identification of bacterial strains

[0021] Culture medium: Potato Dextrose agar (PDA) medium (g / L): 200g potato, 20g glucose, 15g agar, 1000ml distilled water, natural pH.

[0022] Strain source: Bacillus pumilus 21-14 was isolated from soil in a potato field in Yakeshi, Inner Mongolia, by the inventor on November 15, 2022. 2g of soil sample was placed in a 50ml centrifuge tube, 20ml of sterile water was added, and the mixture was shaken thoroughly and allowed to stand until clear. The prepared soil suspension was serially diluted in a clean bench, and 100μL of the 10⁻⁵-fold dilution was spread on LB agar and incubated at 37℃ for 24h. The resulting pure single colonies were transferred to test tube slant agar. The isolated strains were as follows... Figure 2 The colony morphology and strain classification characteristics shown are as follows: the colonies are round and milky white. Under a scanning electron microscope, the cells of this bacterium are rod-shaped (e.g., ...). Figure 3 As shown), the strain is a Gram-positive bacterium (e.g. Figure 4 As shown in the figure, it can decompose arabinose to produce sugar as a carbon source, decompose glycerol and salicin, and utilize trehalose and amygdalin, which is consistent with the physiological and biochemical characteristics of Bacillus strains. The strain was named 21-14.

[0023] Bacterial strain identification method: Genomic DNA was extracted using the TIANGEN bacterial genomic DNA extraction kit. Using the genomic DNA of the test strain as a template, the target fragment was amplified using the universal primers 27F / 1492R for the bacterial 16S rRNA gene. The PCR product was detected by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rRNA gene sequence is shown in SEQ ID NO.1 of the sequence listing. Analysis and sequence comparison identified strain 21-14 as belonging to *Bacillus pumilus*, with NPB34B (KF254 674.1NCBI) as the standard strain.

[0024] This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.31892, deposit date September 6, 2024. The depositary institution is referred to as CGMCC and its address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Example 2

[0026] Physiological and biochemical analysis of strains

[0027] The physiological and biochemical properties of strain 21-14 were tested using the bioMérieux API 20NE non-fermenting Gram-positive bacillus identification kit. Bacillus pumilus 21-14 can utilize arabinose, glucose, xylose, fructose, trehalose, and sucrose. Tests for arbutin, salicin, glycerol, and amygdalin were all positive. Tests for xylitol, potassium gluconate, sorbitol, fucose, rhamnose, and erythritol were all negative. Detailed results are shown in the table below:

[0028] Table 1. Analysis of physiological and biochemical properties

[0029]

[0030] Note: +: positive reaction; -: negative reaction

[0031] Example 3

[0032] Inhibitory effect of strains on Streptomyces scabies

[0033] The pathogen of potato scab, 4.1765 (purchased from the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO. 4.1765), was cultured on potato medium at 28°C for 5 days. Spores and mycelium were scraped off to prepare a pathogen suspension of a certain concentration. Activated strain 21-14 and 100 μl of 4.1765 were used for a plate confrontation experiment, cultured at 28°C, and observed after 3 days. The results were as follows: Figure 5 The experimental results shown indicate that Bacillus pumilus 21-14 effectively inhibited the growth of the potato scab pathogen 4.1765, with an inhibition zone diameter of up to 30 mm (e.g., ...). Figure 6 (As shown).

[0034] Example 4

[0035] Salt tolerance test of strains

[0036] The basal medium was LB liquid medium. NaCl was added to LB solid medium to adjust the salt concentration to 1%, 3%, 5%, 9%, and 11%, and the pH was adjusted to 7.0. The medium was then autoclaved at 121°C for 20 minutes.

[0037] The activated test strain 21-14 was inoculated onto five different salt concentrations of LB liquid medium, with uninoculated medium serving as a blank control. The media were incubated at 37℃ for 24 hours to observe the growth of strain 21-14. OD values ​​were measured. The results show that as the NaCl concentration increased, the concentration of strain 21-14 first increased and then decreased, indicating that 21-14 is more suitable for saline environments. Even when the salt concentration reached 9%, it continued to grow normally. This demonstrates that this strain can be used in saline-alkali land improvement, exhibits salt tolerance, and is suitable as a microbial saline-alkali land conditioner.

[0038] Table 2 Salt tolerance results of strain 21-14

[0039] NaCl 1％ 3％ 5％ 7％ 9％ 11％ OD600 1.453 1.401 1.396 1.386 1.236 0.292

[0040] Example 5

[0041] Determination of strain sensitivity to different pH values

[0042] LB broth was used as the basal medium, with pH values ​​adjusted to 1, 3, 5, 7, 9, and 11. The activated test strain 21-14 was inoculated onto the six pH-sensitive media and incubated at 37°C for 24 hours. The growth of strain 21-14 was observed. The results showed that the concentration of strain 21-14 first increased and then decreased with increasing pH, indicating that 21-14 thrives in a slightly alkaline environment. The OD value of its concentration was measured, and the results showed that the growth of strain 21-14 was inhibited when the pH was less than 5 or greater than 9, but it continued to grow normally at pH 9. This demonstrates that this strain can be used in saline-alkali land improvement, exhibits alkali tolerance, and is suitable as a microbial saline-alkali land conditioner.

[0043] Table 3 pH sensitivity determination of strain 21-14

[0044] pH 1 3 5 7 9 11 OD600 0.160 0.306 1.350 1.362 1.401 0.136

[0045] Example 7

[0046] Results of cellulase activity test of strains

[0047] Carboxymethyl cellulose sodium (CMC-Na) medium: CMC-Na 15 g, disodium hydrogen phosphate 2.5 g, potassium dihydrogen phosphate 1.5 g, peptone 2.5 g, agar powder 20 g, diluted with distilled water to 1000 mL, pH 7.2–7.5.

[0048] The purified bacterial strain was inoculated onto sodium carboxymethyl cellulose (CMC-Na) medium and incubated upside down at 30°C for 48 hours. After staining with 0.5% Congo red for 30 minutes and destaining with 1 mol / L NaCl for 30 minutes, Congo red formed a red complex with CMC-Na, but did not react with the hydrolyzed cellobiose and glucose. The relative levels of bacterial cellulase production were preliminarily determined based on the size of the clear zone on the antagonistic bacterial plate. Figure 7 As shown, the diameter of the cellulose-degrading ring in 21-14 is 27 mm, indicating a strong cellulose-degrading ability.

[0049] Example 8

[0050] Determination of the growth-promoting effect of the strain on potato plants

[0051] Strain 21-14 was cultured in King's medium for 24 hours. The supernatant was mixed with the colorimetric solution, and the mixture was observed to be pink, indicating that strain 21-14 could produce IAA. The IAA yield of strain 21-14 was calculated to be 1.67 mg / L according to the standard curve.

[0052] Prepare a control group and a control group with inoculum, using water as the control inoculum. Set up three parallel groups, with three seedlings in each group. After centrifuging the antagonistic bacteria fermentation broth, suspend the bacterial cells in tap water and apply them to the potato seedlings. After 60 days, record the relative values ​​of plant height, stem diameter, leaf chlorophyll content (SPAD), and the weight of potato tubers after harvest.

[0053] Table 4. Comparison of plant height between the control group and the inoculum group (unit: cm)

[0054]

[0055]

[0056] Table 5. Comparison of SPDA between the control group and the inoculum group (unit: cm)

[0057]

[0058] Table 6. Comparison of stem diameter between the control group and the fungal agent group (unit: mm)

[0059]

[0060] Table 7. Comparison of tuber weight between the control group and the inoculum group (unit: g)

[0061]

[0062] The results showed that plants irrigated with strain 21-14 exhibited significantly better plant height, stem diameter, SPDA (split diameter), and tuber weight than the control group, increasing by 15.13%, 26.95%, 9.57%, and 32.50% respectively compared to the control. These results indicate that strain 21-14 has a good growth-promoting effect (e.g., ...). Figure 9 (As shown).

[0063] Example 9

[0064] Pot experiment on the control of potato scab by strain

[0065] Tissue culture seedlings of the potato variety Shaboti were transplanted into vermiculite pots (17cm in diameter). After 15 days of growth, seedlings with uniform growth were selected and inoculated with 100mL of Streptomyces scabii spore suspension (1×10⁻⁶) using the root irrigation method. 7 Add CFU / mL to the flowerpot; after 20 days of regrowth, inoculate with 100 mL of Bacillus pumilus strain 21-14 fermentation broth (1×10⁻⁶ CFU / mL). 8 The control group received no biocontrol agents (CFU / mL), and the control group received no biocontrol agents. Each treatment was repeated three times. After potato harvest, the incidence rate, disease index, and control effectiveness were statistically analyzed according to the scab disease grading standards.

[0066] Statistical results showed that only potatoes treated with the pathogen had a disease incidence rate of 100% and a disease index of 77.78, while potatoes treated with 21-14 had a disease incidence rate of 45.45% and a disease index of only 27.27. The control efficacy of strain 21-14 against potato scab reached 64.94%.

[0067] Potato scab incidence statistics: Grading criteria:

[0068] Grade 0: Healthy potato skin, no disease spots

[0069] Grade 1: The affected area is 0-1 / 6 of the total area.

[0070] Grade 2: The affected area is 1 / 6-1 / 3 of the total surface area.

[0071] Grade 3: The affected area is 1 / 3 to 1 / 2 of the total surface area.

[0072] Grade 4: Lesions cover more than 1 / 2 of the body surface area.

[0073] Incidence rate (%) = Number of diseased tubers / Total number of tubers surveyed × 100

[0074] Disease index = (Number of tubers at each disease level × Representative value of that disease level) / (Total number of surveyed individuals × Highest disease level) × 100

[0075] Relative efficacy (%) = (Disease index of control group – Disease index of treatment group) × 100 / Disease index of control group

[0076] Table 4 Results of the pot experiment

[0077] Diseased potato level CK (Water) Pathogen (4.1765) Pathogen (4.1765+21-14) Level 0 8 0 6 Level 1 0 0 2 Level 2 0 2 2 Level 3 0 4 1 Level 4 0 3 0 Total number of potatoes 8 9 11 Total potato weight 89.23g 130.47g 128.94g Total number of diseased potatoes 0 9 5 Total diseased potato weight 0g 130.47g 120.03g Incidence rate 0 100％ 45.45％ Disease index 0 77.78 27.27 Prevention - - 64.94％

[0078] Example 10

[0079] Experiment on the effect of strain on potato tuber yield

[0080] Two treatments were set up in the greenhouse flowerpots:

[0081] CK: Regular watering:

[0082] T: After regular fertilization, apply Bacillus pumilus 21-14 via irrigation, with each milliliter of bacterial solution containing 1×10⁻⁶. 8 CFU live bacteria.

[0083] Each treatment was repeated three times. After three months of potato growth, tuber growth was observed. As shown in Table 7, strain 21-14 exhibited a significant growth-promoting effect and can be used in growth-promoting agents.

[0084] Example 11

[0085] Field trials of strains for the control of potato scab

[0086] Field efficacy verification was conducted on potato fields in Hailar District, Inner Mongolia Autonomous Region, where potatoes had been continuously cropped for three years. The results are shown in Table 5. The incidence rate in the CK group was 91.93%, and the disease index was 42.48. The incidence rate in the 21-14 treatment group was 37.43%, the disease index was 21.23, and the efficacy was 51.75%.

[0087] Table 5. Results of field plot experiments in Hailar District, Inner Mongolia Autonomous Region

[0088]

[0089] The results of the community trial in Huidong County, Guangdong Province are shown in Table 6. The incidence rate in the CK group was 91.84%, and the disease index was 58.51; the incidence rate in the 21-14 treatment group was 45.18%, the disease index was 29.12, and the control efficacy was 49.05%.

[0090] Table 6. Results of field plot experiments in Huidong County, Guangdong Province

[0091]

[0092] The statistical data from the field plot trials in both locations were consistent with the trend of the pot experiment in terms of disease prevention efficacy, indicating that the trials had a good effect on promoting growth and preventing disease.

[0093] The Bacillus pumilus 21-14 provided by this invention is simple to cultivate, has a short cycle, is non-ecotoxic, salt-tolerant, and has a rich variety of available carbon sources. It can decompose insoluble phosphorus in the soil and produce cellulase, siderophores, etc. At the same time, it can effectively inhibit the growth of potato scab pathogens. It has very broad application prospects in the biological control of potato scab, saline-alkali soil improvement, and growth-promoting bacteria.

Claims

1. Bacillus pumilus 21-14, with accession number CGMCC NO.31892.

2. The use of Bacillus pumilus 21-14 according to claim 1 in the preparation of an agent to inhibit potato scab pathogens.

3. The application of Bacillus pumilus 21-14 according to claim 1 in the preparation of microbial agents for improving saline-alkali land.

4. The application of Bacillus pumilus 21-14 according to claim 3 in the preparation of microbial agents for improving saline-alkali land, characterized in that, The NaCl concentration in the saline-alkali land is no higher than 9%.

5. The application of Bacillus pumilus 21-14 according to claim 3 in the preparation of microbial agents for improving saline-alkali land, characterized in that, The pH range of the saline-alkali land is 5-9.

6. The use of Bacillus pumilus 21-14 according to claim 1 in the preparation of a microbial agent that converts ineffective phosphorus into available phosphorus that can be absorbed by plants.

7. The use of Bacillus pumilus 21-14 according to claim 1 in the preparation of cellulose-decomposing bacterial agents.

8. The use of Bacillus pumilus 21-14 according to claim 1 in the preparation of a fungal agent for the prevention and control of potato scab.

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