Screening and application of salt-tolerant plant growth-promoting bacillus rhizosphere
By using Bacillus pumilus ZF526 and its metabolites, the problem of insufficient arable land caused by soil salinization was solved, the growth of cucumbers and soil improvement were promoted, and salt stress was alleviated and soil nutrients were improved.
Patent Information
- Application Number
- CN202510930863.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-17
AI Technical Summary
Soil salinization has led to insufficient arable land and reduced agricultural productivity. Existing technologies make it difficult to effectively reduce soil salinity and increase soil nutrients to promote plant growth.
Bacillus pumilus ZF526 and its metabolites were used to prepare bacterial agents and applied to salinized soil, utilizing their functions of promoting plant growth, solubilizing phosphate, potassium, fixing nitrogen and producing protease to alleviate salt stress.
It significantly increased the leaf area, plant height, stem diameter, fresh weight and dry weight of cucumber, effectively alleviated salt stress, increased soil enzyme activity and microbial richness, and improved soil structure.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to screening of a salt-tolerant plant rhizosphere growth-promoting Bacillus and application thereof. BACKGROUND
[0002] The shortage of arable land and the decline of agricultural productivity caused by soil salinization have become an important factor restricting the productivity of arable land, and the improvement of salinized soil has important economic and strategic significance. At present, in view of the problems of high salt content, strong alkalinity, poor physical structure and nutrient deficiency of salinized soil, scholars have proposed many improvement measures such as physical, chemical and biological measures. Among them, biological improvement is considered as the most ecological measure because it has the effects of lasting desalination, stability and being conducive to water and soil conservation and state balance.
[0003] Microbial inoculant is a specific fertilizer containing microorganisms, which is a kind of active bacterial preparation made by artificial breeding and proliferation of one or several beneficial microorganisms isolated from soil. It is an auxiliary fertilizer. After application, the life activities of microorganisms in soil can directly or indirectly provide the required nutrients for plant growth, improve soil nutrients and soil enzyme activity, and also improve the absorption of nutrients by plants, thereby promoting plant growth and improving quality. It has been found that the application of functional microbial inoculants can improve microbial functional diversity and soil enzyme activity. It has been found that the application of microbial inoculants can not only improve the yield of greenhouse eggplants, but also improve soil enzyme activity. The application of microbial inoculants can not only promote the growth of tobacco, but also improve microbial richness and regulate microbial structure. SUMMARY
[0004] The main problem to be solved by the present application is to reduce the salt content of soil, improve soil nutrients and promote plant growth by using biological methods.
[0005] In order to solve the above problems, the present application provides a Bacillus pumilus.
[0006] The Bacillus pumilus provided by the present application is Bacillus pumilus ZF526, which has a preservation number of CGMCC No.32942 in China General Microbiological Culture Collection Center.
[0007] The present application also provides a microbial inoculant containing the aforementioned Bacillus pumilus and / or metabolites of the Bacillus pumilus.
[0008] The present application also provides a culture of the aforementioned Bacillus pumilus, which is a substance obtained by culturing the aforementioned Bacillus pumilus in a bacterial culture medium.
[0009] In the above-mentioned microbial agent, the microbial agent contains a carrier in addition to the active ingredient. The carrier can be a carrier commonly used in the field of pesticides and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material, or a high molecular compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomite; the plant material can be at least one of corn flour, soybean flour, and starch; the high molecular compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil, or water; and the organic solvent can be decane and / or dodecane.
[0010] In the above-mentioned microbial agent, the dosage form of the microbial agent can be various dosage forms, such as a liquid, an emulsion, a suspension, a powder, a granule, a wettable powder, or a water dispersible granule.
[0011] If necessary, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH regulator, etc. can also be added to the microbial agent.
[0012] In the specific embodiment of the present application, the microbial agent is a fermentation broth of the Bacillus pumilus ZF526 with a CFU / mL of 10 8 CFU / mL. Further, it is prepared according to a method comprising the following steps: inoculating the Bacillus pumilus ZF526 into a bacterial culture medium and culturing to obtain a broth with a concentration of 10 8 CFU / mL, i.e. the microbial agent.
[0013] The term "metabolite" refers to a primary metabolite and / or a secondary metabolite produced in the process of microbial metabolism. The primary metabolism refers to the process in which a microorganism absorbs various nutrients from the outside world, and through catabolism and anabolism, generates substances and energy to maintain life activities. The product of primary metabolism is a primary metabolite, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. The secondary metabolism refers to the process in which a microorganism synthesizes some substances that have no clear function for life activities of the microorganism using the primary metabolites as precursors during a certain growth period. The product of secondary metabolism is a secondary metabolite, which is mostly a compound with a relatively complex molecular structure. According to its function, it can be classified into antibiotics, hormones, alkaloids, toxins, etc.
[0014] In the above, the metabolite can be obtained from a fermentation broth of the Bacillus pumilus. The metabolite can be a sterile metabolite of the Bacillus pumilus or a metabolite containing the Bacillus pumilus. The sterile metabolite of the Bacillus pumilus (sterile fermentation filtrate) can be prepared specifically as follows: the Bacillus pumilus is cultured in a liquid medium, and the Bacillus pumilus in the liquid culture (fermentation broth) is removed by filtration to obtain the sterile metabolite of the Bacillus pumilus. The metabolite containing the Bacillus pumilus can be prepared specifically as follows: the Bacillus pumilus is cultured in a liquid fermentation medium, and the fermentation broth is collected, which is the metabolite containing the Bacillus pumilus.
[0015] Further, the bacterial culture medium is LB liquid medium.
[0016] The term "culture" refers to the collective term for the liquid or solid product (all materials in the culture vessel) that has been inoculated and grown by man. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes the subculture obtained by subculturing microorganisms, which can be a culture of a certain generation, or a mixture of several generations.
[0017] The present application also provides the use of the Bacillus pumilus or the bacterial agent or the culture as described above in at least one of the following:
[0018] (a1) promoting plant growth;
[0019] (a2) preparing a product for promoting plant growth;
[0020] (a3) dissolving phosphorus;
[0021] (a4) preparing a product for dissolving phosphorus;
[0022] (a5) dissolving potassium;
[0023] (a6) preparing a product for dissolving potassium;
[0024] (a7) nitrogen fixation ability;
[0025] (a8) preparing a product for nitrogen fixation ability;
[0026] (a9) producing protease;
[0027] (a10) preparing a product for producing protease;
[0028] (a11) producing ACC deaminase;
[0029] (a12) preparing a product for producing ACC deaminase.
[0030] In the above application, the plant growth promotion can be all or part of: the Bacillus pumilus ZF526 strain fermentation liquid is used to treat cucumber seedlings, and in normal soil, the leaf area, plant height, stem diameter, fresh weight and dry weight of cucumber are promoted by 26.96%, 13.65%, 5.54%, 12.80% and 22.74% respectively; in saline soil, the leaf area, plant height, stem diameter, fresh weight and dry weight of cucumber are promoted by 11.21%, 28.86%, 12.75%, 29.27% and 31.92% respectively.
[0031] The application also provides the use of the above-mentioned Bacillus pumilus in the preparation of a product or a soil conditioner for relieving salt stress of plants.
[0032] The salt component in the salt stress can be NaCl, Na2SO4, KNO3, MgCl2, NaHCO3 and Na2CO3.
[0033] The application also provides a preparation method of the above-mentioned bacterial agent, comprising the following steps: taking the Bacillus pumilus in claim 1 as an active ingredient to obtain the bacterial agent.
[0034] The application also provides a bio-organic fertilizer, characterized in that the bio-organic fertilizer contains the above-mentioned Bacillus pumilus or the above-mentioned bacterial agent or the above-mentioned culture.
[0035] The application also provides a method for relieving salt stress of plants, comprising the following steps: under high salt conditions, applying the above-mentioned Bacillus pumilus or the above-mentioned bacterial agent or the above-mentioned culture to a test plant, so as to relieve the salt stress of the plant.
[0036] In the above application or method, the plant is a dicotyledonous plant.
[0037] In the above application or method, the dicotyledonous plant is a Cucurbitaceae plant.
[0038] Further, the Cucurbitaceae plant is a cucumber.
[0039] Compared with other salt-tolerant strains, the Bacillus pumilus provided in the present application has a wide salt tolerance range (most other strains only tolerate NaCl or a single other salt), and can adapt to different salinization soils caused by different salt contents.
[0040] In this experiment, rhizosphere bacteria were isolated from different areas of salinized soil, and salt-tolerant and growth-promoting strains were mainly screened out. The strain Bacillus pumilus ZF526 which can effectively alleviate the salt stress of cucumber was screened out by repeated screening method of living pot culture. Its taxonomic status was determined through morphological characteristics, Biolog determination and sequence analysis of multiple genes. At the same time, the effect of salt-tolerant strain on alleviating salt stress was determined, in order to provide technical reference for the improvement of secondary salinization.
[0041] DEPOSIT ACCOUNT
[0042] Bacillus pumilus
[0043] Bacillus pumilus
[0044] ZF526
[0045] China General Microbiological Culture Collection Center
[0046] CGMCC
[0047] No. 3, Beichen West Road, Beijing, China
[0048] 2024.12.06
[0049] CGMCC No. 32942 BRIEF DESCRIPTION OF DRAWINGS
[0050] Figure 1 The growth of ZF534 and ZF526 after treatment was observed under salt water. 1 is distilled water treatment; 2 is distilled water and ZF534 bacterial suspension treatment; 3 is distilled water and ZF526 treatment; 4 is 200 mmol / L salt water treatment; 5 is salt water and ZF534 bacterial suspension treatment; 6 is salt water and ZF526 bacterial suspension treatment.
[0051] Figure 2 The effects of ZF534 and ZF526 on physiological indicators of cucumber under salt stress. (a) is the effect of strain treatment on cucumber leaf area; (b) is the effect of strain on cucumber plant height; (c) is the effect of strain treatment on cucumber stem diameter; (d) is the effect of strain treatment on cucumber fresh weight; (e) is the effect of strain treatment on cucumber dry weight.
[0052] Figure 3 The morphological characteristics of strain ZF526 were observed. A is the plate growth of strain ZF526; B is the single colony growth of ZF526 strain.
[0053] Figure 4 The phylogenetic tree of strain ZF526 was constructed.
[0054] Figure 5 The effects of various treatments on salt stress of cucumber. Wherein a is distilled water control; b is salt water control; c is ZF526 strain treatment; d is saline-alkali land improvement biological bacteria treatment; e is soil conditioner treatment; f is saline-alkali land microbial inoculant treatment; g is saline-alkali land special inoculant treatment; h is salt reduction and growth promoting agent treatment.
[0055] Figure 6 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to NaCl.
[0056] Figure 7 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to KNO3.
[0057] Figure 8 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to Na2SO4.
[0058] Figure 9 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to MgCl2.
[0059] Figure 10 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to NaHCO3.
[0060] Figure 11 The growth curve analysis of salt-tolerant bacteria ZF526 resistant to Na2CO3.
[0061] Figure 12 The growth-promoting property detection of strain ZF526. Wherein A is ACC deaminase production detection; B is phosphorus solubilization function detection; C is nitrogen fixation function detection; D is potassium solubilization function detection; E is protease production detection. DETAILED DESCRIPTION
[0062] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.
[0063] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.
[0064] The quantitative tests in the following examples, unless otherwise specified, are set up with three repeated experiments, and the results are averaged.
[0065] The test soil samples in the following examples were collected from 30 samples of rhizosphere soil of plants growing vigorously in salinization areas in Tongliao City, Inner Mongolia, Pingluo County, Ningxia, Tibet Autonomous Region, Dongying City, Shandong, and Hohhot, Inner Mongolia.
[0066] The test variety in the following examples was Zhongnong No. 6 (Zhongshu Seed Industry Technology Co., Ltd.).
[0067] The test medium in the following examples was prepared as follows:
[0068] 1) LB medium: tryptone 10 g, yeast extract 5 g, NaCl 10 g, distilled water 1000 mL, 20 g agar powder, pH 7.0
[0069] 2) PD, PDA medium: peeled potato 200 g, glucose 20 g, distilled water 1000 mL, 20 g agar powder, pH 7.0
[0070] 3) PVK medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium sulfate 5 g / L, agar 15 g / L, pH range 7-7.5.
[0071] 4) Organophosphorus-degrading medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium sulfate 5 g / L, lecithin 0.2 g / L, agar 15 g / L, adjust pH range 7-7.5.
[0072] 5) Ashby medium (purchased from Solarbio): potassium hydrogen sulfate 0.2 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium sulfate dihydrate 0.1 g / L, sodium chloride 0.2 g / L, calcium carbonate 5.0 g / L, mannitol 10 g / L, agar 18 g / L, pH adjusted to 7.0.
[0073] 6) Protease detection medium: tryptone 5 g, yeast extract 3 g, glucose 1 g, distilled water 1000 mL, 15 g agar powder, pH 7.0, 121°C high pressure sterilization for 30 min, 10% 60°C preheated skimmed milk was added when pouring the plate.
[0074] 7) Potassium-degrading medium: sucrose 5 g, diammonium sulfate 0.5 g, yeast extract 0.5 g, magnesium sulfate heptahydrate 0.3 g, disodium hydrogen phosphate 2 g, ferric sulfate heptahydrate 0.03 g, manganese sulfate 0.03 g, potassium feldspar 2 g, agarose 15 g.
[0075] 8) Selective medium: Tryptone 10 g, yeast extract 5 g, NaCl 100 g, distilled water 1000 mL, pH 7.0
[0076] 9) Nitrogen-free medium: sucrose 10 g, NaCl 0.12 g, K2HPO4·3H2O 0.5 g, CaCO3 1 g, MgSO4·7H2O 0.2 g, distilled water 1000 mL, pH 7.2.
[0077] 10) DF medium: KH2PO4 4.0 g, Na2HPO4 6.0 g, MgSO4·7H2O 0.2 g, glucose 2.0 g, sodium gluconate 2.0 g, citric acid 2.0 g, (NH4)2SO4 2.0 g, component one and component two solution each 0.1 mL, H2O 1000 mL, pH 7.2; wherein component one: H3BO3 10 mg, MnSO4·H2O 11.19 mg, ZnSO4·7H2O 124.6 mg, CuSO4·5H2O 78.22 mg, MoO3 10 mg, dissolved in 100 mL of sterilized distilled water; component two: FeSO4·7H2O 100 mg dissolved in 10 mL of sterilized distilled water.
[0078] 11) ADF medium: ACC is dissolved in ultrapure water, filtered and sterilized, and added to sterilized DF medium without (NH4)2SO4, with a final concentration of 3.0 mmol·L -1 .
[0079] 12) Other salt-containing medium: based on LB medium, adjust the salt content with NaCl, Na2SO4, KNO3, MgCl2, NaHCO3 and Na2CO3.
[0080] The following examples use Excel 2021 for data collation, statistics and analysis, SPSS 24.0 for difference significance test, and MEGA 11 and other software for multi-gene phylogenetic tree construction. Each test is set up with 3 replicates.
[0081] Example 1, isolation and preservation of bacillus in soil
[0082] 1. Isolation and screening of bacillus in soil
[0083] According to the five-point sampling method, 30 strains of different shapes, colors and sizes were isolated from the rhizosphere soil of 30 plants growing vigorously in the salinization areas of Tongliao City, Inner Mongolia, Pingluo County, Ningxia, Tibet Autonomous Region, Dongying City, Shandong and Hohhot, Inner Mongolia.
[0084] Take 5 g of soil sample into a flask containing sterile water (45 mL / 100 mL), shake culture at 180 r / min, 28℃ for 0.5 h, and then treat in a 80℃ water bath for 15 min to obtain soil suspension. Dilute according to a 10-fold gradient, select 10 -3 , 10 -4 , 10 -5 , 10 -6 four concentration gradients, and then culture on LB solid medium. Set 3 repeats for each concentration, place the coated plates in a 28℃ bacterial incubator for 2 d, pick single colonies with inconsistent morphology for purification and reculture, a total of 359 strains, of which 59 strains can grow in 10% NaCl LB medium. Number the single colonies and store in a -40℃ refrigerator using 40% glycerol at a ratio of 1:1 with the bacterial solution.
[0085] 2. Culture of test Bacillus sp. and preparation of selection medium
[0086] Culture of Bacillus sp. and preparation of bacterial suspension: the Bacillus sp. isolated from soil is activated using LB solid medium, cultured in a 28℃ bacterial incubator for 2 d, and single colonies are picked using a sterile toothpick in a sterile environment and placed in a flask containing 200 mL of LB liquid medium for culture for 36 h, with a shaking bed condition of 28℃ and a rotation speed of 180 r / min. Dilute with sterile water to 1×10 8 CFU / mL bacterial suspension.
[0087] Preparation of selection medium: prepare 10% NaCl LB liquid medium (10 g of tryptone, 5 g of yeast powder, 100 g of NaCl, 1000 mL of distilled water, pH 7.0), high-pressure steam sterilization, and store at 4℃ for standby.
[0088] 3. Screening of salt-tolerant Bacillus sp.
[0089] Screen high-salt-tolerant strains using the method of culturing in the selection medium. Add 10 μL of bacterial suspension (1×10 8 CFU / mL) to a flask containing 3 mL of 10% NaCl LB liquid medium and shake culture, use only 3 mL of 10% NaCl LB liquid medium as a control, observe whether the medium is turbid after 24 h, and measure the OD 600 value of the medium, with 3 repeats for each treatment. Then store the strains with obvious turbidity and high OD 600 values, purify by streaking on LB medium for 5 times to obtain pure culture strains, culture again in 10% NaCl LB medium to obtain genetically stable salt-tolerant strains, and store at -80℃ using 25% glycerol.
[0090] 4. Screening of salt-tolerant growth-promoting strains
[0091] Primary screening: the salt-tolerant Bacillus strains obtained from the screening were activated on LB medium, and then 1 x 10 8 CFU / L bacterial suspension was prepared for use. Then, cucumber seedlings with consistent growth (two leaves and one new) were transplanted into 12-hole plug trays, and then the bacterial suspension was diluted 100 times, 300 ml of which was irrigated into the 12-hole plug trays, which were placed in a constant-temperature light incubator for culture, with 3 repeats for each treatment and 6 repeats in total. Sterile water was irrigated as a control. After 3 days, 200 mmol / L salt water was quantitatively irrigated into each group at 300 ml, with only sterile water irrigation as a healthy control and only salt water irrigation as a salt water control, with 3 repeats for each control. After the cucumber grew to 2-3 true leaves, the leaf area and plant height were investigated.
[0092] Secondary screening: cucumber seedlings with consistent growth (two leaves and one new) were transplanted into 12-hole plug trays, which were then divided into 4 groups, A: only irrigated with clean water, B: irrigated with the bacterial suspension diluted 100 times, C: only irrigated with 200 mmol / L salt water, and D: irrigated with the bacterial suspension diluted with salt water, with 3 repeats for each group, which were placed in a constant-temperature light incubator for culture. When the cucumber grew to 2-3 true leaves, the leaf area, stem diameter, plant height, and fresh and dry weights of the aboveground part were investigated and measured.
[0093] Through pot screening, 2 strains (ZF524 and ZF526) were screened from the 59 salt-tolerant bacteria, which could significantly alleviate the growth conditions of cucumber under salt stress. Figure 1 ).
[0094] ZF524 promoted the leaf area and plant height of cucumber by 17.52% and 19.23% respectively under normal conditions, and promoted the stem diameter, fresh weight, and dry weight of cucumber by 15.46%, 17.07%, and 17.96% respectively under salt stress. Among them, ZF526 had the most obvious alleviating effect on salt damage to cucumber, and promoted the leaf area, plant height, stem diameter, fresh weight, and dry weight of cucumber.
[0095] Under normal conditions, ZF526 promoted the leaf area, plant height, stem diameter, fresh weight, and dry weight by 26.96%, 13.65%, 5.54%, 12.80%, and 22.74% respectively, and under salt stress, it promoted the leaf area, plant height, stem diameter, fresh weight, and dry weight by 11.21%, 28.86%, 12.75%, 29.27%, and 31.92% respectively, which formed a significant difference Figure 2 ).
[0096] The above results show that inoculation with ZF526 can better improve the leaf area, plant height, stem diameter, fresh weight, and dry weight of cucumber, and inoculation with ZF526 has a better effect in alleviating salt stress than inoculation with ZF524.
[0097] Example 2, Identification and preservation of salt-tolerant strain ZF526
[0098] 1. Morphological and molecular biological identification of salt-tolerant strain ZF526
[0099] Morphological characteristics identification of strain ZF526: The salt-tolerant growth-promoting strain ZF526 was cultured on LB solid medium, and the colony morphology, color state, transparency, etc. were observed after 36h of culture.
[0100] The results are as follows: ZF526 grew well on LB medium, with a milky yellow color, opaque, smooth and moist colony surface, central elevation, irregular colony edge. Figure 3 ).
[0101] Physiological and biochemical characteristics determination of strain ZF526: The physiological and biochemical characteristics of strain ZF526 were determined according to the Eighth Edition of Bergey's Manual of Determinative Bacteriology
[19] and the Manual of Systematic Identification of Common Bacteria
[20] , and the determination of unique carbon source utilization of strain ZF526 was performed by China Agricultural Microbial Culture Collection Center using BIOLOG GEN III kit.
[0102] Physiological and biochemical determination: Strain ZF526 grew well at 28-37℃, had strong salt tolerance, and could grow normally under the condition of 10% NaCl, and could utilize glycerol, D-trehalose, sucrose, D-mannose, D-mannitol, etc. Its physiological and biochemical characteristics were consistent with the previously reported standard B. pumilus strain (Table 1).
[0103] Table 1. Physiological and biochemical identification of strain ZF526 by BIOLOG GEN III reagent strip
[0104]
[0105]
[0106]
[0107] Multi-gene amplification and sequence analysis of strain ZF526: The genomic DNA of strain ZF526 was extracted using bacterial genomic DNA extraction kit (Beijing Tiangeng Biochemical Technology Co., Ltd.), and 16S rRNA and gyrA, gyrB, rpoB gene sequences were used as targets for amplification (Table 2). The PCR reaction system was 50μL: 2×Taq PCR Mix 25μL, 10μL mol·L -11 μL of forward and reverse primers, 1 μL of genomic DNA, and ddH2O was added to make up to 50 μL. The reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 50 °C annealing for 30 s, 72 °C extension for 1 min, 34 cycles; 72 °C extension for 10 min. The reaction products were sent to Beijing Bomaide Biotechnology Co., Ltd. for sequencing. The 16S rRNA and gyrA, gyrB, rpoB gene sequences of strain ZF516 and the strains in the NCBI database were compared, and the sequencing results were analyzed using MEGA11, Sequence Matrix, Seaview4 and other software to construct a phylogenetic tree and determine the taxonomic status of strain ZF526.
[0108] Table 2. Primer information for constructing the multi-gene phylogenetic tree of strain ZF526
[0109]
[0110] After PCR amplification, the 16S rDNA gene sequence of strain ZF526 (nucleotide sequence is SEQ ID No: 1 in the sequence listing), the gyrA gene sequence (nucleotide sequence is SEQ ID No: 2 in the sequence listing), the gyrB gene sequence (nucleotide sequence is SEQ ID No: 3 in the sequence listing), and the rpoB gene sequence (nucleotide sequence is SEQ ID No: 4 in the sequence listing) were obtained by sequencing. In the GenBank database, strains with high similarity and Bacillus were downloaded, and a multi-gene phylogenetic tree of ZF526 was constructed using the neighbor-joining method. The strain was clustered with the model strain Bacillus pumilus DSM 27, so it was preliminarily determined to be Bacillus pumilus. Therefore, combined with the morphological observation, physiological and biochemical determination results, Biolog determination results, and multi-gene phylogenetic tree results of strain ZF526, it was determined that strain ZF526 belongs to Bacillus pumilus. Figure 4
[0111] 3. Preservation of Bacillus pumilus strain ZF526
[0112] Bacillus pumilus ZF526 was preserved in the China General Microbiological Culture Collection Center on December 6, 2024, and the preservation number is CGMCC No. 32942. Hereinafter, it is referred to as Bacillus pumilus CGMCC No. 32942 or strain ZF526.
[0113] Example 3. Comparison of the effects of strain ZF526 and commercial microbial inoculants on alleviating salt stress
[0114] Five commercial products, four microbial inoculants and one chemical preparation were selected to compare the alleviating effects of strain ZF526 and the five preparations on salt stress of cucumber. Two-leaf-one-heart cucumber seedlings were irrigated with 200 mmol / L NaCl salt water, and then ZF526 bacterial suspension and diluted commercial preparations were irrigated after 24 h. The concentration of strain ZF526 was 10 8 cfu / mL, and the specific treatments of commercial preparations were shown in Table 3. Subsequently, the cucumber seedlings were continuously irrigated with salt water.
[0115] Table 3, Treatment of ZF526 and commercial preparations
[0116]
[0117] The experiment was set up with three replicates, each with 24 cucumber seedlings. Desalinization and growth promoting agent was selected as the chemical control, and only water was irrigated as the water control. The cucumber seedlings were irrigated with only salt water as the salt water control. The salt water control was observed every day, and the salt injury index and salt stress alleviating effect were calculated after the salt injury occurred.
[0118] Table 4, Salt injury index and alleviating effect of cucumber of each treatment
[0119]
[0120]
[0121] The salt injury index of cucumber of each treatment was investigated after 15 days of application (Table 4), and the results were as follows: the alleviating effect of strain ZF526 on salt stress of cucumber was the best, reaching 35.37%, and the alleviating effect of desalinization and growth promoting agent, the control preparation, on salt stress of cucumber reached 28.05%. The effect of strain ZF526 was significantly higher than that of commercial microbial inoculants. Among the four selected commercial preparations, the alleviating effect of saline-alkali land improvement biological bacteria on salt stress of cucumber reached 24.39%, which was significantly higher than that of other microbial commercial preparations. Figure 5
[0122] Example 4, Tolerance range of salt-tolerant growth promoting bacteria ZF526 to different salt components
[0123] The common salt components (NaCl, Na2SO4, KNO3, MgCl2, NaHCO3 and Na2CO3) causing secondary salinization were selected to be added to the LB medium, and different concentrations of salt-containing medium were prepared in the LB medium. Then 200 μL of each was taken and added to a 96-well plate, and 2 μL of bacterial suspension was added. The plates were placed in an Agilent biotek log phase 600 full-automatic microbial monitor, and the rotation speed was set to 600 rpm. Three parallel holes were set for each treatment, and the culture was performed for 72 h. The growth curves of ZF526 in different salt components were determined with LB liquid medium as the CK.
[0124] The results show that when the salt component is NaCl, more than 10% of the salt concentration will delay the growth of the strain, and the lag time will increase with the increase of the concentration. The lag time is shortest at 10% and longest at 16%, with 8 h and 24 h, respectively. At 48 h, the growth of the bacteria at different salt concentrations enters the stationary phase in turn. Figure 6 Therefore, ZF526 can still tolerate 16% NaCl and grow normally.
[0125] When the salt component is KNO3, the growth of ZF526 at each gradient starts to increase from 8 h, and all gradients enter the stationary phase at 32 h. The growth of the strain is almost unaffected below 14% concentration. The growth of the strain decreases at 32 h and tends to be stable at 16% and 18% concentrations. Figure 7 ZF526 can still tolerate 20% KNO3.
[0126] When the salt component is Na2SO4, the growth of ZF526 at each gradient starts to increase from 8 h, and the growth of the strain is basically not affected below 8% concentration, and the growth curve is similar to that of CK. The growth rates of the strains at 10% and 12% concentrations are different, and the time lag to enter the stationary phase is delayed. Figure 8 ZF526 is in the stationary phase at 64 h at 14% and 16% concentrations, so the strain has a certain tolerance to 16% Na2SO4.
[0127] When the salt component is MgCl2, the growth of ZF526 at each gradient starts to increase from 8 h, and the growth of the strain is basically not affected below 8% concentration, and the growth curve is similar to that of CK. The growth rates of the strains at 10% and 12% concentrations are different, and the time lag to enter the stationary phase is delayed. 600 The OD values at 8% and 10% concentrations remain at a low level. Figure 9 Therefore, ZF526 has a certain tolerance to 6% MgCl2.
[0128] When the salt is NaHCO3, the growth of ZF526 shows an upward trend at each gradient strain from 8h, and 4% and 6% enter the stable period at 32h, 8% enters the stable period at 56h, and the growth rate decreases at 10% and 12% concentrations, and the OD 600 value remains at a low level. Figure 10 Therefore, it is shown that the strain is resistant to NaHCO3 within 8% and can grow normally.
[0129] When the salt is Na2CO3, the growth curve of ZF526 at a concentration of 2% is basically consistent with CK, and both show an upward trend at 4h and enter the stable period at 28h. The strain growth is delayed at 4%, starts to rise at 16h, and enters the stable period at 56h Figure 11 . At a concentration of 6%, there is no obvious change in OD 600 value, so the strain is resistant to 4% Na2CO3.
[0130] Example 5, Analysis of the growth-promoting effect of Bacillus pumilus ZF526
[0131] 1. ACC deaminase production determination
[0132] According to the method of Penrose et al., the isolated strain ZF526 is inoculated into 3mL liquid nitrogen-free medium, and cultured at 30°C, 200r / min for 24h; 0.1mL of the above culture is inoculated into 3mL DF medium and shaken for 24h; 0.1mL of the above culture is inoculated into 3mL ADF medium and shaken for 24-48h; the strain grown in ADF is repeatedly transferred and cultured, and ADF medium is used as a negative control. The strain that can grow with ACC as the only nitrogen source is an ACC deaminase positive strain.
[0133] ADF medium is used as a negative control Figure 12 (A1), strain ZF526 can grow in ADF medium Figure 12 (A2), and can also grow after repeated transfer Figure 12 (A3), indicating that strain ZF526 can grow with ACC as the only nitrogen source, proving that the strain produces ACC deaminase.
[0134] 2. Determination of phosphorus solubilization ability
[0135] 5μL of ZF526 bacterial suspension is uniformly added to 3 points on the inorganic phosphorus detection medium (PVK) plate, 3 replicates are set for each strain, and cultured in a 28°C incubator for 48h. Whether a hydrolysis ring is produced is observed. The colony diameter (d) of strain ZF526 and the size of the hydrolysis ring diameter (D) produced are determined, and the ratio (D / d) is calculated. The size of the ratio is used to roughly determine the size of the strain's phosphoric acid degradation ability.
[0136] Strain ZF526 can produce obvious transparent hydrolysis circle on inorganic phosphorus plate (PVK) medium Figure 12 The ratio of the diameter of degradation circle to the diameter of colony is 1.75, indicating that the strain has the ability to dissolve phosphorus.
[0137] 3. Determination of nitrogen fixation ability
[0138] 5 μL of ZF526 bacterial suspension was uniformly dropped on Ashby medium plate in 3 points, 3 repeats were set for each bacteria, and it was placed in a 28°C incubator for 48h. Whether the strain could survive on the medium was observed.
[0139] Strain ZF526 can grow on Ashby medium and form white colonies Figure 12 The ratio of the diameter of degradation circle to the diameter of colony is 1.75, indicating that the strain has the ability to dissolve phosphorus.
[0140] 4. Determination of potassium solubilizing ability
[0141] 5 μL of bacterial suspension of the screened strain was inoculated on potassium solubilizing detection medium, and incubated at 28°C for 48h. After 3 repeated experiments, if colonies can grow on the plate and the edge is smooth and transparent oil droplet, it is proved that the strain has the ability to solubilize potassium.
[0142] Strain ZF526 grows rapidly on potassium solubilizing medium, and the edge is smooth and oil droplet Figure 12 The ratio of the diameter of degradation circle to the diameter of colony is 1.75, indicating that the strain has the ability to dissolve phosphorus.
[0143] 5. Determination of protease
[0144] 5 μL of ZF526 bacterial suspension was uniformly dropped on protease detection medium in 3 points, 3 repeats were set for each bacteria, and it was placed in a 28°C incubator for 48h. Whether transparent circle was produced was observed. The D / d value was used to judge the strength of the strain producing protease.
[0145] Strain ZF526 grows on protease detection medium and produces obvious transparent circle Figure 12 The ratio of the diameter of degradation circle to the diameter of colony is 3.83, indicating that strain ZF526 has the ability to produce protease.
[0146] In this study, 359 strains of Bacillus and 59 strains of salt-tolerant bacteria were isolated and purified from saline soils all over the country. The 59 strains were subjected to pot experiment and growth promoting test. After various functional tests and comprehensive consideration, Bacillus pumilus ZF526 was finally determined as the best strain.
[0147] When screening the strains, the salt components are added for screening, the solid culture medium is used for dilution coating at the beginning, the salt concentration is screened, and no positive result is obtained. Finally, the liquid culture medium is used for screening, and the content of 10% NaCl is added. When screening the strains, the salt stress screening is carried out by means of tissue culture, water culture and other methods for simulating salt damage treatment, and no positive result is obtained. Finally, the method of irrigating salt water is determined for simulating salt damage.
[0148] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application.
Claims
1. Bacillus pumilus, characterized in that: The Bacillus pumilus is Bacillus pumilus ZF526, and its deposit number in the General Microbiology Center of China Culture Collection Administration is CGMCC No. 32942.
2. A bacterial agent, characterized in that: The bacterial agent contains the Bacillus pumilus according to claim 1 and / or a metabolite of the Bacillus pumilus.
3. The culture product of Bacillus pumilus according to claim 1, which is obtained by culturing the Bacillus pumilus according to claim 1 in a bacterial culture medium.
4. Use of the Bacillus pumilus according to claim 1, the bacterial agent according to claim 2, or the culture according to claim 3 in at least one of the following: a1) promoting plant growth; a2) preparing products that promote plant growth; a3) Phosphate dissolution; a4) preparing a phosphate-dissolved product; a5) potassium solution; a6) preparing a potassium-solubilizing product; a7) Nitrogen fixation capacity; a8) preparing products capable of nitrogen fixation; a9) Protease production; a10) preparing a protease-producing product; a11) produces ACC deaminase; a12) preparing a product for producing ACC deaminase.
5. Use of the Bacillus pumilus according to claim 1, the bacterial agent according to claim 2, or the culture according to claim 3 in preparing a product for alleviating plant salt stress or a soil conditioner.
6. A method for preparing the microbial agent according to claim 2, comprising the following steps: using the Bacillus pumilus according to claim 1 as an active ingredient to obtain the microbial agent.
7. Bio-organic fertilizer, characterized by: The biological organic fertilizer contains the Bacillus pumilus according to claim 1 or the bacterial agent according to claim 2 or the culture according to claim 3.
8. A method for alleviating plant salt stress, comprising the steps of: applying the Bacillus pumilus of claim 1, the bacterial agent of claim 2, or the culture of claim 3 to a test plant under high salt conditions, thereby alleviating the plant salt stress.
9. The use according to any one of claims 4-5 or the method according to claim 8, characterized in that: The plant is a dicotyledonous plant.
10. The use or method according to claim 9, characterized in that: The dicotyledonous plant is cucumber.