Bacillus CAU-D1, biological agent and application

Abstract

The invention discloses a bacillus CAU-D1, a biological agent and application. The bacillus CAU-D1 comprises the bacillus CAU-D1, and a bacterial colony of the bacillus CAU-D1 on an LB culture medium is yellow in white, opaque, round or nearly round, unsmooth in edge and obvious in surface wrinkles; the physiological and biochemical characteristics of the bacillus CAU-D1 are gram staining positive, and the bacillus CAU-D1 has the functions of fixing nitrogen and dissolving inorganic phosphorus and potassium and has no chemotaxis; the bacterium content of the bacillus CAU-D1 in the biological agent is 1 * 10 < 8 > cfu / ml. The bacillus CAU-D1 provided by the invention has the effects of fixing nitrogen, dissolving phosphorus and dissolving potassium on a flat plate, and has an obvious promoting effect on soybean growth and root configuration improvement under a pot experiment condition, so that a high-quality resource is provided for bacillus resource research and development and application of adaptive biocontrol strains.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a strain of Bacillus CAU-D1, a biological bacterial agent and application. Background Art

[0002] As one of the most important food and oil crops for mankind, the demand for soybeans in my country has been increasing with the development of my country's economy and society, but there is currently a shortage of soybeans in my country. Against the backdrop of limited arable land in my country, increasing the yield per unit area is the fundamental way to develop my country's soybean industry. If the problem of nutrient supply in the soybean production process cannot be effectively solved during this period, it will seriously hinder the development of my country's soybean industry.

[0003] At present, more and more studies have shown that crops can improve root health, promote nutrient nutrition and enhance resistance to adversity by inoculating beneficial microorganisms. Compared with the deterioration of soil structure and eutrophication of water bodies caused by the long-term application of traditional chemical fertilizers, microbial fertilizers that use microbial life activities and metabolites to stimulate plant growth are more environmentally friendly. In the context of my country's green agricultural development becoming a national strategy, microbial fertilizers will naturally become the mainstream of future agricultural production.

[0004] Leguminosae is a typical representative of microbe-plant interaction. Its most notable feature is that they have nodules on their roots, which are formed by a type of nitrogen-fixing bacteria that establishes a symbiotic relationship with plants after infection. This type of nitrogen-fixing bacteria is called rhizobia. The symbiotic system of rhizobia and soybeans shapes the unique root structure of soybeans. For soybeans, the biggest role of this type of root structure is to fix nitrogen in the atmosphere through biological nitrogen fixation for the host to use. If this symbiotic nitrogen fixation model is used well, it can not only provide more nitrogen sources for plants. It can be inferred that inoculating beneficial microorganisms to improve soybean root structure through changes in rhizosphere microorganisms will become one of the effective ways to increase soybean production. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a strain of Bacillus CAU-D1, a biological agent and an application. Bacillus CAU-D1 has been proven through potted plant experiments to have an obvious growth-promoting effect on soybean growth, thereby providing high-quality resources for Bacillus resource research and the development and application of adaptive biocontrol strains.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A Bacillus CAU-D1, comprising: Bacillus CAU-D1, wherein the colonies of the Bacillus CAU-D1 on LB medium are white with yellowish color, opaque, round or nearly round, with rough edges and obvious wrinkles on the surface;

[0008] The physiological and biochemical characteristics of the Bacillus CAU-D1 are Gram-positive staining, nitrogen fixation, inorganic phosphorus dissolution and potassium dissolution functions, and no chemotaxis.

[0009] As a further technical solution of the present invention, the Bacillus CAU-D1 is preserved in the General Microbiology Center of China Microorganism Culture Collection Administration, and the address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the strain preservation number is CGMCC NO.30738, the preservation classification name is: Bacillus piscis; the preservation date is: May 23, 2024.

[0010] A biological agent of Bacillus CAU-D1, the biological agent includes Bacillus CAU-D1, and the bacterial content of Bacillus CAU-D1 in the biological agent is 1×10 8 cfu / ml.

[0011] The invention discloses an application of Bacillus CAU-D1, specifically, an application of Bacillus CAU-D1 in promoting soybean growth and nodulation.

[0012] The beneficial effects of the present invention are:

[0013] The Bacillus CAU-D1 provided by the invention has the effects of nitrogen fixation, phosphorus dissolution and potassium dissolution on a plate, and has an obvious promoting effect on soybean growth and root structure improvement under pot test conditions, thereby providing high-quality resources for Bacillus resource research and the development and application of adaptive biocontrol strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the colony morphology of Bacillus CAU-D1;

[0015] Figure 2 To construct a phylogenetic tree for the 16S rRNA of Bacillus CAU-D1;

[0016] Figure 3 The nitrogen fixation function of Bacillus CAU-D1;

[0017] Figure 4 Solubilized inorganic phosphorus of Bacillus CAU-D1;

[0018] Figure 5 It is the potassium-solubilizing function of Bacillus CAU-D1;

[0019] Figure 6 This is the test of the growth-promoting and yield-increasing function of Bacillus CAU-D1 on soybean. The three pots on the left are CK, and the three pots on the right are treated with CAU-D1 bacterial solution.

[0020] Figure 7 This is the effect of Bacillus CAU-D1 on soybean root morphology. DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0022] A Bacillus CAU-D1, comprising: Bacillus CAU-D1 (Bacillus piscis in Latin), wherein the colonies of the Bacillus CAU-D1 on LB medium are white with yellowish color, opaque, round or nearly round, with rough edges and obvious wrinkles on the surface;

[0023] The physiological and biochemical characteristics of Bacillus CAU-D1 are Gram-positive, nitrogen fixation, inorganic phosphorus dissolution and potassium dissolution, and no chemotaxis.

[0024] In a preferred embodiment, the Bacillus CAU-D1 is stored in the General Microbiology Center of China Culture Collection Administration, and the address of the depository is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the strain deposit number is CGMCC NO.30738, the deposit classification name is: Bacillus piscis; the deposit date is: May 23, 2024.

[0025] A biological agent of Bacillus CAU-D1, the biological agent includes Bacillus CAU-D1, and the bacterial content of Bacillus CAU-D1 in the biological agent is 1×10 8 cfu / ml.

[0026] The invention discloses an application of Bacillus CAU-D1, specifically, an application of Bacillus CAU-D1 in promoting soybean growth and nodulation.

[0027] The Bacillus CAU-D1 is stored in the General Microbiology Center of the China Culture Collection Administration, and the address of the collection unit is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the strain collection number is CGMCC NO.30738, the collection classification name is: Bacillus piscis; the collection date is: May 23, 2024.

[0028] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Identification of Bacillus CAU-D1 strain (referred to as CAU-D1):

[0031] The CAU-D1 strain in the laboratory strain library was inoculated onto solid LB medium by three-zone streaking on a plate and cultured in a constant temperature incubator at 28°C for 1-2 days. If colonies of different morphologies appeared on the culture medium, a pure single colony should be picked out and streaked again until a pure CAU-D1 strain was obtained.

[0032] Pick out a single colony and inoculate it into a triangular flask containing liquid LB medium, shake and culture it at 180r / min in a constant temperature shaker at 37℃ for 8h, and take a sample under sterile conditions and read the OD value of the culture solution under a UV spectrophotometer. 600 Value, when OD 600 The value is close to 1.00, that is, the colony concentration is about 1×10 9 The culture was stopped when the concentration of 500 cfu / mL was 0.

[0033] The genomic DNA of antagonistic bacteria was extracted using TaKaRa Mini BEST Bacteria Genomic DNA Extraction Kit Ver.3.0.

[0034] PCR amplification was performed using bacterial 16S rDNA universal primers 27F and 1492R; PCR reaction program: 94℃ pre-denaturation for 5min; 94℃ denaturation for 60s; 53℃ annealing for 60s; 72℃ extension for 2min; 35 cycles; final extension at 72℃ for 7min, stored at 4℃. After the reaction, 5μL of the reaction product was taken, subjected to 1.0% agarose gel electrophoresis, and observed in a gel imaging system.

[0035] The amplified products were purified and recovered by agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rRNA sequencing. The sequencing results were compared with the gene sequences of related species in the NCBI database by BLAST search. The model strain sequences with high homology were selected as reference objects, and the phylogenetic tree was constructed using MEGA7.0 using the neighbor-joining analysis method. The bootstrap value was set to 1000, and the rest were the default values.Among them, the nucleotide sequence of the 16S of strain CAU-D1 is specifically as follows: ATTCGGGCCAGCACCGC GGTAATCGTAGGTGCAAGCGTGTCCGGAATTATGGGCGTAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTCACAGAGAT。

[0036] Example 2

[0037] Functional identification of CAU-D1 strain

[0038] (1) Identification of nitrogen fixation function of CAU-D1 strain

[0039] Nitrogen-free culture medium (1L): KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O0.1g, CaCO3 5.0g, glucose 10.0g, agar 20g, pH 7.0.

[0040] The purified CAU-D1 strain was picked up and placed on a 5 mL nitrogen-free culture medium plate and placed in an incubator at 28°C and 180 r / min. The ability of the strain to grow on the culture medium indicated that it had nitrogen fixation.

[0041] (2) Determination of phosphorus dissolving function (organic phosphorus and inorganic phosphorus)

[0042] Organophosphorus medium (1L): glucose 10.0g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, KCl 0.3g, MnSO4·7H2O0.03g, CaCO3 5.0g, lecithin 1.0g (lecithin was first dissolved in 75% ethanol when used), agar 20g, pH 7.2-7.4.

[0043] Inorganic phosphorus culture medium (1L): glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl0.3g, MgSO4·7H2O0.1g, MnSO4·H2O 0.03g, FeSO4 30mg, Ca3(PO4)25g, yeast powder 0.5g, agar 20g, pH 7.0.

[0044] Using the transparent zone method, the CAU-D1 strain was inoculated on the organophosphate culture medium and placed in a constant temperature incubator at 28°C for 4 days. When a transparent zone appeared at the edge of the bacterial colony on the organophosphate culture medium, the bacterial strain had the ability to degrade organophosphate. Each group of experiments was repeated 3 times.

[0045] Experimental method for inorganic phosphorus solubilization: Inoculate the bacterial strain with an inoculation loop on the inorganic phosphorus culture medium, culture it in a constant temperature incubator at 28°C for 4 days and observe. If bacterial colonies are formed on the inorganic phosphorus culture medium, the strain has the ability to solubilize inorganic phosphorus. Each group of experiments is repeated 3 times.

[0046] (3) Determination of potassium-dissolving function

[0047] Potassium-dissolving medium (1L): glucose 10g, potassium feldspar 2.5g, Na2HPO4 0.2g, MgSO4·7H2O 0.2g, NaCl0.2g, CaCO3 5g, CaSO4·7H2O 0.1g, agar 20g, pH 7.0.

[0048] The strain was inoculated onto a potassium-solubilizing medium and cultured in a constant temperature incubator at 28°C for 3 days. If colonies grew on the surface of the medium, the strain had the ability to solubilize potassium.

[0049] (4) Chemotaxis function assay

[0050] TB medium (1 L): peptone 10 g, agar 3 g, NaCl 10 g.

[0051] Pour the prepared TB culture medium into a 6-well culture plate, add 5 mL of TB culture medium to each well, and after solidification, pick up the CAU-D1 strain with a pipette tip and inoculate it to the center of the culture medium. Incubate it at 28°C for 8 hours. If the strain spreads radially to the surrounding areas, it has chemotaxis function.

[0052] Example 3

[0053] The actual growth-promoting effect of CAU-D1 on soybean

[0054] Select a batch of full and complete soybean seeds, soak them in 3% sodium hypochlorite solution in a tissue culture bottle for 2 minutes, and then rinse them with sterile water 5 times to achieve the disinfection effect. After the disinfection, the seeds are soaked in saturated calcium sulfate solution for 6 hours under ventilation conditions, placed on a sterilized tray covered with moist gauze, and germinated in a light-shading environment for 2 days. Soybeans with a root length of about 2 cm are selected for sowing.

[0055] The CAU-D1 strain in the laboratory strain library was inoculated onto solid LB medium by streaking, and cultured in a constant temperature incubator at 28°C for 1-2 days. After the colonies grew, the colony morphology was observed, and pure single colonies were picked out and streaked again until the morphology of all colonies on the solid LB medium was consistent.

[0056] The activated CAU-D1 strain was inoculated into a test tube containing 5 ml of liquid LB medium and cultured in a constant temperature shaker at 28°C at 180 r / min for 16 h to obtain a concentration of about 1×10 8 cfu / mL of bacterial suspension.

[0057] Potted soybeans were placed in the greenhouse of the West Campus of China Agricultural University, with 3 replicates for each treatment. Four seeds were planted in each pot during sowing, and thinning was carried out 5 days after emergence, with 2 seedlings of similar growth retained in each pot. After thinning, 5 ml of bacterial solution was poured on the roots of soybeans, and the inoculated strain number was marked on the pots. Samples were collected after 50 days of growth, and the above-ground and underground parts of the plants were collected separately to determine the corresponding indicators, and the average value of two plants in each pot was taken for each indicator.

[0058] Table 1 CAU-D1 growth-promoting and yield-increasing indexes of soybean

[0059] Plant height(cm) Stem and leaf dry weight (g) Pod dry weight (g) Dry weight of beans (g) CK 36.8±2.25 3.08±0.4 2.36±0.27 1.4±0.2 CAU-D1 49.08±6.27 4.08±0.16 2.88±0.12 1.66±0.1

[0060] Table 2 CAU-D1 improves soybean root architecture

[0061]

[0062] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the Bacillus CAU-D1 provided by the present invention has the effects of nitrogen fixation, phosphorus solubilization and potassium solubilization on the plate, and has a significant promoting effect on soybean growth and root architecture improvement under pot test conditions, thereby providing high-quality resources for Bacillus resource research and the development and application of adaptive biocontrol strains.

[0063] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0064] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus strain CAU-D1, characterized in that include: Bacillus CAU-D1, wherein the colonies of the Bacillus CAU-D1 on LB medium are white with a yellowish tint, opaque, round or nearly round, with rough edges and obvious wrinkles on the surface; The physiological and biochemical characteristics of the Bacillus CAU-D1 are Gram-positive staining, nitrogen fixation, inorganic phosphorus dissolution and potassium dissolution functions, and no chemotaxis.

2. The Bacillus CAU-D1 according to claim 1, characterized in that The Bacillus CAU-D1 is stored in the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, and the culture collection number is CGMCC NO.30738.

3. A biological agent of Bacillus CAU-D1, characterized in that: The biological agent includes Bacillus CAU-D1, and the bacterial content of Bacillus CAU-D1 in the biological agent is 1×10 8 cfu / ml.

4. The use of Bacillus CAU-D1 according to claim 1, characterized in that: Specifically, it is the application of Bacillus CAU-D1 in promoting soybean growth and nodulation.

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