Bacillus CAU-D1, biological agent and application

Abstract

The invention discloses a strain of Bacillus CAU-D1, a biological microbial agent and an application thereof, comprising Bacillus CAU-D1, wherein the colony color of the Bacillus CAU-D1 on an LB culture medium is white with yellowish color, is opaque, round or nearly round, has an unsmooth edge and has obvious wrinkles on the surface; the physiological and biochemical characteristics of the Bacillus CAU-D1 are Gram-positive, have the functions of nitrogen fixation, inorganic phosphorus dissolution and potassium dissolution, and have no chemotaxis; the bacterial content of the Bacillus CAU-D1 in the biological microbial agent is 1×10 8 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 structure improvement under pot test conditions, thereby providing high-quality resources for Bacillus resource research and the 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. Incubate it in a 37°C constant temperature shaker at 180 r / min for 8 h. Take a sample under sterile conditions and read the OD value of the culture solution using 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 product was purified and recovered by agarose gel electrophoresis and sent to Beijing Qingke Biological 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 sequence with high homology was selected as the reference object, and the phylogenetic tree was constructed using MEGA7.0 using the neighbor-joining analysis method. Among them, the bootstrap value was set to 1000, and the rest were the default values. Among them, the nucleotide sequence of CAU-D1 strain 16S is as follows: ATTCGGGCCAGCACCGC

[0036] GGTAATCGTAGGTGCAAGCGTGTCCGGAATTATGGGCGTAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTCACAGAGAT。

[0037] Example 2

[0038] Functional identification of CAU-D1 strain

[0039] (1) Identification of nitrogen fixation function of CAU-D1 strain

[0040] Nitrogen-free culture medium (1 L): KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, CaCO3 5.0 g, glucose 10.0 g, agar 20 g, pH 7.0.

[0041] 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.

[0042] (2) Determination of phosphorus dissolving function (organic phosphorus and inorganic phosphorus)

[0043] Organophosphorus culture medium (1L): glucose 10.0g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, KCl 0.3g, MnSO4·7H2O 0.03g, CaCO3 5.0g, lecithin 1.0g (lecithin should be dissolved in 75% ethanol before use), agar 20g, pH 7.2-7.4.

[0044] Inorganic phosphorus culture medium (1L): glucose 10g, (NH4)2SO40.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O0.1g, MnSO4·H2O 0.03g, FeSO430mg, Ca3(PO4)25g, yeast powder 0.5g, agar 20g, pH7.0.

[0045] 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.

[0046] The experimental method for inorganic phosphorus solubilization is as follows: the bacterial strain is inoculated with an inoculation loop on the inorganic phosphorus culture medium, and the culture is placed in a constant temperature incubator at 28°C for 4 days and then observed. 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.

[0047] (3) Determination of potassium-dissolving function

[0048] Potassium-dissolving medium (1L): glucose 10g, potassium feldspar 2.5g, Na2HPO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaCO3 5g, CaSO4·7H2O 0.1g, agar 20g, pH 7.0.

[0049] The strain was inoculated onto 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.

[0050] (4) Chemotaxis function assay

[0051] TB medium (1 L): peptone 10 g, agar 3 g, NaCl 10 g.

[0052] 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.

[0053] Example 3

[0054] The actual growth-promoting effect of CAU-D1 on soybean

[0055] Select a batch of plump 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] Table 1 CAU-D1 growth-promoting and yield-increasing indexes of soybean

[0060]

[0061] Table 2 CAU-D1 improves soybean root architecture

[0062]

[0063] 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.

[0064] 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.

[0065] 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 strain of Bacillus piscis CAU-D1, characterized in that: The colonies of the Bacillus CAU-D1 on the 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; 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.

2. 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, the strain collection number is CGMCC NO. 30738.

3. 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, and the strain collection number is CGMCC NO. 30738.

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