Microbial composite agent capable of promoting growth and increasing production, and preparation method and application thereof

Abstract

The present invention belongs to the technical field of agricultural microorganisms, and in particular, relates to a cold-resistant Brevibacterium with the function of promoting growth and increasing production, a composite bacterial agent, and a preparation method and application thereof. The cold-resistant Brevibacterium is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCM2023822. Cold-resistant Brevibacterium has the functions of nitrogen fixation, iron carrier production or growth factor production; at the same time, when it is used in combination with Sinorhizobium meliloti Z12 and Vibrio vulgaris Cb1, it has the beneficial effects of promoting alfalfa growth, reducing the application of chemical fertilizers, and increasing the yield and quality of alfalfa. It is an environmentally friendly multifunctional composite bacterial agent with good development prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to a microbial composite agent with growth-promoting and yield-increasing effects, and a preparation method and application thereof. Background Art

[0002] Alfalfa (Medicago sativa L.) is a perennial leguminous plant. It is rich in nutrients such as crude protein and vitamins, has extremely high nutritional value and good palatability. It is mainly used for livestock feeding. It is high-yield, high-quality, and has high economic benefits. It is recognized by the world as the "king of forage" and is one of the forage varieties vigorously promoted in my country. However, due to inconsistent planting times, non-standard planting techniques, and extensive field management, the quality of alfalfa has been uneven in recent years, with a large number of inferior products and serious waste. The yield and quality have dropped significantly, seriously affecting the development of my country's animal husbandry. With the continuous advancement of policies such as "grain to feed" and "revitalizing the dairy industry", the demand for alfalfa in my country's animal husbandry market has greatly increased, and the production problem of alfalfa needs to be solved urgently.

[0003] Microbial agents are a new type of application technology that can give full play to the potential of microorganisms, and have the excellent characteristics of high efficiency, stability and cost saving. At present, they have been widely used in vegetable crops such as cucumbers and tomatoes. Compared with single agents, compound agents have more comprehensive and diverse functions. They contain specific types of microbial cells, which can balance the soil ecology to a certain extent, improve the soil environment, and have safer and more stable field effects. They can promote the growth and development of plants in many ways, are easy to prepare, have no pollution to the human body and the environment, and are not easy to develop drug resistance. After application, it can promote the growth and development of alfalfa, increase alfalfa yield and improve quality, which is of great significance to the development of my country's forage industry.

[0004] The purpose of the present invention is to isolate new strains to prepare an environmentally friendly composite bacterial agent with the effect of promoting growth and increasing production. Summary of the invention

[0005] Based on the above objectives, the present invention screened and obtained non-antagonistic superior strains with the ability to fix nitrogen, dissolve inorganic phosphorus, and secrete plant growth hormone (IAA), and developed a multifunctional composite bacterial agent.

[0006] In a first aspect, the present invention provides a cold-resistant Brevibacterium having the function of promoting growth and increasing production, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC M 2023822.

[0007] The second aspect of the present invention provides a use of the cold-resistant Brevibacterium in nitrogen fixation, siderophore production or elongation production.

[0008] The third aspect of the present invention provides a microbial composite agent for promoting growth and increasing production, which is a compound of the cold-resistant Brevibacterium, Sinorhizobium meliloti Z12, and Phagephagophora cb1.

[0009] Among them, Sinorhizobium meliloti Z12 and Phage Cb1 are both deposited in the China Center for Type Culture Collection, and the deposit number of Sinorhizobium meliloti Z12 is CCTCC M 2023820, and the deposit number of Phage Cb1 is CCTCC M2023821.

[0010] Preferably, the Sinorhizobium meliloti Z12, the phagocytes Cb1 and the cold-resistant Brevibacterium S14 are compounded in equal proportions according to the number of effective live bacteria.

[0011] In a fourth aspect, the present invention provides a method for preparing the microbial composite agent, comprising the following steps:

[0012] S1, inoculating the Chinese rhizobium meliloti Z12, the phagocyte Cb1 and the cold-resistant Brevibacterium into YMA solid culture medium for cultivation and activation respectively;

[0013] S2. The three activated strains were inoculated into TY liquid medium and cultured until the logarithmic growth phase;

[0014] S3. Centrifuge the culture fluid of each strain obtained in S2, collect three kinds of bacteria respectively, mix and suspend them to obtain the microbial composite agent.

[0015] Preferably, the suspension is performed so that the total number of effective viable bacteria in the suspension of the obtained microbial composite bacterial agent is 2×10 8 ~2×10 9 CFU / mL.

[0016] Preferably, in S1, the activation is performed at 28-30° C. for 2-3 days.

[0017] Preferably, in S3, the centrifugation is performed at 4°C and 10,000 rpm for 10 to 15 min.

[0018] The fifth aspect of the present invention provides a use of the microbial composite agent in nitrogen fixation, inorganic phosphorus solubilization and / or growth factor production.

[0019] Preferably, the microbial composite agent is used for promoting the growth and increasing the yield of alfalfa.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The present invention provides a cold-resistant brevis bacillus, which has the functions of nitrogen fixation, siderophore production and growth factor production. At the same time, the cold-resistant brevis bacillus, together with Sinorhizobium meliloti Z12 and Phagephagnum glutinosarum Cb1, can promote the growth of alfalfa, increase alfalfa yield, and achieve the beneficial effects of reducing the application of chemical fertilizers, increasing alfalfa yield and improving quality. The cold-resistant brevis bacillus is an environmentally friendly multifunctional bacterial agent with good development prospects.

[0022] Biomaterial deposit information:

[0023] Brevibacterium frigoritolerans S14, Latin name: Brevibacterium frigoritolerans, was deposited in the China Center for Type Culture Collection on May 25, 2023, with the accession number: CCTCC M 2023822.

[0024] Sinorhizobium meliloti Z12, Latin name: Ensifer meliloti, was deposited in the China Center for Type Culture Collection on May 25, 2023, with the accession number: CCTCC M 2023820.

[0025] Variovorax boronicumulans Cb1, Latin name: Variovorax boronicumulans, was deposited in the China Center for Type Culture Collection on May 25, 2023, with the accession number: CCTCC M 2023821.

[0026] The address of the depository is: Wuhan University, Wuhan BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the phosphate solubilizing capacity of the three strains;

[0028] Figure 2 is the nitrogen-fixing ability of the three strains;

[0029] Figure 3 is the siderophore-producing capacity of the three strains;

[0030] Figure 4 It is the antagonism of the three strains. DETAILED DESCRIPTION

[0031] The present invention is further illustrated by examples. It should be understood that all the examples listed are to help those skilled in the art understand and implement the present invention, and are not any form of any limitation to the present invention. All simple improvements and adjustments based on the present invention, as well as equivalent or equivalent replacement of the raw materials therein, which fail to produce new substantive features and significant progress, should be within the scope of protection of the present invention.

[0032] Example 1

[0033] Isolation, purification and identification of three strains constituting a composite bacterial agent

[0034] 1. Isolation, identification and purification of strain Z12

[0035] The Z12 strain was isolated from the surface-sterilized nodules of Davidia involucrata Baill. using YMA solid medium. The specific operation process was as follows:

[0036] The surface of the freshly collected nodules was disinfected, and they were crushed and spread on YMA solid culture medium with tweezers, and cultured at 28°C for 48h (YMA solid culture medium contained 10.0g mannitol, 3.0g yeast powder, 0.25g K2HPO4, 0.25g KH2PO4, 0.20g MgSO4·7H2O, 0.10g NaCl, 1L distilled water, 15-20g agar, pH was adjusted to 6.8-7.0, and sterilized at 121°C for 30min), single colonies were selected for isolation according to morphological characteristics, and strains were purified by the three-line method, and purification was performed continuously for 3 times. The purified strain was sequenced by 16S rRNA gene (see SEQ ID NO.1) and identified as Ensifer meliloti by EZ Biocloud comparison. The pure culture of the strain was deposited in the China Center for Type Culture Collection on May 25, 2023, with the accession number: CCTCC M 2023820.

[0037] SEQ ID NO.1:

[0038] TACCCGTGGTTAGCTGCCTCCTTGCGGTTAGCGCACTACCTTCGGGTAGAAC

[0039] CAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA

[0040] CCGCAGCATGCTGATCTGCGATTACTAGCGATTCCAACTTCATGCACTCGAG

[0041] TTGCAGAGTGCAATCCGAACTGAGATGGCTTTTGGAGATTAGCTCGACCTC

[0042] GCGGTCTCGCTGCCCACTGTCACCACCATTGTAGCACGTGTGTAGCCCAGC

[0043] CCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCTTATC

[0044] ACCGGCAGTCCCCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGGGCGA

[0045] GGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC

[0046] GACAGCCATGCAGCACCTGTCTCCGATCCAGCCGAACTGAAGGATACGATC

[0047] TCTCGTATCCGCGATCGGGATGTCAAGGGCTGGTAAGGTTCTGCGCGTTGCT

[0048] TCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTT

[0049] GAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGTTTAATGCGTTAGC

[0050] TGCGCCACCGAACAGTAAACTGCCCGACGGCTAACATTCATCGTTTACGGC

[0051] GTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCA

[0052] GCGTCAGTTCCAGACCAGTGAGCCGCCTTCGCCACTGGTGTTCCTCCGAAT

[0053] ATCTACGAATTTCACCTCTACACTCGGAATTCCACTCACCTCTTCTGGACTCT

[0054] AGATTGCCAGTATGAAAGGCAGTTCCAGGGTTGAGCCCTGGGATTTCACCC

[0055] CTCACTTAACAATCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAAC

[0056] GCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCT

[0057] TCTTCTCCGGTTACCGTCATTATCTTCACCGGTGAAAGAGCTTTACAACCCT

[0058] AGGGCCTTCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGT

[0059] CCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCC

[0060] CAATGTGGCTGATCATCCTCTCAGACCAGCTATGGATCGTCGCCTTGGTAGG

[0061] CCTTTACCCCACCAACTAGCTAATCCAACGCGGGCTCATCCTTTCCCGATAA

[0062] ATCTTTCCCCCGAAGGGCTCATACGGTATTAGCACAAGTTTCCCTGCGTTATT

[0063] CCGTAGAAAAGGGTAGATTCCCACGCGTTACTCACCCGTCTGCCGCTCCCC

[0064] TTGCGGGGCGCTCGACTTGCATGTGT

[0065] 2. Isolation, identification and purification of Cb1 strain

[0066] The Cb1 strain was isolated from the surface-sterilized nodules of Melilotus officinalis (L.) Pall. using YMA solid medium. The specific operation was the same as that of the Z12 strain. The purified strain was sequenced by 16S rRNA (see SEQID NO.2) and identified as Variovorax boronicumulans by EZ Biocloud comparison. The pure culture of the strain was deposited in the China Center for Type Culture Collection on May 25, 2023. The deposit number is: CCTCC M 2023821.

[0067] SEQ ID NO.2:

[0068] TTTTAATCGTCGACATGTCAGACGGCAGCGCGGGAGCAATCCTGGCGGCGA

[0069] GTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAATCGTGGGGGATAA

[0070] CGCAGCGAAAGCTGTGCTAATACCGCATACGATCTACGGATGAAAGCAGGG

[0071] GATCGCAAGGCCTTGCGCGAATGGAGCGGCCGATGGCAGATTAGGTAGTTG

[0072] GTGAGGTAAAGGCTCACCAAGCCTTCGATCTGTAGCTGGTCTGAGAGGACG

[0073] ACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC

[0074] AGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTG

[0075] CAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAACGGT

[0076] CTTTTCTAATACAGAAGGCTAATGACGGTACCGTAAGAATAAGCACCGGCTA

[0077] ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAA

[0078] TTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGTTGTGAAATC

[0079] CCCGGGCTCAACCTGGGAACTGCATCTGTGACTGCATAGCTAGAGTACGGT

[0080] AGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAG

[0081] GAACACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCAC

[0082] GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAA

[0083] ACGATGTCAACTGGTTGTTGGGTCTTCACTGACTCAGTAACGAAGCTAACG

[0084] CGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGG

[0085] AATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCA

[0086] ACGCGAAAAACCTTACCCACCTTTGACATGTACGGAATTCGCCAGAGATGG

[0087] CTTAGTGCTCGAAAGAGAACCGTAACACAGGTGCTGCATGGCTGTCGTCAG

[0088] CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTC

[0089] ATTAGTTGCTACATTCAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACC

[0090] GGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCT

[0091] ACACACGTCATACAATGGCTGGTACAAAGGGTTGCCAACCCGCGAGGGGG

[0092] AGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGAC

[0093] TGCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGTCACGGTGAAT

[0094] ACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTCT

[0095] GCCAGAAGTAGTTAGCTTAACCGCAAGGAGGGCGATACCGTCG

[0096] 3. Isolation, identification and purification of S14 strain

[0097] The S14 strain was isolated from the surface-sterilized nodules of Astragalus adsurgens Pall. using YMA solid medium. The specific operation was the same as that of the Z12 strain. The purified strain was sequenced by 16S rRNA gene (see SEQ ID NO.3) and identified as Brevibacterium frigoritolerans by EZ Biocloud comparison. The pure culture of the strain was deposited in the China Center for Type Culture Collection on May 25, 2023. The deposit number is: CCTCC M 2023822.

[0098] SEQ ID NO.3:

[0099] CGTACTACTGCAGTCGAGCGAATCGACGGGAGCTTGCTCCCTGAGATTAGC

[0100] GGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTATAAGACTGGGATAAC

[0101] TTCGGGAAACCGGAGCTAATACCGGATACGTTCTTTTCTCGCATGAGAGAA

[0102] GATGGAAAGACGGTTTACGCTGTCACTTATAGATGGGCCCGCGGCGCATTA

[0103] GCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTG

[0104] AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG

[0105] GAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAA

[0106] CGCCGCGTGAACGAAGAAGGCCTTCGGGTCGTAAAGTTCTGTTGTTAGGGA

[0107] AGAACAAGTACCAGAGTAACTGCTGGTACCTTGACGGTACCTAACCAGAAA

[0108] GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCG

[0109] TTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTCCTTAAGTCTGA

[0110] TGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACT

[0111] TGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTA

[0112] GAGATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTG

[0113] ACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTA

[0114] GTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTA

[0115] GTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGG

[0116] CTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG

[0117] GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGA

[0118] CAACCCTAGAGATAGGGCTTTCCCCTTCGGGGGACAGAGTGACAGGTGGTG

[0119] CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCT GCAAACCTGCGAAGGTAAGCGAATCCCATAAAGCCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTCAGGAGCCCA

[0120] Example 2

[0121] Determination of the growth-promoting properties of three strains constituting the composite microbial agent

[0122] 1. Determination of phosphorus dissolving capacity

[0123] 50ul of the activated bacterial solution was transferred to YMA solid medium, cultured at 28℃ for 48h, and then transferred to solid phosphate-soluble medium by dot inoculation. Four replicates were inoculated on each plate, cultured at 28℃ for 5d, and the size of the halo was observed. 0: no halo; 1: halo thickness ≤2.5mm; 2: halo thickness between 2.5 and 5mm; 3: halo thickness >5mm.

[0124] 2. Qualitative determination of siderophore production capacity

[0125] The activated bacterial solution was transferred to YMA solid medium and cultured at 28℃ for 48h, and then transferred to CAS solid medium by dot inoculation. Each culture dish was inoculated with 4 replicates and cultured at 28℃ for 5d. The size of the halo was observed, and the thickness of the halo indicated the secretion degree of siderophore. 0: no halo; 1: halo thickness ≤ 2.5mm; 2: halo thickness between 2.5 and 5mm; 3: halo thickness > 5mm.

[0126] 3. Determination of IAA production capacity

[0127] The activated bacterial solution was transferred to TY liquid medium, cultured at 28°C for 72h, and centrifuged at 4°C, 10000rpm for 10min. The supernatant was mixed vigorously with Salkowski's reagent, reacted at room temperature in the dark for 30min, and the absorbance at 530nm was measured using an enzyme marker. Deionized water was used instead of the supernatant of the cultured strain to perform the same reaction as a blank control, and a standard curve with an IAA concentration range of 0-100μg / mL was prepared, and the IAA concentration in the supernatant was calculated based on this.

[0128] 4. Qualitative determination of nitrogen fixation capacity

[0129] The activated bacterial liquid was transferred to YMA solid medium and cultured at 28℃ for 48h, and then transferred to nitrogen-fixed solid medium with dye added by dot inoculation. Four replicates were inoculated in each culture dish and cultured at 28℃ for 5d. The size of the halo was observed. 0: no halo; 1: halo thickness ≤ 2.5mm; 2: halo thickness between 2.5 and 5mm; 3: halo thickness > 5mm.

[0130] The above culture medium and reagent formulas are:

[0131] Solid phosphate-dissolving medium: glucose 10.0 g, (NH4)2SO4 0.5 g, yeast extract 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, Fe2SO4 0.03 g, MnSO4 0.03 g, Ca3(PO4) 25.0 g, agar 20.0 g, and distilled water 1000 ml.

[0132] SM liquid culture medium: glucose 5.0 g, NH4NO3 1.0 g, KH2PO4 0.5 g, Na2HPO4 1.5 g, NaCl 1.0 g, MgSO4·7H2O 0.2 g, distilled water 1000 ml, adjust pH to 7.2, sterilize at 121℃ for 20 min.

[0133] CAS solid medium:

[0134] Solution A: Dissolve 0.012 g CAS in 10 ml deionized water and mix with 2 ml 1 mmol / LFeCl3;

[0135] Solution B: Dissolve 0.015 g HDTMA (hexadecyltrimethylammonium bromide) in 8 ml deionized water;

[0136] Dye solution C: Slowly add A into B, mix well and sterilize at 121℃ for 20 minutes.

[0137] Culture medium D: 20ml MM9 solution, 6.04g anhydrous piperazine are added to a conical flask containing 150ml deionized water, mixed and adjusted to pH 6.8 with hydrochloric acid, then 3.2g agar powder is added, mixed and sterilized at 121℃ for 20min. Then 0.2ml 1mmol / LCaCl2, 4ml 1mmol / LMgSO4·7H2O, 2ml 20% glucose, 6ml 10% acid hydrolyzed casein are added to D, and the dye solution is added along the wall of the flask, and gently shaken.

[0138] Salkowski's reagent: 0.5mol / LFeCl3·6H2O 2ml, 35% HClO4 98 ml.

[0139] TY liquid medium: 5.0 g tryptone, 3.0 g yeast extract, 0.7 g CaCl2·6H2O, 1000 ml distilled water.

[0140] MM9 solution: KH2PO4 15.0 g, NaCl 2.5 g, NH4Cl 5.0 g, Na2HPO4 30.0 g, deionized water 500 ml.

[0141] Nitrogen-fixing medium: mannitol 10.0 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·7H2O 0.1 g, CaCO3 5.0 g, agar 18-20 g, 0.5% Congo red solution 10 ml, distilled water 1000 ml.

[0142] The results of the growth-promoting properties test showed that (see Figures 1 to 3 ), the nitrogen fixation ability of the rhizobium EnsifermelilotiZ12 isolated from the nodules of Amorpha fruticosa was 1, the siderophore production ability was 1, and the content of levofloxacin produced was 4.94 mg / L. The inorganic phosphorus solubility ability, nitrogen fixation ability, siderophore production ability, and siderophore production of the growth-promoting bacterium Variovorax boronicumulans Cb1 isolated from the nodules of Rhizophora meliloti were 3, 2, 3, and 6.74 mg / L. The nitrogen fixation ability, siderophore production ability, and siderophore production of the growth-promoting bacterium Brevibacteriumfrigoritolerans S14 isolated from the nodules of Astragalus meliloti were 2, 2, and 22.83 mg / L.

[0143] Example 3

[0144] Antagonism assay

[0145] The activated bacterial solution was spread on a YMA solid plate and cultured at 28°C for 48 hours. Then, activated soybean-sized strains of one type were inoculated on a YMA solid plate that had been coated with another type of bacteria. After cultured at 28°C for 72 hours, the inoculated strains were observed to see whether they could grow normally and whether isolation zones were produced. If they could not grow normally and isolation zones were produced, it indicated that there was antagonism between the strains.

[0146] like Figure 4 As shown, there was no antagonism between the three strains.

[0147] Example 4

[0148] Preparation of composite bacterial agent for promoting growth and increasing yield of alfalfa

[0149] Based on the growth-promoting properties of the above strains and the results of no antagonism, a composite bacterial agent with the function of promoting growth and increasing yield of alfalfa was prepared according to the following steps:

[0150] a. Ensifer meliloti Z12, Variovorax boronicumulans Cb1 and Brevibacterium frigoritolerans S14 were inoculated into YMA solid medium and activated at 28-30℃ for 3 days;

[0151] b. The three activated strains were inoculated into TY liquid medium and cultured in a shaking incubator at 28°C until the logarithmic growth phase;

[0152] c. Centrifuge the culture solution in step (b) at 4°C, 10,000 rpm for 15 min, remove the supernatant, and collect the bacteria. Wash the bacteria twice with sterile distilled water by centrifugation, and then suspend them in sterile distilled water. Adjust the OD value to 0.8-1.0 at a wavelength of 600 nm. Mix the suspensions of the three strains with adjusted OD values ​​in equal proportions according to the number of effective live bacteria (the total number of effective live bacteria in the bacterial suspension is 2×10 8 ~2×10 9 CFU / mL), and obtain a composite bacterial agent.

[0153] The YMA solid culture medium contains 10.0 g of mannitol, 3.0 g of yeast powder, 0.25 g of K2HPO4, 0.25 g of KH2PO4, 0.20 g of MgSO4·7H2O, 0.10 g of NaCl, 1 L of distilled water, and 15-20 g of agar. After the pH is adjusted to 6.8-7.0, it is sterilized at 121°C for 30 min.

[0154] The TY liquid culture medium contains 5.0 g of tryptone, 3.0 g of yeast extract powder, 0.70 g of CaCl2·6H2O, and 1 L of distilled water. After the pH is adjusted to 6.8-7.0, it is sterilized at 121°C for 30 min.

[0155] Example 4

[0156] Effect of alfalfa inoculation under limited bacteria conditions

[0157] 1. Plant materials

[0158] Alfalfa WL319HQ was purchased from Beijing Zhengdao Seed Co., Ltd. It has the excellent characteristics of strong cold resistance, many branches, high yield and quality, many leaves, and long service life. The seeds were sterilized with 75% ethanol for 30 seconds, then soaked in 5% HClO4 for 5 minutes, then rinsed with sterile water for 3 times, and placed in a water agar culture dish for germination.

[0159] 2. Hoagland's nutrient solution

[0160] The macroelements include Ca(NO3)2·4H2O 1417mg / L; KNO3607 mg / L; MgSO4493 mg / L; (NH4)3PO4115 mg / L; the trace elements include H3BO32.86 mg / L; MnCl2·4H2O 1.81mg / L; ZnSO4·7H2O 0.22mg / L; CuSO4·5H2O 0.08mg / L; H2MnO4·H2O 0.02mg / L; FeSO450mg / L. The nutrient solution is prepared with sterile deionized water and adjusted to pH 7.0±0.2 with NaOH or HCl (1mg / L).

[0161] 3. Experimental treatment

[0162] This experiment was conducted in the artificial climate chamber of the Grassland and Grassland College of Northwest Agriculture and Forestry University in April 2023. Perlite and vermiculite were used as the cultivation medium. Perlite and vermiculite were mixed in a mass ratio of 3:2 and put into a cultivation bag, and then filled with the prepared Hoagland's nutrient solution, sterilized at 121℃ for 20 minutes for use.

[0163] Use tweezers to select 2 to 3 germinated alfalfa seeds and bury them on the surface of the cultivation medium. Tie the cultivation bag tightly after leaving an air vent.

[0164] Each treatment was repeated 8 times, and the alfalfa cultivation bag without inoculation was used as the control treatment. After the cotyledons of the alfalfa seedlings broke through the seed coat, the prepared composite bacterial agent was inoculated. During the growth period of alfalfa, the growth of the alfalfa seedlings was paid attention to at any time, and the size of the air outlet was changed. After 40 days of treatment, the biomass, plant height and root length of alfalfa were measured.

[0165] 4. Determination of nitrogen content in alfalfa plants

[0166] Weigh 0.1g (accurate to 0.0001g) of plant sample into a digestion tube, add 5ml of 98% concentrated sulfuric acid, place for 30-60min, add 2ml of 30% hydrogen peroxide, set the digestion chamber to 260℃, boil for 30min, then heat to 350℃, take out the sample after boiling for 1h, cool for 10min, slowly add 2ml of 30% hydrogen peroxide, set the digestion chamber to 350℃, boil the sample until it is clear and transparent, then stop digestion. Make up the digestion liquid in a 100ml volumetric flask, dilute to 20 times, and then use a continuous flow analyzer to determine the nitrogen content of the plant.

[0167] 5. Results

[0168] 5.1. Effects on fresh weight and dry weight of alfalfa

[0169] After 40 days of growth treatment, compared with the control treatment, the application of this composite inoculant significantly increased the fresh weight and dry weight of alfalfa (P<0.05). The fresh weight and dry weight of alfalfa inoculated with this inoculant increased by 186% and 115% respectively compared with the control, see Table 1.

[0170] Table 1 Effects of compound bacterial inoculation on fresh weight and dry weight of alfalfa

[0171]

[0172] Note: CK is the control, not inoculated with bacteria; Z12+S14+Cb1 is inoculated with compound bacteria. The same lowercase letters in the table indicate no significant difference between the control and the treatment, and different lowercase letters indicate significant difference between the control and the treatment.

[0173] 5.2 Effects on root length and plant height of alfalfa

[0174] After 40 days of growth treatment, compared with the control treatment, the application of this composite bacterial agent significantly increased the root length of alfalfa and the plant height also increased significantly (P<0.05). The root length and plant height of alfalfa inoculated with this bacterial agent increased by 38.3% and 30.8% respectively compared with the control, as shown in Table 2.

[0175] Table 2 Effects of inoculation with composite bacterial agent on root length and plant height of alfalfa

[0176]

[0177] Note: CK is the control, not inoculated with bacteria; Z12+S14+Cb1 is inoculated with compound bacteria. The same lowercase letters in the table indicate no significant difference between the control and the treatment, and different lowercase letters indicate significant difference between the control and the treatment.

[0178] 5.3 Effect on the number of alfalfa nodules

[0179] After 40 days of growth treatment, compared with the control treatment, the nodulation ability of alfalfa was significantly enhanced and the number of nodules was significantly increased after the application of the composite bacterial agent (P<0.05), as shown in Table 3.

[0180] Table 3 Effect of inoculation with composite bacterial agent on the number of alfalfa nodules

[0181]

[0182] Note: CK is the control, not inoculated with bacteria; Z12+S14+Cb1 is inoculated with compound bacteria. The same lowercase letters in the table indicate no significant difference between the control and the treatment, and different lowercase letters indicate significant difference between the control and the treatment.

[0183] 5.4. Effect of the bacterial agent of the present invention on the total nitrogen content of alfalfa plants

[0184] After 40 days of growth treatment, the total nitrogen content of alfalfa plants increased significantly after the application of the composite microbial agent compared with the control treatment (P<0.05). The total nitrogen content of alfalfa plants inoculated with the composite microbial agent increased by 84.7% compared with the control, as shown in Table 4.

[0185] Table 4 Effect of inoculation with composite microbial agent on total nitrogen content of alfalfa plants

[0186]

[0187] Note: CK is the control, not inoculated with bacteria; Z12+S14+Cb1 is inoculated with compound bacteria. The same lowercase letters in the table indicate no significant difference between the control and the treatment, and different lowercase letters indicate significant difference between the control and the treatment.

[0188] Tables 1 to 4 show that the composite bacterial agent of the present invention has a significant promoting effect on the growth of alfalfa, mainly promoting the growth of alfalfa plant height and root system, and promoting the increase of alfalfa plant biomass and nitrogen content.

[0189] The above data show that the composite bacterial agent provided by the present invention has the following advantages:

[0190] The composite bacterial agent provided by the present invention has the ability of fixing nitrogen, producing iron carriers and producing growth hormones, can promote the improvement of alfalfa yield and quality in areas with soil nutrient deficiency, and has good promotion and application value.

[0191] The composite bacterial agent provided by the present invention is easy to prepare, has no pollution to the human body and the environment, is not easy to produce drug resistance, has a significant growth-promoting effect on alfalfa, and has a comprehensive and diverse function compared with a single bacterial agent product, and has obvious advantages.

[0192] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A microbial composite agent for promoting growth and increasing production, characterized in that: It is caused by Brevibacterium frostii ( Brevibacterium frigoritolerans ) and Sinorhizobium meliloti ( Sinorhizobium meliloti )Z12、Voracious phage( Variovorax boronicumulans ) Cb1 is a compound of three kinds of bacteria; Among them, cold-resistant Brevibacterium, Sinorhizobium meliloti Z12 and Phage Cb1 are all deposited in the China Center for Type Culture Collection, and the preservation number of Sinorhizobium meliloti Z12 is CCTCC NO: M2023820, the preservation number of Phage Cb1 is CCTCC NO: M2023821, and the preservation number of cold-resistant Brevibacterium is CCTCC NO:M2023822.

2. The microbial composite agent according to claim 1, characterized in that: The Chinese rhizobium meliloti Z12, the phagocyte Cb1 and the cold-resistant Brevibacterium are compounded in equal proportions according to the number of effective live bacteria.

3. A method for preparing the microbial composite agent according to claim 1, characterized in that: The following steps are involved: S1, inoculating the Chinese rhizobium meliloti Z12, the phagocyte Cb1 and the cold-resistant Brevibacterium into YMA solid culture medium for cultivation and activation respectively; S2, the three activated strains were inoculated into TY liquid medium and cultured to the logarithmic growth phase; S3. Centrifuge the culture fluid of each strain obtained in S2, collect three kinds of bacteria respectively, mix and suspend them, and obtain the microbial composite agent.

4. The preparation method according to claim 3, characterized in that: The suspension is to make the total number of effective live bacteria in the suspension of the obtained microbial composite bacterial agent to be 2×10 8 ~2×10 9 CFU / mL.

5. The preparation method according to claim 3, characterized in that: In S1, the activation is performed at 28-30° C. for 2-3 days.

6. The preparation method according to claim 3, characterized in that: In S3, the centrifugation is carried out at 4°C and 10,000 rpm for 10 to 15 min.

7. Use of the microbial composite agent according to claim 1 in nitrogen fixation, inorganic phosphorus solubilization, iron carrier production or growth factor production.

8. The use according to claim 7, characterized in that The microbial composite agent is used for promoting the growth and increasing the yield of alfalfa.

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