A functional complex microbial propagation liquid, a complex microbial inoculant containing the liquid, and a preparation method and application of the inoculant

Abstract

The present application relates to the technical field of microbial inoculant, and provides a functional complex microbial propagation liquid, a complex microbial inoculant containing the propagation liquid, and a preparation method and application of the inoculant, wherein the functional complex microbial propagation liquid comprises Burkholderia WQ-6 liquid, Streptomyces JX-1 liquid and Rhizobium ZM-3 liquid. The three strains do not cross-antagonize each other, can play a synergistic effect, improve the effect of producing indole acetic acid, and promote the growth of crops. The complex microbial inoculant comprises the functional complex microbial propagation liquid, soybean powder and peanut straw, and when the complex microbial inoculant is applied to an intercropping system in farmland, the growth of plants can be effectively promoted, and the yield of intercropped crops can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial agents, and in particular to a functional composite microbial propagation liquid, a composite microbial agent containing the liquid, and a preparation method and application of the agent. Background Art

[0002] In agricultural production, long-term continuous cropping and the extensive use of chemical fertilizers have led to red soil acidification, reduced soil fertility, loss of biodiversity, and an increased risk of soil-borne diseases. This series of problems seriously hinders the sustainable development of agricultural production. Therefore, it is crucial to explore more optimal planting models and more environmentally friendly fertilization methods to improve the soil ecological environment and increase crop yield and quality.

[0003] Crop diversification improves nutrient use efficiency, soil structure, soil health, and productivity, making it a key measure for sustainable agricultural development. Intercropping legumes with non-legumes, leveraging their functional characteristics, is a typical cropping system that combines nutrient use with nutrients. This system can enhance soil carbon and nitrogen fixation capacity and steadily improve dryland crop productivity while reducing the need for exogenous nitrogen fertilizer.

[0004] As a kind of biological fertilizer, microbial agents have unique biological activity and are environmentally friendly, and are receiving more and more attention. Microbial agents can improve soil structure, promote plant growth, and play a positive role in enhancing plant health and increasing crop yields. Agricultural microbial agents mainly include single microbial agents and composite microbial agents. Compared with single microbial agents, composite microbial agents usually have certain synergistic effects, are more diverse in types, and have more complete functions. For example, Chinese patent CN116769680A discloses a composite microbial agent for promoting wheat production and its preparation method. By using nitrifying bacteria, phosphate-solubilizing bacteria, nitrogen-fixing bacteria, chitosanase-producing bacteria, and soluble phosphate-producing bacteria, combined with microbial fermentation substrates, a composite microbial agent is prepared, which can effectively promote wheat growth, reduce the use of chemical fertilizers, and help promote the development of green agriculture and achieve large-scale wheat production increases.

[0005] However, few researchers have previously combined the two in production. Most composite microbial agents have been developed for single crops, and there are very few reports on composite microbial agents for promoting growth and increasing yields in diversified cropping systems. Therefore, the development of a composite microbial agent suitable for diversified cropping systems in dryland farmland, combining diversified cropping with composite microbial agents to increase crop yield and quality, improve the soil environment, and achieve a "1+1>2" effect, is a key goal of this research. Summary of the Invention

[0006] The application aims to provide a functional complex microbial propagation bacterial liquid, a complex microbial agent containing the bacterial liquid, and a preparation method and application of the agent.

[0007] To achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides a functional complex microbial propagation bacterial liquid, which comprises Burkholderia WQ-6 bacterial liquid, Streptomyces JX-1 bacterial liquid, and Rhizobium ZM-3 bacterial liquid.

[0009] The preservation name of the Burkholderia WQ-6 is Caballeronia concitans WQ-6, the preservation date is January 31, 2024, the preservation unit is the General Microbiological Center of the Chinese Microorganism Bacterial Strain Preservation Management Committee, and the preservation number is CGMCC No. 29821.

[0010] The preservation name of the Streptomyces JX-1 is Streptacidiphilus monticola JX-1, the preservation date is January 31, 2024, the preservation unit is the General Microbiological Center of the Chinese Microorganism Bacterial Strain Preservation Management Committee, and the preservation number is CGMCC No. 29822.

[0011] The preservation name of the Rhizobium ZM-3 is Rhizobium changzhiense ZM-3, the preservation date is January 31, 2024, the preservation unit is the General Microbiological Center of the Chinese Microorganism Bacterial Strain Preservation Management Committee, and the preservation number is CGMCC No. 29823.

[0012] Preferably, the mass ratio of the Burkholderia WQ-6 bacterial liquid, the Streptomyces JX-1 bacterial liquid, and the Rhizobium ZM-3 bacterial liquid is 1:1.1-1.3:1.4-1.6.

[0013] The application further provides a preparation method of the functional complex microbial propagation bacterial liquid, which comprises the following steps.

[0014] (1) inoculating Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 into solid culture media respectively to obtain activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0015] (2) inoculating the activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 into liquid culture media respectively to obtain single-strain propagation bacterial liquids of Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0016] (3) The OD600 of the single strain propagation culture liquid of Burkholderia WQ-6, Streptomyces JX-1 and Rhizobium ZM-3 was uniformly adjusted to 0.03-0.05 to obtain Burkholderia WQ-6 culture liquid, Streptomyces JX-1 culture liquid and Rhizobium ZM-3 culture liquid;

[0017] (4) The Burkholderia WQ-6 bacterial solution, the Streptomyces JX-1 bacterial solution and the Rhizobium ZM-3 bacterial solution were mixed in a mass ratio of 1:1.1-1.3:1.4-1.6 to obtain a functional composite microbial propagation bacterial solution.

[0018] Preferably, the culture conditions in step (1) are constant temperature static culture at 26-30° C. for 22-26 hours, and the solid culture medium is TY solid culture medium.

[0019] Preferably, the culture conditions in step (2) are constant temperature shaking culture at 26-30° C. and 160-200 rpm / min for 22-26 hours, and the liquid culture medium is TY liquid culture medium.

[0020] The present invention also provides a composite microbial agent, which comprises the functional composite microbial propagation solution, soybean powder, and peanut straw;

[0021] Preferably, the mass ratio of the functional composite microbial propagation solution, soybean powder and peanut straw is 1:9-11:190-210.

[0022] The present invention also provides a method for preparing the composite microbial agent, comprising the following steps: mixing a functional composite microbial propagation solution, soybean powder, and peanut straw in a mass ratio of 1:9-11:190-210, mixing them evenly, and fermenting and culturing them to obtain the composite microbial agent;

[0023] Preferably, the content of effective strains in the composite microbial agent is 1 to 9×10 8 / kg compound microbial agent.

[0024] Preferably, the fermentation temperature is 24-26° C., the fermentation time is 5-7 days, and the culture is stirred 1-5 times a day during the fermentation period.

[0025] The present invention also provides an application of the composite microbial agent in promoting growth and increasing yield in a farmland intercropping system, wherein the farmland intercropping system is a diversified planting system containing leguminous peanuts.

[0026] The present invention also provides a method for using the composite microbial agent, which comprises burying 50 to 100 g of the composite microbial agent around each plant.

[0027] Preservation Instructions

[0028] Burkholderia concitans WQ-6, deposited as Caballeronia concitans WQ-6, deposit date: January 31, 2024, deposited at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 29821;

[0029] Streptomyces JX-1, deposited as Streptacidiphilus monticola JX-1, deposit date: January 31, 2024, deposited at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 29822;

[0030] Rhizobium ZM-3, deposited as Rhizobium changzhiense ZM-3, deposit date: January 31, 2024, deposited at: General Microbiology Center, China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 29823. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a characteristic diagram of the Burkholderia WQ-6 strain, where a in the figure is a colony of WQ-6 and b in the figure is the Gram stain of WQ-6;

[0032] Figure 2 is the neighbor-joining phylogenetic tree of Burkholderia WQ-6;

[0033] Figure 3 This is a characteristic diagram of the strain of Streptomyces JX-1, where a in the figure is a colony of JX-1 and b in the figure is the Gram stain of JX-1;

[0034] Figure 4 is the Neighbor-joining phylogenetic tree of Streptomyces JX-1;

[0035] Figure 5 This is a characteristic diagram of the strain of Rhizobium ZM-3, where a in the figure is a colony of ZM-3 and b in the figure is the Gram stain of ZM-3;

[0036] Figure 6 is the Neighbor-joining phylogenetic tree of Rhizobium ZM-3;

[0037] Figure 7This is a diagram of the cross-antagonism and coexistence of three bacterial strains;

[0038] Figure 8 This is a diagram showing the coexistence of functional composite microbial propagation solutions and the production of indoleacetic acid. FIGURE a shows the gel electrophoresis of the three bacterial strains under co-culture conditions, and FIGURE b shows the comparison of the indoleacetic acid production between the composite propagation solution and the three single bacterial solutions.

[0039] Figure 9 : This is a graph showing the growth-promoting effect of the functional composite microbial propagation solution on cassava tissue culture seedlings, wherein a in the figure represents the effect of the composite propagation solution on the number of lateral roots of cassava tissue culture seedlings; b in the figure represents the effect of the composite propagation solution on the number of primordia of cassava tissue culture seedlings; c in the figure represents the effect of the composite propagation solution on the root diameter of cassava tissue culture seedlings; d in the figure represents the effect of the composite propagation solution on the root biomass of cassava tissue culture seedlings;

[0040] Figure 10 This is a diagram showing the beneficial effects of composite microbial agents on crops in the intercropping system in farmland, where a in the figure represents the yield of cassava and peanuts in the cassava-peanut intercropping system, b in the figure represents the plant height of peanuts and corn in the peanut-corn intercropping system, c in the figure represents the underground biomass of peanuts and corn in the peanut-corn intercropping system, and d in the figure represents the aboveground biomass of peanuts and corn in the peanut-corn intercropping system. DETAILED DESCRIPTION

[0041] The present invention provides a functional composite microbial propagation bacterial solution, which comprises: Burkholderia WQ-6 bacterial solution, Streptomyces JX-1 bacterial solution and Rhizobium ZM-3 bacterial solution;

[0042] The Burkholderia WQ-6 is deposited as Caballeronia concitans WQ-6, on January 31, 2024, at the General Microbiology Center of China Culture Collection Administration, with a deposit number of CGMCC No. 29821.

[0043] The deposited name of Streptomyces JX-1 is Streptacidiphilus monticola JX-1, the deposit date is January 31, 2024, the depositary unit is General Microbiology Center of China Culture Collection Administration of Microorganisms, and the deposit number is CGMCC No. 29822;

[0044] The preservation name of the rhizobium ZM-3 is Rhizobium changzhiense ZM-3, the preservation date is January 31, 2024, the preservation unit is the General Microbiology Center of the China Microorganism Culture Collection Administration, and the preservation number is CGMCC No. 29823.

[0045] In the present invention, the mass ratio of the Burkholderia WQ-6 bacterial solution, the Streptomyces JX-1 bacterial solution and the Rhizobium ZM-3 bacterial solution is preferably 1:1.1-1.3:1.4-1.6, and more preferably 1:1.2:1.5.

[0046] The present invention also provides a method for preparing the functional composite microbial propagation solution, comprising the following steps:

[0047] (1) Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into solid culture medium to obtain activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0048] (2) The activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into liquid culture medium to obtain single strain propagation liquids of Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0049] (3) The OD600 of the single strain propagation culture liquid of Burkholderia WQ-6, Streptomyces JX-1 and Rhizobium ZM-3 was uniformly adjusted to 0.03-0.05 to obtain Burkholderia WQ-6 culture liquid, Streptomyces JX-1 culture liquid and Rhizobium ZM-3 culture liquid;

[0050] (4) The Burkholderia WQ-6 bacterial solution, the Streptomyces JX-1 bacterial solution and the Rhizobium ZM-3 bacterial solution were mixed in a mass ratio of 1:1.1-1.3:1.4-1.6 to obtain a functional composite microbial propagation bacterial solution.

[0051] In the present invention, the culture temperature in step (1) is preferably 26-30°C, more preferably 27-29°C, and further preferably 28°C, the constant temperature static culture time is preferably 22-26h, more preferably 23-25h, and further preferably 24h, and the solid culture medium is preferably TY solid culture medium.

[0052] In the present invention, the culture temperature in step (2) is preferably 26-30°C, more preferably 27-29°C, and further preferably 28°C, the constant temperature shaking speed is preferably 160-200 rpm / min, more preferably 170-190 rpm / min, and further preferably 180 rpm / min, the culture time is preferably 22-26h, more preferably 23-25h, and further preferably 24h, and the liquid culture medium is preferably TY liquid culture medium.

[0053] The present invention also provides a composite microbial agent, which comprises the functional composite microbial propagation solution, soybean powder, and peanut straw;

[0054] In the present invention, the mass ratio of the functional composite microbial propagation solution, soybean powder, and peanut straw is preferably 1:9-11:190-210, and more preferably 1:10:200.

[0055] The present invention also provides a method for preparing the composite microbial agent, comprising the following steps: mixing a functional composite microbial propagation solution, soybean powder, and peanut straw preferably in a mass ratio of 1:9-11:190-210, mixing uniformly, and fermenting to obtain the composite microbial agent;

[0056] In the present invention, the content of effective strains in the composite microbial agent is further 1 to 9×10 8 / kg composite microbial agent, more preferably 2 to 8 × 10 8 / kg composite microbial agent, and further preferably 3 to 7 × 10 8 / kg composite microbial agent, and further preferably 5×10 8 / kg compound microbial agent.

[0057] Preferably, the fermentation culture temperature is preferably 24-26°C, more preferably 25°C, the fermentation time is preferably 5-7 days, more preferably 6 days, and the fermentation period is preferably stirred 1-5 times a day, more preferably 2-4 times, and even more preferably 3 times.

[0058] The present invention also provides an application of the composite microbial agent in promoting growth and increasing yield in a farmland intercropping system, wherein the farmland intercropping system is a diversified planting system containing leguminous peanuts.

[0059] The present invention also provides a method for using the composite microbial agent, preferably burying 50 to 100 g of the composite microbial agent around each plant, further preferably burying 60 to 90 g of the composite microbial agent around each plant, further preferably burying 70 to 80 g of the composite microbial agent around each plant, and further preferably burying 75 g of the composite microbial agent around each plant.

[0060] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0062] Example 1 Strain Type and Cross-Antagonistic Properties

[0063] 1. Isolation, screening and identification of Burkholderia WQ-6

[0064] 1. Isolation, screening and purification of Burkholderia WQ-6

[0065] Peanut rhizosphere soil from a peanut-corn intercropping system was collected and, under aseptic operation, shaken and diluted with sterile water. The soil was then spread on LB solid plate medium using a spreader rod. Single colonies were obtained after multiple isolation and purification and stored.

[0066] The colonies of strain WQ-6 cultured on TY medium for 24 hours are ivory white, opaque, oval, with wavy edges, rough and dry surface, and 1 to 3 mm in diameter. Figure 1 .a), microscopic examination showed that the bacteria were rod-shaped and Gram-positive ( Figure 1 .b).

[0067] 2. Molecular identification of Burkholderia WQ-6

[0068] The DNA of the strain WQ-6 was used as a template, and 16S rDNA universal primers were used for amplification and the sequence was determined. The sequence is shown in SEQ ID NO.1.

[0069] The 16S universal gene primers are: 27F: AGAGTTTGATCCTGGCTCAG; 1492R: TACGGCTACCTTGTTACGACGACTT. The 16S rDNA sequencing results of strain WQ-6 were entered into the NCBI database for BLAST comparison to obtain related model strains. After preliminary processing of the target sequence and the model strain sequence using MEGA7.0, a phylogenetic tree was constructed using the neighbor-joining method. The results are shown in Figure 2. Figure 2 As shown, the 16S rRNA genes of strain WQ-6 were highly similar to those of Caballeronia concitans strain LMG 29315, so strain WQ-6 was identified as Caballeronia concitans, belonging to the Burkholderiaceae family.

[0070] 16S rDNA sequence of WQ-6 (441bp, SEQ ID No.1):

[0071] GGGTAGGCATCGTGCATGAGCGTCAGTGTTGGCCCAGGAGGCTGCCTTCGCCATCGGTATTCCTCCACATCTCTACGCATTTCACTGCTACACGTGGAATTCTACCTCCCTCTGCCACACTCTAGCCTGCCAGTCACCAATGCAGTTCCCAGGTTAAGCCCGGGGATTTCACATCGGTCTTAACAGACCGCCTGCGCACGCTTTACGCCCAGTAATTCCGA TTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTTCCGGTACCGTCATCCTCCATCCATATTAGGGACGAAGTTTTCTTTCCGGACAAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATTGCTGGATCAGGGTTGCCCCCATTGTCCAAAATTCCCCACTGCTGCCCCGGGAGGGGAGTAA.

[0072] 3. Preservation of Burkholderia WQ-6

[0073] The screened strain Burkholderia WQ-6 was deposited, and the depository unit of the Burkholderia WQ-6 is the General Microbiology Center of the China Culture Collection Administration (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 31, 2024, and the deposit number is CGMCC No. 29821.

[0074] 2. Isolation, screening and identification of Streptomyces JX-1

[0075] 1. Isolation, screening and purification of Streptomyces JX-1

[0076] Peanut rhizosphere soil from a peanut-corn intercropping system was collected and, under aseptic operation, shaken and diluted with sterile water. The soil was then spread on LB solid plate medium using a spreader rod. Single colonies were obtained after multiple isolation and purification and stored.

[0077] The colonies of strain JX-1 cultured on TY medium for 24 hours are orange, opaque, oval, with neat edges, smooth and moist surface, and a diameter of 1 mm ( Figure 3 .a), microscopic examination showed that the bacteria were spherical and Gram-negative ( Figure 3 .b).

[0078] 2. Molecular identification of Streptomyces JX-1

[0079] The DNA of the strain JX-1 was used as a template, and 16S rDNA universal primers were used for amplification and the sequence was determined. The sequence is shown in SEQ ID NO.2.

[0080] The 16S universal gene primers are: 27F: AGAGTTTGATCCTGGCTCAG; 1492R: TACGGCTACCTTGTTACGACGACTT. The 16S rDNA sequencing results of strain JX-1 were entered into the NCBI database for BLAST comparison to obtain related model strains. After preliminary processing of the target sequence and the model strain sequence using MEGA7.0, a phylogenetic tree was constructed using the neighbor-joining method. The results are shown in Figure 2. Figure 4 As shown, the 16S rRNA genes of strain JX-1 were highly similar to those of Streptacidiphilus monticola strain NEAU-SW11, thus confirming that strain JX-1 was a Streptomyces sp.

[0081] 16S rDNA sequence of JX-1 (417bp, SEQ ID No.2):

[0082] GAGCAGCTCGCTCTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCACACTCCAGCCTGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGGGCTTTCACATCCGACGCGACAGGCCGCCTACGAGCTCTTTACGCCCAA TAATTCCGGACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCGCGTAGGAGTA.

[0083] 3. Preservation of Streptomyces JX-1 strains

[0084] The screened strain Streptomyces JX-1 was deposited, and the depository unit of the Streptomyces JX-1 is the General Microbiology Center of the China Culture Collection Administration (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 31, 2024, and the deposit number is CGMCC No. 29822.

[0085] 3. Isolation, screening and identification of rhizobium ZM-3

[0086] 1. Isolation, screening and purification of Rhizobium ZM-3

[0087] Peanut rhizosphere soil from a peanut-corn intercropping system was collected and, under aseptic operation, shaken and diluted with sterile water. The soil was then spread on LB solid plate medium using a spreader rod. Single colonies were obtained after multiple isolation and purification and stored.

[0088] The colonies of strain ZM-3 cultured on TY medium for 24 hours are orange, opaque, round, with neat edges, smooth and moist surface, and 1 mm in diameter. Figure 5 .a), microscopic examination showed that the bacteria were spherical and Gram-negative ( Figure 5 .b).

[0089] 2. Molecular identification of rhizobium ZM-3

[0090] The DNA of the strain ZM-3 was used as a template, and 16S rDNA universal primers were used for amplification and the sequence was determined. The sequence is shown in SEQ ID NO.3.

[0091] The 16S universal gene primers are: 27F: AGAGTTTGATCCTGGCTCAG; 1492R: TACGGCTACCTTGTTACGACGACTT. The 16S rDNA sequencing results of strain ZM-3 were entered into the NCBI database for BLAST comparison to obtain related model strains. After preliminary processing of the target sequence and the model strain sequence using MEGA7.0, a phylogenetic tree was constructed using the neighbor-joining method. The results are shown in Figure 2. Figure 6 As shown, the 16S rRNA genes of strain ZM-3 were highly similar to those of Rhizobiumchangzhiense strain WYCCWR 11279, so strain ZM-3 was identified as a rhizobium.

[0092] 16S rDNA sequence of ZM-3 (451bp, SEQ ID NO.3):

[0093] TTACTCCTACCCGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGG CGGATCGATCAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCGGGTAGTCCAAA.

[0094] 3. Preservation of Rhizobium ZM-3

[0095] The screened strain Rhizobium ZM-3 was deposited, and the depository unit of the Rhizobium ZM-3 is the General Microbiology Center of the China Culture Collection Administration (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 31, 2024, and the deposit number is CGMCC No. 29823.

[0096] 4. Cross-antagonistic effect of the three strains

[0097] After activation of Burkholderia WQ-6, Streptomyces JX-1 and Rhizobium ZM-3, single colonies were picked and streaked onto TY solid plate medium. The culture was kept at 28°C for 2 days to investigate the cross-antagonistic properties of the three strains. The results are as follows: Figure 7 shown.

[0098] The results showed that the three strains had no cross-antagonistic effect and could be used simultaneously.

[0099] Example 2 Preparation of functional composite microbial propagation solution

[0100] The specific preparation method is as follows:

[0101] (1) Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into TY solid medium and cultured at 28°C for 24 h to obtain activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0102] (2) The activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into TY liquid medium, respectively, and cultured at 180 rpm / min and 28°C for 1 day to obtain single strain propagation liquids of Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3;

[0103] (3) The OD600 of the single strain propagation culture liquid of Burkholderia WQ-6, Streptomyces JX-1 and Rhizobium ZM-3 was uniformly adjusted to 0.03-0.05 to obtain Burkholderia WQ-6 culture liquid, Streptomyces JX-1 culture liquid and Rhizobium ZM-3 culture liquid;

[0104] (4) The bacterial solution of Burkholderia WQ-6, the bacterial solution of Streptomyces JX-1 and the bacterial solution of Rhizobium ZM-3 were mixed in a mass ratio of 1:1.2:1.5 to obtain a functional composite microbial propagation bacterial solution.

[0105] Example 3 Preparation of composite microbial agent

[0106] The specific preparation method is as follows:

[0107] The functional composite microbial propagation liquid, soybean powder and peanut straw are mixed in a mass ratio of 1:10:200, mixed evenly, and then put into a production tank for fermentation and culture. The mixture is fermented at room temperature for 5 days, and stirred once a day during the fermentation period to obtain a composite microbial agent.

[0108] Example 4 Coexistence of functional composite microbial propagation liquid

[0109] The functional composite microorganism propagation liquid obtained in Example 2 was further placed in a constant temperature shaking culture at 180 rpm / min and 28°C for 2 days. The composite microorganism propagation liquid after culturing for 2 days was subjected to PCR amplification using primers WQ-6, JX-1 and ZM-3, respectively, and then subjected to gel electrophoresis. The primers are shown in the table below. The results are shown in the table below. Figure 8 .a shown.

[0110] Table 1 PCR amplification primers for WQ-6, JX-1, and ZM-3

[0111] Primer name Primer sequences serial number WQ-6F CTGCCTTCGCCATCGGTATTCC SEQ ID NO.4 WQ-6R ATCCAGCAATGCCGCGTGTG SEQ ID NO.5 JX-1F CAGCGTCAGTAATGGCCCAGAG SEQ ID NO.6 JX-1R CGTAGGGTGCGAGCGTTGTC SEQ ID NO.7 ZM-3F CGCAAGCCTGATCCAGCCATG SEQ ID NO.8 ZM-3R TGGACCAGTGAGCCGCCTTC SEQ ID NO.9

[0112] The results showed that the three bacterial strains were able to grow normally under co-culture conditions.

[0113] Example 5 Effect of functional composite microbial expansion on indoleacetic acid production

[0114] 500 μl of WQ-6, JX-1, ZM-3 single strain propagation liquid and 500 μl of composite microbial propagation liquid were added to 5 g of soil respectively. After incubation at 28 ° C for two days, 0.15 g of soil was taken out and placed in a 2 ml centrifuge tube. 1.35 ml of PBS solution was added and vortexed evenly. Centrifuged at 2500 r / min and 4 ° C for 15 min, the supernatant was taken and the indoleacetic acid content was determined using an Elisa (ml147100) kit to obtain the indoleacetic acid production situation. Figure 8 .b shown.

[0115] The results showed that compared with a single strain, the soil with the addition of composite microbial enrichment solution had a higher indoleacetic acid content. The indoleacetic acid content of the soil with the addition of WQ-6, JX-1, ZM-3 and composite bacterial solution was 61.25 nmol / L, 54.44 nmol / L, 55.12 nmol / L and 62.71 nmol / L, respectively.

[0116] Example 6 Growth-promoting effect of functional composite microbial propagation solution

[0117] Cassava tissue culture seedlings were used as experimental subjects to study the growth-promoting effect of the functional composite microbial propagation solution. The specific steps were as follows:

[0118] Cultivation method of cassava tissue culture seedlings: Select cassava tissue culture seedling stem segments and place them on 1 / 2MS culture medium, with 3 stem segments placed in each bottle, and place them in a light incubator with a light intensity of 16000lx and a constant temperature of 28℃.

[0119] 100 μL of the obtained functional composite microbial propagation liquid was inoculated into the root system of cassava tissue culture seedlings grown for 30 days, and 100 μL of sterile water was inoculated as a control. The cells were placed in a light incubator with a light intensity of 16000 lx and a constant temperature of 28°C for 21 days. The growth-promoting effect of the functional composite microbial propagation liquid on cassava tissue culture seedlings was statistically obtained. The results are as follows: Figure 9 shown.

[0120] The results showed that when the functional composite microbial propagation solution was inoculated into cassava tissue culture seedlings, the number of cassava lateral roots, the number of primordia, the root diameter and the underground biomass increased by 31%, 166%, 88% and 70% respectively.

[0121] Beneficial effects of the composite microbial agent described in Example 7 on crops in the intercropping system

[0122] The field experiment was conducted at the Red Soil Ecological Experimental Station of the Chinese Academy of Sciences in Yingtan, Jiangxi Province, China (28°15′N, 116°5′E). The soil type in this area is classified as acidic loamy clay derived from Quaternary red soil (eluvial red soil in the Chinese soil classification system and ferroalloy soil in the FAO classification system), with a soil pH (in water) of 4.9. At the top 25 cm of the soil profile, the soil organic carbon (SOC) content was 10.23 g / kg, the total nitrogen (TN) content was 0.90 g / kg, the available phosphorus (AP) content was 34.15 mg / kg, and the available potassium (AK) content was 235.11 mg / kg. Each experimental plot was 20×5 m and belonged to the cassava-peanut intercropping system and the peanut-corn intercropping system. Three replicates were set up for each treatment. The composite microbial inoculant prepared according to the method of Example 2 was buried around the plants, with an application rate of 50 g per plant; no inoculant was applied to the control treatment.

[0123] (1) During the harvest season, the yield levels of cassava and peanuts in the cassava-peanut intercropping system were calculated.

[0124] (2) Three months after sowing, the growth status of peanuts and corn in the peanut-corn intercropping system (including plant height, aboveground biomass and underground biomass) was counted.

[0125] The results are as follows Figure 10 shown.

[0126] Depend on Figure 10 .a The yield results showed that the application of composite microbial agents in cassava-peanut and peanut-corn intercropping systems increased the yield of cassava and peanut in the cassava-peanut intercropping system by 8% and 79% respectively.

[0127] Depend on Figure 10 The results shown in .b~d show that in the peanut-corn intercropping system, the plant height, aboveground and underground biomass of peanut increased by 15%, 55% and 100%, respectively, and the plant height and aboveground biomass of corn increased by 143% and 13%, respectively.

[0128] As can be seen from the above embodiments, the present invention provides a functional composite microbial propagation liquid and a composite microbial agent containing the liquid, as well as a preparation method and application of the agent, wherein the functional composite microbial propagation liquid includes Burkholderia WQ-6 liquid, Streptomyces JX-1 liquid and Rhizobium ZM-3 liquid. There is no cross-antagonism between the three strains, and they can play a synergistic role, improve the indoleacetic acid production effect, and promote the growth of crops. The composite microbial agent includes a functional composite microbial propagation liquid, soybean powder and peanut straw. The composite microbial agent can be applied to diversified planting bodies containing leguminous peanuts. The composite microbial agent can effectively promote the growth of multiple crops in the intercropping system, and comprehensively improve the yield of intercropped crops for multiple crops.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A functional composite microbial propagation solution, characterized in that: The functional composite microbial propagation liquid includes: Burkholderia WQ-6 liquid, Streptomyces JX-1 liquid and Rhizobium ZM-3 liquid; The Burkholderia WQ-6 is deposited as Caballeronia concitans WQ-6, on January 31, 2024, at the General Microbiology Center of China Culture Collection Administration, with a deposit number of CGMCC No. 29821. The deposited name of Streptomyces JX-1 is Streptacidiphilus monticola JX-1, the deposit date is January 31, 2024, the depositary unit is General Microbiology Center of China Culture Collection Administration of Microorganisms, and the deposit number is CGMCC No. 29822; The preservation name of the rhizobium ZM-3 is Rhizobium changzhiense ZM-3, the preservation date is January 31, 2024, the preservation unit is the General Microbiology Center of the China Microorganism Culture Collection Administration, and the preservation number is CGMCC No. 29823.

2. The functional composite microbial propagation solution according to claim 1, characterized in that: The mass ratio of the Burkholderia WQ-6 bacterial solution, the Streptomyces JX-1 bacterial solution and the Rhizobium ZM-3 bacterial solution is 1:1.1-1.3:1.4-1.

6.

3. The method for preparing the functional composite microbial propagation solution according to claim 1, characterized in that: The following steps are included: (1) Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into solid culture medium to obtain activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3; (2) The activated Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3 were inoculated into liquid culture medium to obtain single strain propagation liquids of Burkholderia WQ-6, Streptomyces JX-1, and Rhizobium ZM-3; (3) The OD600 of the single strain propagation culture liquid of Burkholderia WQ-6, Streptomyces JX-1 and Rhizobium ZM-3 was uniformly adjusted to 0.03-0.05 to obtain Burkholderia WQ-6 culture liquid, Streptomyces JX-1 culture liquid and Rhizobium ZM-3 culture liquid; (4) The Burkholderia WQ-6 bacterial solution, the Streptomyces JX-1 bacterial solution and the Rhizobium ZM-3 bacterial solution were mixed in a mass ratio of 1:1.1-1.3:1.4-1.6 to obtain a functional composite microbial propagation bacterial solution.

4. The method for preparing a functional composite microbial propagation solution according to claim 3, characterized in that: The culture conditions in step (1) are constant temperature static culture at 26-30° C. for 22-26 hours, and the solid culture medium is TY solid culture medium.

5. The method for preparing a functional composite microbial propagation solution according to claim 3, characterized in that: The culture conditions in step (2) are constant temperature shaking culture at 26-30° C. and 160-200 rpm / min for 22-26 hours, and the liquid culture medium is TY liquid culture medium.

6. A composite microbial agent, characterized in that: The composite microbial agent comprises the functional composite microbial propagation solution according to claim 1 or 2, soybean powder, and peanut straw; The mass ratio of the functional composite microorganism propagation liquid, soybean powder and peanut straw is 1:9-11:190-210.

7. The method for preparing the composite microbial agent according to claim 6, characterized in that: The method comprises the following steps: mixing a functional composite microbial propagation liquid, soybean powder and peanut straw in a mass ratio of 1:9-11:190-210, mixing the mixture evenly and performing fermentation culture to obtain a composite microbial agent; The content of effective strains in the composite microbial agent is 1 to 9×10 8 / kg compound microbial agent.

8. The preparation method according to claim 7, characterized in that The fermentation culture temperature is 24-26° C., the fermentation time is 5-7 days, and the fermentation is stirred 1-5 times a day.

9. Use of the composite microbial agent according to claim 6 in promoting growth and increasing yield in intercropping systems in farmland, characterized in that: The farmland intercropping system is a diversified planting system containing leguminous peanuts.

10. The method for using the composite microbial agent according to claim 6, characterized in that: Bury 50-100g of compound microbial agent around each plant.

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