Fermentation production method for arc microbial agent capable of coupling toxin control, nitrogen fixation and yield increase

Abstract

The present invention relates to the field of microbial agents. Provided is a fermentation production method for an ARC microbial agent capable of toxin control, nitrogen fixation and yield increase. The method sequentially comprises the following steps: performing strain domestication, so as to obtain a domesticated bacterial liquid of each strain contained in the ARC microbial agent; performing seed liquid preparation: using the domesticated bacterial liquid of each strain contained in the ARC microbial agent to prepare a seed liquid of each strain contained in the ARC microbial agent; performing fermentation production: fermenting the seed liquid of each strain contained in the ARC microbial agent to produce a batch fermentation liquid of each strain contained in the ARC microbial agent; performing bacterial agent combination: combining the batch fermentation liquids of the strains contained in the ARC microbial agent or combining bacterial powders prepared from the batch fermentation liquids of the strains contained in the ARC microbial agent obtained by the fermentation production, so as to obtain a microbial composition containing the strains. The present invention is used for batch production of the ARC microbial agent capable of coupling toxin control, nitrogen fixation and yield increase, is simple to use, achieves remarkable social and ecological benefits, and facilitates popularization and use.

Description

ARC microbial agent fermentation production method for coupling toxicity control and nitrogen fixation with yield increase Technical Field

[0001] The present invention relates to the field of microbial agents, and in particular to a fermentation production method of an ARC microbial agent for coupled toxicity control and nitrogen fixation and yield increase. Background Art

[0002] Oilseeds are important sources of fat and plant protein, two of the three major nutrients for the human body. They play a crucial role in ensuring national grain and oil security, maintaining people's nutritional health, and promoting rural revitalization. Soybeans and peanuts are important grain crops, oil crops, and feed crops in my country, and are fundamental, critical, and strategic industries that are crucial to the national economy and people's livelihood.

[0003] The soybean and peanut industries face two common challenges. First, peanuts and soybeans are susceptible to contamination with highly toxic and carcinogenic aflatoxins, which not only reduce quality and production, but also pose a serious threat to people's health and safety. Aflatoxin B1, for example, is 10 times more toxic than potassium cyanide and is classified as a Class I carcinogen by the WHO's International Agency for Research on Cancer. It contributes to 28.2% of liver cancer worldwide. Peanut aflatoxin contamination is a major risk factor for the peanut and other industries. Existing methods for controlling aflatoxin in peanuts and soybeans primarily rely on temperature and humidity control during storage, transportation, and processing, which is energy-intensive and difficult to control. Aflatoxin contamination prevention and control remains a global challenge.

[0004] Secondly, legume crops like soybeans and peanuts, while nodulating and fixing nitrogen symbiotically with soil rhizobia, naturally have few nodules, a short nitrogen fixation period (it's generally believed that no nodules or nitrogen fixation occur in the first month after sowing, and no new nodules form during the pod-filling or fruit-full stages, while existing nodules begin to wither), resulting in low efficiency. Research on biological nitrogen fixation using rhizobia has a history of over 100 years, establishing the classically recognized AON theory—that plants self-regulate nodule number and growth while maintaining total energy conservation. Excessive nodulation inevitably comes at the expense of plant growth. Current approaches primarily rely on selecting and applying optimized rhizobia adapted to specific production environments. This approach is geographically limited and constrained by the AON theory, resulting in limited improvements in nodulation and nitrogen fixation efficiency, typically around 30%. Achieving a doubling of nodulation and nitrogen fixation efficiency while also significantly increasing growth (a challenge to the AON theory) is difficult. Improving the nodulation and nitrogen fixation efficiency of legumes like peanuts and soybeans remains a hotly debated and challenging issue internationally.

[0005] To address these challenges, the inventors' team has conducted over 20 years of continuous research, uncovering the spatiotemporal correlation between the abundance and toxicity of aflatoxin-producing fungi, geography, climate, and 53 other factors, and the occurrence of aflatoxin. They have developed highly sensitive detection and early warning technologies, identified the source of aflatoxin contamination in the soil, constructed a library of aflatoxin-producing strains, and for the first time proposed the scientific concept of coupling soil-source control of aflatoxin with nodulation and nitrogen fixation. By analyzing the microbial population structure within the peanut rhizosphere, isolating and identifying the strains and constructing a library of probiotic strains, and assembling a large number of combinations, they completed laboratory and field screening and identification, thus opening up research and exploration into the coupling of soil-source control of aflatoxin and induced nodulation and nitrogen fixation. Over the past five years, the successful development of the ARC microbial agent has enabled green control of aflatoxin at the source while simultaneously promoting efficient nodulation and nitrogen fixation in soybeans and peanuts, significantly increasing yields. This microbial agent, known as ARC (Aspergillus flavus / Aflatoxins and Rhizobia Coupling), combines its effectiveness in controlling aflatoxin contamination with promoting nodulation and nitrogen fixation. This agent is simple to use, low-cost, and highly effective. It boasts significant advantages: two fixes (nitrogen and carbon fixation), three increases (yield, efficiency, and safety), and five reductions (reducing toxicity, damage, weight, costs, and carbon emissions). This agent has demonstrated significant application potential and has been demonstrated and validated in field trials across major soybean, peanut, and pea producing regions across China. It holds significant promise for boosting soybean oilseed production capacity and promoting green, low-carbon, and efficient production in my country. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides a fermentation production method for an ARC microbial agent for coupled toxicity control and nitrogen fixation and yield increase, which can be used for batch production of an ARC microbial agent for coupled toxicity control and nitrogen fixation and yield increase, is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] On the one hand, the present invention provides an ARC microbial agent for coupled toxicity control and nitrogen fixation and yield increase, which is a microbial composition with a coupled toxicity control and nitrogen fixation effect, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, and contains all the gene sequences in DNA sequences 1 to 4 shown in SEQ ID No. 1-4.

[0009] According to the above scheme, the ARC microbial agent has an inhibitory effect on Aspergillus flavus and / or its toxins. Furthermore, the inhibition rate of Aspergillus flavus can reach more than 60%, and the inhibition rate of aflatoxin can reach more than 80%. The inhibition of Aspergillus flavus and / or toxins can be detected using other conventional detection methods in the prior art, or the inhibition analysis of Aspergillus flavus and / or its toxins can be performed by inhibition analysis of marker molecules of Aspergillus flavus toxin-producing fungi. Specifically, the inhibition analysis of ARC microbial agent on Aspergillus flavus and / or its toxins can be performed by inhibition analysis of marker molecules of Aspergillus flavus toxin-producing fungi.

[0010] According to the above scheme, the ARC microbial agent of the present invention significantly inhibits the expression of the Aspergillus flavus PAB-01 protein, the amino acid sequence of which is shown in SEQ ID No. 5. The inhibition rate is greater than 90%, preferably greater than 95%, reflecting the excellent antibacterial and antitoxic effects of the ARC microbial agent of the present invention. The method for determining the inhibition rate of Aspergillus flavus PAB-01 protein expression can be referenced in the following literature: Protein biomarker for early diagnosis of microbial toxin contamination: Using Aspergillus flavus as an example, Food Frontiers. 2023, 4, 2013-2023, DOI: 10.1002 / fft2.295.

[0011] According to the above scheme, preferably, the ARC microbial agent is a combination of three or more microorganisms.

[0012] According to the above scheme, the ARC microbial agent has an inhibitory effect on aflatoxin / its toxins.

[0013] According to the above scheme, the ARC microbial agent has an inhibitory effect on one or more pathogens (pathogenic factors) / toxins of soil-borne plant pathogens such as Penicillium, Aspergillus other than Aspergillus flavus, Fusarium, Sclerotium sclerotiorum, Pseudomonas solanacearum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani.

[0014] According to the above scheme, the ARC microbial agent can promote early nodulation of leguminous crops such as peanuts and soybeans and prolong the nodulation and nitrogen fixation time.

[0015] The DNA sequence genes shown in SEQ ID No. 1-4 may vary to a certain extent in different strains. When the degree of variation is small, such as no more than 10% base variation, preferably no more than 5% base variation, and more preferably no more than 1% base variation, that is, the identity is more than 90%, preferably more than 95%, and more preferably more than 99%, and when they have corresponding biological activity functions, these are called functional equivalents of the DNA sequences shown in SEQ ID No. 1-4, and containing these sequences is equivalent to containing the corresponding sequences of DNA sequences 1 to 4. Microbial compositions containing the DNA sequences shown in SEQ ID NO. 1 to 4 or their functional equivalents, and having the coupled effects of toxicity control and nitrogen fixation, and having the effects of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops are all ARC microbial agents of the present invention.

[0016] The aforementioned DNA sequences 1-4 are specific sequences obtained after comparison with Genbank genome data. They are directly or indirectly related to the function of the microbial agent of this patent. When containing all the gene sequences of the aforementioned DNA sequences 1-4, they have a coupled effect of controlling toxicity and nitrogen fixation, and have the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops and increasing the number of nodules in legume crops, thus forming the ARC microbial agent of the present invention. These genes may vary to a certain extent in different strains. If the degree of variation is small, such as no more than 10% base variation, and the corresponding biological activity is maintained, containing them is equivalent to containing the gene sequences shown in SEQ ID Nos. 1-4.

[0017] According to the above scheme, preferably, the above-mentioned ARC microbial agent can be, but is not limited to, a composition of the following four strains of microorganisms: Bacillus laterosporus with a preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595.

[0018] Bacillus laterosporus H-CB4802, deposit date is September 27, 2023, deposit number is CCTCC NO: M 20231815, classification name is: Brevibacillus laterosporus strain H-CB4802, the depository is China Center for Type Culture Collection, address is Wuhan University, Wuhan, China.

[0019] Bacillus amylolyticus AR1004, deposited on September 4, 2023, with a deposit number of CCTCC NO: M 20231598, is classified as Bacillus amylolyticus AR1004, and is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0020] Bacillus mucilaginosus JZ2013, deposited on September 27, 2023, with a deposit number of CCTCC NO: M 20231817, and a classification name of Bacillus mucilaginosus strain JZ2013. The depository institution is China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0021] Enterobacter ludwigii AR1001, deposit date is September 4, 2023, deposit number is CCTCC NO: M 20231595, classification name is: Enterobacter ludwigiiAR1001, deposit unit name is China Center for Type Culture Collection, address is Wuhan University, Wuhan, China.

[0022] According to the above scheme, preferably, the above-mentioned ARC microbial agent can be a combination of one or more microorganisms selected from Bacillus laterosporus with a preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595, and other microorganisms, so that the combined microbial agent satisfies the requirements of containing all gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 4 or their functional equivalents, has a coupled effect of toxicity control and nitrogen fixation, has the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops, thereby constituting the ARC microbial agent of the present invention.

[0023] According to the above scheme, the proportion of the number of viable bacteria of any one strain in the above microbial agent, i.e. the mixed microbial composition, is greater than or equal to 1%.

[0024] The ARC microbial agent of the present invention is a microbial composition. The synergistic action of the various microorganisms in the microbial composition produces a coupled effect of toxicity control and nitrogen fixation, exerting a coupled effect of toxicity control and nitrogen fixation. When used in crop production, it has a coupled effect of toxicity control and nitrogen fixation, plays a coupled role in preventing aflatoxin and its toxin pollution and promoting nodulation and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops and increasing the number of nodules in legume crops. Although it is not a rhizobium itself, it can regulate and increase the abundance of rhizobia in the rhizosphere of legume crops, increase the number of nodules in legume crops, and improve the nitrogenase activity of a single plant. It can increase the abundance of rhizobia in the rhizosphere of legume crops by at least 15%, and increase the number of nodules by more than 2 times. It can promote early nodulation of peanuts and soybeans and prolong the time of nodulation and nitrogen fixation.

[0025] The ARC microbial agent can be prepared by the following method: the microorganisms in the above microbial agent are combined and fermented. The fermentation route can adopt conventional fermentation routes of bacteria disclosed in the prior art, including existing literature.

[0026] The above-mentioned ARC microbial agent can be used in the production of leguminous crops as follows: to improve the quality and safety level of leguminous crop products; to promote nodulation and nitrogen fixation of leguminous crops; to increase the yield level of leguminous crops; to recruit indigenous rhizobia and increase the abundance of rhizobia in the rhizosphere soil of leguminous crops; to promote early nodulation of leguminous crops and prolong the nodulation and nitrogen fixation time; to prevent leguminous crops from premature aging due to lack of fertilizer during maturity; to increase the number of leguminous crop pods; to increase the fullness of leguminous crop pods and reduce the rate of shrunken pods; to promote early flowering and early pod formation of leguminous crops; to reduce the occurrence of peanut fruit rot; to reduce the occurrence of bacterial wilt of leguminous crops; to reduce the occurrence of powdery mildew of leguminous crops; to reduce the occurrence of leaf spot of leguminous crops; to reduce the occurrence of root nematode disease of leguminous crops; to reduce Used to reduce the occurrence of root rot in leguminous crops; used to reduce the occurrence of root nematode disease in leguminous crops; used to reduce the occurrence of blight in leguminous crops; used to reduce the occurrence of sclerotinia rot in leguminous crops; used to reduce the occurrence of downy mildew in leguminous crops; used to reduce the occurrence of wilt in leguminous crops; used to reduce the occurrence of white rot in leguminous crops; used to reduce the incidence of soybean green spondylosis; used to reduce the incidence of corn ear rot and Fusarium toxin; used to reduce the abundance of wheat fusarium and reduce the incidence of fusarium toxin; used to promote carbon emission reduction in leguminous crops, which is beneficial to soil improvement; used to promote the increase of total biomass of leguminous crops; used to reduce the abundance of pests such as Aspergillus terreus and Fusarium in the rhizosphere of leguminous crops, which is beneficial to improving the soil microbial population structure; used to reduce the surface spots of peanuts and increase commercial value; used to promote soybean production in saline-alkali land.

[0027] Another aspect of the present invention provides a fermentation production method of ARC microbial agent for controlling toxicity and fixing nitrogen coupled with increasing yield, which comprises the following steps in sequence:

[0028] Strain domestication: strain domestication to obtain the bacterial liquid of each strain of the combined ARC microbial agent for controlling toxicity and fixing nitrogen and increasing production;

[0029] Seed solution preparation: The seed solution of each strain of the ARC microbial agent for combined toxicity control and nitrogen fixation coupled with yield increase is prepared with the acclimated bacterial solution of each strain;

[0030] Fermentation production: The seed liquid of each strain of the combined ARC microbial agent for controlling toxicity and nitrogen fixation is fermented to obtain batch fermentation liquid of each strain of the combined ARC microbial agent;

[0031] Bacterial agent combination: The batch fermentation liquid of each strain of the above-mentioned combined toxic control and nitrogen fixation coupled with increased production ARC microbial agent or the bacterial powder prepared from the batch fermentation liquid of each strain of the combined toxic control and nitrogen fixation coupled with increased production ARC microbial agent obtained by fermentation production is combined to obtain a microbial composition containing the above-mentioned strains.

[0032] According to the above scheme, the domestication of the above-mentioned strains refers to the domestication of each strain of the assembled ARC microbial agent separately, and the domestication steps are as follows: 1) the frozen samples of each strain of the assembled ARC microbial agent are inoculated into liquid LB culture medium containing not less than 5% peanut powder or soybean powder, and cultured on a shaking table to obtain the zero bacterial liquid of the above-mentioned strain; 2) the above-mentioned zero bacterial liquid is streaked onto a conventional solid LB plate, and cultured in an incubator to obtain single colonies of the above-mentioned strain; 3) the single colonies of the above-mentioned strains are taken from the above-mentioned LB plate, inoculated into a conventional bacterial liquid fermentation liquid, and cultured at 30~37℃ for more than 15 hours to obtain the domesticated bacterial liquid of each strain of the above-mentioned assembled ARC microbial agent.

[0033] According to the above scheme, in the acclimation step, the shaking culture in step 1) is a shaking culture at a constant temperature of 37°C and a speed of 200 r / min for more than 15 hours; the culture in step 2) is a constant temperature culture at 37°C overnight.

[0034] Taking the ARC bacterial agent composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC M 20231595 as an example, the acclimation steps are as follows: 1) Frozen samples of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC M 20231595 in the above ARC bacterial agent are inoculated into liquid LB culture containing not less than 5% peanut powder or soybean powder, preferably, shaken and cultured at 37°C for 15 minutes at a constant temperature shaker and a speed of 200 r / min. h or more to obtain bacterial liquid zero of the above four strains; 2) streaking the above four bacterial liquid zeros onto conventional solid LB plates, preferably, placing them in an incubator and culturing them at a constant temperature of 37°C overnight to obtain single colonies of the above four strains; 3) taking single colonies of the above strains from the above LB plates, inoculating them into conventional bacterial liquid fermentation broth, preferably, culturing them at 30-37°C for more than 15 h, to obtain bacterial liquids after acclimation of the four strains of the above ARC bacterial agent.

[0035] According to the above scheme, the seed solution is prepared by: fermenting the acclimated bacterial solution of each strain of the above-mentioned assembled ARC microbial agent in a conventional seed fermentation tank step by step to obtain the seed solution of each strain of the above-mentioned assembled ARC microbial agent.

[0036] According to the above scheme, the fermentation production is as follows: the seed liquid of each strain of the assembled ARC microbial agent obtained above is respectively mixed with conventional bacterial fermentation medium at a volume ratio of 1:200~800 for scaled-up fermentation production, the temperature is 30~37°C, the tank pressure is 0.03~0.06 MPa, the initial speed is 150~300 r / min, the ventilation ratio is 0.8~1.5 V / V∙m, and the fermentation is carried out for more than 15 hours to obtain batch fermentation liquid of each strain of the assembled ARC microbial agent.

[0037] According to the above scheme, the above bacterial powder is prepared by using fermentation liquid powder spraying and drying equipment to spray the batch fermentation liquid of each strain of the ARC microbial agent separately by conventional powder spraying method to make the bacterial powder of the above four strains of the ARC microbial agent.

[0038] According to the above scheme, the above-mentioned microbial agent combination is: the batch fermentation liquid or bacterial powder of each strain of the above-mentioned ARC microbial agent is mixed uniformly according to a certain proportion to form a microbial composition containing the above-mentioned strains, namely the ARC microbial agent, which can be a liquid composition or a powdered composition.

[0039] According to the above scheme, the above certain ratio means that the ratio of the number of viable bacteria of any one of the strains in the composition of the ARC microbial inoculant is not less than 1%.

[0040] According to the above scheme, the above preparation method also includes preparation of dosage forms, which are prepared by adding a conventional protective agent to a liquid composition containing each strain of the assembled ARC microbial agent, i.e., a microbial composition obtained by combining batch fermentation broths of each strain of the assembled ARC microbial agent, to prepare an ARC microbial agent liquid dosage form; or adding a conventional protective agent to a microbial composition obtained by combining a powdered composition containing each strain of the assembled ARC microbial agent, i.e., a microbial powder prepared from batch fermentation broths of each strain of the assembled ARC microbial agent, to prepare an ARC microbial agent powder; or mixing the above liquid composition or powdered composition with a conventional carrier and then preparing an ARC microbial agent granular agent by conventional means such as drum granulation.

[0041] The fermentation process of the ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing yield of the present invention can be used for the fermentation production of the constituent strains of the ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing yield, and for the production of different dosage forms of the ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing yield. The ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing yield produced above can be used in the production of leguminous crops such as soybeans and peanuts. On the one hand, it can reduce the damage caused by pests such as Aspergillus flavus, and at the same time, it can promote nodulation and nitrogen fixation, increase yield per unit area, and reduce carbon emissions.

[0042] The beneficial effects of the present invention are:

[0043] 1. The ARC microbial inoculant fermentation process for toxin control and nitrogen fixation coupled with yield increase can be used for the fermentation production of ARC microbial inoculant strains and the production of ARC microbial inoculants in different dosage forms. It is easy to use, low-cost, and highly profitable. 2. The ARC microbial inoculant strains have undergone acclimation to the soybean or peanut environment during the fermentation process and are better able to adapt to the environment after being released into the field. 3. The ARC microbial inoculant for toxin control and nitrogen fixation coupled with yield increase can be used to increase the yield of legume crops such as peanuts and soybeans. Through a single technology, it achieves green control of aflatoxin sources while inducing and promoting efficient nodulation and nitrogen fixation in soybeans and peanuts, as well as significantly increasing yields. 4. It is of great significance to promoting the expansion of my country's soybean oilseed production capacity and promoting green, low-carbon, and efficient production. Modes for Carrying Out the Invention

[0044] Whole plants of peanuts, soybeans, peas, broad beans, cowpeas, and alfalfa, along with rhizosphere soil samples, were ground and mixed. Strains were isolated using conventional bacterial isolation methods and subsequently identified using conventional 16S rDNA analysis. Bacillus amyloliquefaciens, Brevibacillus laterosporus, Bacillus mucilaginosus, and Enterobacter ludwigii were obtained through these procedures. Details are shown in Table 1.

[0045] Table 1. Strains isolated and identified from mixtures of peanut, soybean and other major legume crops

[0046] Strain Code or Deposit Number Strain Name Strain Code or Deposit Number Strain Name CCTCC M 20231815 Brevibacillus laterosporus strain 202308 Bacillus amyloliquefaciens CCTCC M 20231598 Bacillus amyloliquefaciens strain 202311 Brevibacillus laterosporus CCTCC M 20231817 Bacillus mucilaginosus strain 202312 Bacillus mucilaginosus CCTCC M 20231595 Enterobacter ludwigii strain 202326 Enterobacter ludwigii strain 202330 Brevibacillus laterosporus Bacillus mucilaginosus

[0047] The ARC bacterial agent (agents 1, 2, 3, and 4) composed of Bacillus laterosporus (CCTCC M 20231815), Bacillus amyloliquefaciens (CCTCC M 20231598), Bacillus mucilaginosus (CCTCC M 20231817), and Enterobacter ludwigii (CCTCC M 20231595) is used as an example for specific description (see Examples 1-6). Other bacterial agents can be prepared similarly.

[0048] Example 1: Acclimation of the constituent strains of the ARC microbial agent

[0049] Taking the ARC bacterial agent composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter ludwigii with a deposit number of CCTCC M 20231595 as an example, the acclimation steps are as follows:

[0050] 1) Inoculate a frozen sample of Bacillus laterosporus (CCTCC M 20231815) into liquid LB medium containing at least 5% peanut powder and culture in a 37°C shaker at 200 rpm for at least 15 hours to obtain a strain zero of the strain.

[0051] 2) Inoculate a frozen sample of Bacillus amyloliquefaciens (CCTCC M 20231598) into liquid LB medium containing at least 5% peanut powder and culture in a 37°C shaker at 200 rpm for at least 15 hours to obtain a culture medium of the strain number zero.

[0052] 3) Inoculate a frozen sample of Bacillus mucilaginosus (accession number CCTCC M 20231817) into liquid LB medium containing at least 5% soybean meal and culture in a 37°C shaker at 200 rpm for at least 15 h to obtain a culture medium of the strain number zero.

[0053] 4) Inoculate a frozen sample of Enterobacter ludwigii (accession number CCTCC M 20231595) into liquid LB medium containing at least 5% soybean meal and culture in a 37°C shaker at 200 rpm for at least 15 h to obtain a strain zero of the strain.

[0054] 5) Streak each of the four No. 0 bacterial cultures onto different conventional solid LB plates and incubate overnight at 37°C in an incubator to obtain single colonies of each of the four strains.

[0055] 6) Take a single colony of each of the above strains from the LB plate, inoculate it into a conventional bacterial liquid fermentation broth, and culture it at 30-37°C for at least 15 hours to obtain the acclimated bacterial broth of each of the four strains of the ARC agent.

[0056] Example 2 Preparation of Seed Liquid of Four Strains of ARC Bacterial Agent

[0057] Taking the ARC bacterial agent composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter ludwigii with a deposit number of CCTCC M 20231595 as an example, the seed solution preparation steps are as follows:

[0058] 1) Preparation of primary seed solution: The bacterial solution of the four strains of the above-mentioned ARC agent after acclimation can be regarded as four primary seed solutions.

[0059] 2) Preparation of secondary seed solution: 10–30 mL of the acclimated bacterial solution of each of the four ARC strains was added to 6 L of conventional bacterial fermentation liquid medium. Fermentation conditions were as follows: temperature: 30–37°C, tank pressure: 0.03–0.06 MPa, initial rotation speed: 150–300 r / min, aeration ratio: 0.8–1.5 V / V∙m. Fermentation was continued for at least 15 h to obtain secondary seed solutions of each of the four ARC strains.

[0060] 3) Preparation of tertiary seed solution: The operation was similar to step 2. The culture medium accounted for approximately 60% of the tank volume. The volume ratio of the secondary seed solution to the culture medium was controlled between 1:200 and 800. The fermentation conditions were: temperature: 30-37°C, tank pressure: 0.03-0.06 MPa, initial rotation speed: 150-300 r / min, ventilation ratio: 0.8-1.5 V / V∙m, and fermentation for more than 15 h. Tertiary seed solutions of the four strains of the ARC inoculum were obtained.

[0061] Example 3 Fermentation production of ARC bacterial agent using four strains

[0062] Taking a 500 L fermenter and an ARC inoculum composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter ludwigii with a deposit number of CCTCC M 20231595 as an example, the fermentation production steps of the four strains of ARC inoculum are as follows:

[0063] 500-1500 mL of seed solution of each of the four strains of the ARC microbial agent obtained above was respectively added to different fermenters containing 300 L of conventional bacterial fermentation medium for scale-up fermentation production. The fermentation conditions were as follows: temperature: 30-37°C, tank pressure: 0.03-0.06 MPa, initial speed: 150-300 r / min, ventilation ratio: 0.8-1.5 V / V∙m, and fermentation for more than 15 h to obtain batch fermentation broth of the four strains of the ARC microbial agent.

[0064] Example 4 Preparation of ARC bacterial agent powders containing four strains

[0065] Taking the batch fermentation broth of the four strains of the ARC microbial agent as an example, the steps for preparing the microbial powder are as follows:

[0066] Fermentation liquid spray drying equipment is used, using batch fermentation liquid of the four strains with the above-mentioned deposit numbers of CCTCC M 20231815, Bacillus amyloliquefaciens, CCTCC M 20231817, and CCTCC M 20231595 as raw materials, and conventional spray drying method is used to prepare bacterial powders of the four strains of the above-mentioned ARC bacterial agent, and the moisture content of the finished product does not exceed 8%.

[0067] Example 5 ARC bacterial agent combination

[0068] Taking the ARC bacterial agent composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter ludwigii with a deposit number of CCTCC M 20231595 as an example, the preparation steps are as follows:

[0069] The batch fermentation liquid or bacterial powder of the above four strains are mixed uniformly in a certain proportion - any proportion of which is not less than 1% to form a microbial composition containing four strains, which can be a liquid composition or a powdered composition, wherein the feed can be batch fermentation liquid, bacterial powder, or both fermentation liquid and bacterial powder.

[0070] Table 1. ARC microbial agent composition information

[0071] Microbial agent number, agent composition and proportion of viable bacteria of the constituent strains (%) Microbial agent number, agent composition and proportion of viable bacteria of the constituent strains (%) Composition of combination 1: CCTCC M 20231815 / CCTCC M 20231598 / CCTCC M 20231817 / CCTCC M 20231595 Ratio: 33 / 33 / 1 / 33 Composition of combination 7: Strain 202330 / CCTCC M 20231598 / Strain 202334 / CCTCC M 20231595 Ratio: 33 / 1 / 33 / 33 Composition of combination 2: CCTCC M 20231815 / CCTCC M 20231598 / CCTCC M 20231817 / CCTCC M 20231595 Ratio: 1 / 33 / 33 / 33 Group 8 composition: strain 202330 / CCTCC M 20231598 / strain 202334 / CCTCC M 20231595 Ratio: 1 / 33 / 33 / 33 Group 3 composition: CCTCC M 20231815 / CCTCC M 20231598 / CCTCC M 20231817 / CCTCC M 2023159533 / 33 / 33 / 1 Group 9 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 Ratio: 33 / 33 / 33 / 1 Group 4 composition: CCTCC M 20231815 / CCTCC M 20231598 / CCTCC M 20231817 / CCTCC M 2023159533 / 1 / 33 / 33 Group 10 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 ratio: 33 / 1 / 33 / 33 Group 5 composition: strain 202330 / CCTCC M 20231598 / strain 202334 / CCTCC M 20231595 ratio: 30 / 10 / 30 / 30 Group 11 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 ratio: 33 / 33 / 1 / 33 Group 6 composition: strain / CCTCC M 20231598 / strain / CCTCC M 20231595 Ratio: 10 / 30 / 30 / 30 Combination 12 Composition: Strain 202308 / Strain 202311 / Strain 202312 / Strain 202326 Ratio: 1 / 33 / 33 / 33

[0072] Example 6 Preparation of ARC Inoculant Formulation

[0073] Taking the ARC bacterial agent composed of Bacillus laterosporus with a deposit number of CCTCC M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC M 20231817, and Enterobacter ludwigii with a deposit number of CCTCC M 20231595 as an example, the steps for preparing the dosage form are as follows:

[0074] 1) After compositions 1 to 12 in Table 1 are prepared from batch fermentation broth, conventional protective agents such as potassium sorbate and lactose are added to the compositions and mixed to prepare the ARC microbial agent liquid formulation.

[0075] 2) When compositions 1 to 12 in Table 1 are all composed of bacterial powders, conventional protective agents such as potassium sorbate and protein powder are added to the compositions and mixed to prepare the ARC microbial agent powder formulation.

[0076] 3) The liquid components and powder components are mixed with conventional carriers such as carbon powder, and then granulated by conventional means such as drum granulation to produce ARC microbial inoculant granules.

[0077] Example 7 Production of ARC bacterial agent

[0078] The ARC microbial inoculant fermentation process, i.e., the production method, of this patented technology can be used to produce ARC microbial inoculants in different dosage forms. According to Example 6, 12 liquid dosage forms, 12 powders, and 12 granules were produced in batches, as shown in Table 2.

[0079] Table 2. ARC microbial agent combination information

[0080] Produced inoculant number Combination of strains number Dosage form Produced inoculant number Combination of strains number Dosage form ARC-L1 Inoculant 1 liquid dosage form ARC-L2 Inoculant 2 liquid dosage form ARC-L3 Inoculant 3 liquid dosage form ARC-L4 Inoculant 4 liquid dosage form ARC-L5 Inoculant 5 liquid dosage form ARC-L6 Inoculant 6 liquid dosage form ARC-L7 Inoculant 7 liquid dosage form ARC-L8 Inoculant 8 liquid dosage form ARC-L9 Inoculant 9 liquid dosage form ARC-L10 Inoculant 10 liquid dosage form ARC-L11 Inoculant 11 liquid dosage form ARC-L12 Inoculant 12 liquid dosage form ARC-P1 Inoculant 1 powder ARC-P2 Inoculant 2 powder ARC-P3 Inoculant 3 powder ARC-P4 Inoculant 4 powder ARC-P5 Bacterial agent 5 powder ARC-P6 Bacterial agent 6 powder ARC-P7 Bacterial agent 7 powder ARC-P8 Bacterial agent 8 powder ARC-P9 Bacterial agent 9 powder ARC-P10 Bacterial agent 10 powder ARC-P7 Bacterial agent 11 powder ARC-P8 Bacterial agent 12 powder ARC-S1 Bacterial agent 1 granules ARC-S2 Bacterial agent 2 granules ARC-S3 Bacterial agent 3 granules ARC-S4 Bacterial agent 4 granules ARC-S5 Bacterial agent 5 granules ARC-S6 Bacterial agent 6 granules ARC-S7 Bacterial agent 7 granules ARC-S8 Bacterial agent 8 granules ARC-S9 Bacterial agent 9 granules ARC-S10 Bacterial agent 10 granules ARC-S11 Bacterial agent 11 granules ARC-S12 Bacterial agent 12 granules

[0081] Example 8 Application and Effect of ARC Microbial Agent in the Production of Leguminous Crops Such as Soybeans and Peanuts

[0082] The ARC microbial agent produced above can be used in the production of leguminous crops such as soybeans and peanuts. On the one hand, it can reduce the damage caused by pests such as aflatoxin, and at the same time promote nodulation and nitrogen fixation, increase yield and reduce carbon emissions.

[0083] (1) Determination of the toxicity control and nitrogen fixation effect of ARC microbial agent in peanut production

[0084] Taking peanuts as an example, the steps for determining the toxicity control and nitrogen fixation effects of the above-mentioned microbial agents are as follows.

[0085] On the one hand, the bacterial agents listed in Table 2 were co-cultured with toxigenic Aspergillus flavus strains under the same conditions, the expression level of PAB-01 was determined using the literature method, and the inhibition rate of the bacterial agents on PAB-01 expression was calculated. The results are shown in Table 3.

[0086] Alternatively, the microbial agents listed in Table 2 were applied to the fields along with the peanut sowing base fertilizer, or during the growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 1.5 billion). A control plot was established without any of the microbial agents, while all other plots were managed using conventional field management. Root nodulation in peanut seedlings was investigated 7 to 12 days after emergence; root nodulation in mature peanuts was investigated 1 to 3 days before harvest; and pest control effectiveness was investigated from the flowering stage to around harvest, specifically assessing field disease control and aflatoxin abundance in peanuts after harvest. Peanut yield was also investigated at harvest. The results are shown in Table 3.

[0087] Combined with the results in Table 3 above, the microbial agents in Table 2 also have the following characteristics: 1) The microbial agent contains all four gene sequences in DNA sequences 1 to 4, and these genes may have no more than 10% base variation in different strains; 2) The microbial agent has a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, has an inhibitory effect on Aspergillus flavus, and has an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium and Aspergillus other than Aspergillus flavus, Fusarium, and Pseudomonas solanacearum; 3) Although not a rhizobium itself, the ARC microbial agent can simultaneously regulate and increase the abundance of rhizobia in the peanut rhizosphere and increase the number of peanut root nodules; 4) It can promote early nodulation of peanuts and prolong the time of nodulation and nitrogen fixation; 5) It can significantly increase the yield per unit area of ​​peanuts; and 6) It has a significant carbon emission reduction effect.

[0088] The above-mentioned soil-borne plant pathogens are the pathogenic microorganisms of the above-mentioned corresponding field diseases. Therefore, the field disease prevention and control effect is equivalent to the inhibitory effect of ARC microbial agent on the pathogenic factors / toxins of the above-mentioned soil-borne plant pathogens.

[0089] Table 3. Results of the microbial agent test on the effects of peanut poison control and nitrogen fixation

[0090] Bacterial agent number: PAB-01 inhibition rate; Does it have an inhibitory effect on Aspergillus flavus / toxins?; Does it have an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium, Aspergillus other than Aspergillus flavus, Fusarium, and Pseudomonas solanacearum?; Promotion of nodulation and nitrogen fixation: Percent increase in rhizobium abundance in the rhizosphere / fold increase in the number of nodules; Does it promote early nodulation of peanuts and prolong the nodulation and nitrogen fixation period?; Does it have a significant carbon emission reduction effect?; Yield per mu increased; ARC-L1>95% yes yes >15% / > 2 times yes yes >10%; ARC-L2>95% yes yes >15% / > 2 times yes yes >10%; ARC-L3>95% yes yes >15% / > 2 times yes yes >10%; ARC-L4>95% yes yes >15% / > 2 times yes yes >10%; ARC-L5>95% yes yes >15% / > 2 times yes yes >10%; ARC-L6>95% yes yes >15% 2-fold yes > 10% ARC-L7 > 95% yes > 15% 2-fold yes > 10% ARC-L8 > 95% yes > 15% 2-fold yes > 10% ARC-L9 > 95% yes > 15% 2-fold yes > 10% ARC-L10 > 95% yes > 15% 2-fold yes > 10% ARC-L11 > 95% yes > 15% 2-fold yes > 10% ARC-L12 > 95% yes > 15% 2-fold yes > 10% ARC-P1 > 95% yes > 15% 2-fold yes > 10% ARC-P2 > 95% yes > 15% 2-fold yes > 10% ARC-P3 > 95% yes > 15% 2-fold yes > 10% ARC-P4 > 95% yes > 15% 2-fold yes > 10% ARC-P5 > 95% yes > 15% 2-fold yes > 10% ARC-P6 > 95% yes > 15% 2-fold yes > 10% ARC-P7 > 95% yes > 15% 2-fold yes > 10% ARC-P8 > 95% yes > 15% 2-fold yes > 10% ARC-P9 > 95% yes > 15% 2-fold yes > 10% ARC-P10 > 95% yes > 15% 2-fold yes > 10% ARC-P11 > 95% yes > 15% 2-fold yes > 10% ARC-P12 > 95% yes > 15% 2-fold yes > 10% ARC-S1 > 95% yes > 15% 2-fold yes > 10% ARC-S2 > 95% yes > 15% 2-fold yes > 10% ARC-S3 > 95% yes > 15% 2-fold yes > 10% ARC-S4 > 95% yes > 15% 2-fold yes > 10% ARC-S5 > 95% yes > 15% 2-fold yes > 10% ARC-S6 > 95% yes > 15% 2-fold yes > 10% ARC-S7 > 95% yes > 15%2x is >10% ARC-S8 >95% is >15% 2x is >10% ARC-S9 >95% is >15% 2x is >10% ARC-S10 >95% is >15% 2x is >10% ARC-S11 >95% is >15% 2x is >10% ARC-S12 >95% is >15% 2x is >10%

[0091] The above-mentioned microbial agent has an inhibition rate of more than 60% against aflatoxin / toxin, and further more than 90%; the inhibition rate of one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus, Penicillium, Fusarium, and Pseudomonas solanacearum other than aflatoxin can reach at least 30% or more, preferably more than 60%, and even more preferably 80% or even more than 90%.

[0092] (II) Determination of the toxicity control and nitrogen fixation effect of ARC microbial agent in soybean production

[0093] Taking soybean as an example, the steps for determining the toxicity control and nitrogen fixation effects of the above-mentioned microbial agents are described as follows.

[0094] On the one hand, the bacterial agents listed in Table 2 were co-cultured with toxigenic Aspergillus flavus strains under the same conditions, the expression level of PAB-01 was determined using the literature method, and the inhibition rate of the bacterial agents on PAB-01 expression was calculated. The results are shown in Table 2.

[0095] Alternatively, the microbial agents listed in Table 2 were applied to the fields along with the soybean sowing base fertilizer, or during the soybean growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 1.5 billion cells / acre). A control plot was established without any of the microbial agents. Conventional field management was used for all other plots. Root nodulation was investigated at the seedling stage 7 to 12 days after emergence; at the mature stage 1 to 3 days before harvest; and toxicity control effectiveness (i.e., effectiveness in preventing and controlling pest toxin-induced diseases) was investigated from the flowering and podding stage until harvest. At harvest, the yield improvement per mu (approximately 1.5 billion cells / acre) was investigated. The results are shown in Table 4.

[0096] Based on the above results, the inoculants in Table 2 have the following characteristics: 1) The ARC microbial inoculant contains all four gene sequences in DNA sequences 1 to 4, and these genes may have no more than 10% base variation in different strains; 2) The inoculant has a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, has an inhibitory effect on Aspergillus flavus, and has an inhibitory effect on soil-borne plant pathogens such as Penicillium and Aspergillus other than Aspergillus flavus, Fusarium, Sclerotinia, Phytophthora, Sclerotinia, Pythium, and Rhizoctonia; 3) Although not a rhizobium itself, the inoculant can simultaneously regulate and increase the abundance of rhizobia in the soybean rhizosphere and the number of soybean root nodules; 4) The inoculant can promote early soybean nodulation and prolong the time of nodulation and nitrogen fixation; 5) It can significantly increase the yield level; and 6) It has a significant carbon emission reduction effect.

[0097] Table 4. Results of microbial agents on the control of toxicity and nitrogen fixation in soybeans

[0098] Bacterial agent number: PAB-01 inhibition rate; Does it have an inhibitory effect on Aspergillus flavus / toxins?; Does it have an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium, Aspergillus other than Aspergillus flavus, Fusarium, and Pseudomonas solanacearum?; Promotion of nodulation and nitrogen fixation: Percent increase in rhizobium abundance in the rhizosphere / fold increase in the number of nodules; Does it promote early nodulation of peanuts and prolong the nodulation and nitrogen fixation period?; Does it have a significant carbon emission reduction effect?; Yield per mu increased by ARC-L1>95% yes yes>15% / >2-fold yes yes>8%; ARC-L2>95% yes yes>15% / >2-fold yes yes>8%; ARC-L3>95% yes yes>15% / >2-fold yes yes>8%; ARC-L4>95% yes yes>15% / >2-fold yes yes>8%; ARC-L5>95% yes yes>15% / >2-fold yes yes>8%; ARC-L6>95% yes yes>15% 2-fold yes > 8% ARC-L7 > 95% yes > 15% 2-fold yes > 8% ARC-L8 > 95% yes > 15% 2-fold yes > 8% ARC-L9 > 95% yes > 15% 2-fold yes > 8% ARC-L10 > 95% yes > 15% 2-fold yes > 8% ARC-L11 > 95% yes > 15% 2-fold yes > 8% ARC-L12 > 95% yes > 15% 2-fold yes > 8% ARC-P1 > 95% yes > 15% 2-fold yes > 8% ARC-P2 > 95% yes > 15% 2-fold yes > 8% ARC-P3 > 95% yes > 15% 2-fold yes > 8% ARC-P4 > 95% yes > 15% 2-fold yes > 8% ARC-P5 > 95% yes > 15% 2-fold yes > 8% ARC-P6 > 95% yes > 15% 2-fold yes > 8% ARC-P7 > 95% yes > 15% 2-fold yes > 8% ARC-P8 > 95% yes > 15% 2-fold yes > 8% ARC-P9 > 95% yes > 15% 2-fold yes > 8% ARC-P10 > 95% yes > 15% 2-fold yes > 8% ARC-P11 > 95% yes > 15% 2-fold yes > 8% ARC-P12 > 95% yes > 15% 2-fold yes > 8% ARC-S1 > 95% yes > 15% 2-fold yes > 8% ARC-S2 > 95% yes > 15% 2-fold yes > 8% ARC-S3 > 95% yes > 15% 2-fold yes > 8% ARC-S4 > 95% yes > 15% 2-fold yes > 8% ARC-S5 > 95% yes > 15% 2-fold yes > 8% ARC-S6 > 95% yes > 15% 2-fold yes > 8% ARC-S7 > 95% yes > 15% 2-fold yes > 8% ARC-S8 > 95% yes > 15% / >2-fold is >8%ARC-S9>95% is >15% / >2-fold is >8%ARC-S10>95% is >15% / >2-fold is >8%ARC-11>95% is >15% / >2-fold is >8%ARC-S12>95% is >15% / >2-fold is >8%

[0099] The above-mentioned microbial agent has an inhibition rate of more than 60% against aflatoxin / toxin, and further more than 90%; the inhibition rate of one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus, Penicillium, Fusarium, and Pseudomonas solanacearum other than aflatoxin can reach at least 30% or more, preferably more than 60%, and even more preferably 80% or even more than 90%.

[0100] (III) Determination of the effect of ARC microbial agent on toxicity control and nitrogen fixation in other leguminous crops

[0101] The sampling method and steps were the same as those for peanuts and soybeans mentioned above, and the ARC microbial agent in Table 2 was used to determine the toxicity control and nitrogen fixation effects on other legume crops such as peas, broad beans, cowpeas, and alfalfa, and the results were similar to those in Tables 3 and 4 above.

Claims

1. A method for producing ARC microbial inoculant fermentation by coupling toxicity control and nitrogen fixation with yield increase, characterized by: The following steps are included in sequence: Strain domestication: strain domestication to obtain the bacterial liquid of each strain of the combined ARC microbial agent for controlling toxicity and fixing nitrogen and increasing production; Seed solution preparation: using the acclimated bacterial solution of each strain of the ARC microbial agent to prepare the seed solution of each strain of the ARC microbial agent; Fermentation production: Fermentation of the seed liquid of each strain of the ARC microbial agent to obtain batch fermentation liquid of each strain of the ARC microbial agent; Bacterial agent combination: The batch fermentation broth of each strain of the above-mentioned assembled ARC microbial agent or the bacterial powder prepared from the batch fermentation broth of each strain of the assembled ARC microbial agent obtained by fermentation production is combined to obtain a microbial composition containing the above-mentioned strains. The ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing yield is a microbial composition, has a coupled effect of controlling toxicity and nitrogen fixation, has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, and contains the DNA sequences shown in SEQ ID No. 1-4.

2. The fermentation production method according to claim 1, characterized in that: The strain domestication refers to domesticating each strain of the combined ARC microbial agent separately, and the domestication steps are as follows: 1) Frozen samples of each strain of the combined ARC microbial agent for controlling toxicity and nitrogen fixation coupled with increasing production are inoculated into liquid LB culture medium containing not less than 5% peanut powder or soybean powder, and cultured on a shaking table to obtain the zero bacterial liquid of the above strain; 2) The zero bacterial liquid is streaked onto a conventional solid LB plate, placed in an incubator for culture, and single colonies of the above strain are obtained; 3) Single colonies of the above strains are taken from the above LB plate, inoculated into conventional bacterial liquid fermentation liquid, and cultured at 30-37°C for more than 15 hours to obtain the domesticated bacterial liquid of each strain of the combined ARC microbial agent.

3. The fermentation production method according to claim 1, characterized in that: The seed solution is prepared by: fermenting the acclimated bacterial liquid of each strain of the combined toxicity-controlling and nitrogen-fixing coupled yield-increasing ARC microbial agent in a conventional seed fermentation tank step by step to obtain the seed solution of each strain of the combined toxicity-controlling and nitrogen-fixing coupled yield-increasing ARC microbial agent.

4. The fermentation production method according to claim 1, characterized in that: The fermentation production comprises the following steps: the seed liquid of each strain of the obtained combined ARC microbial agent for controlling toxicity and fixing nitrogen coupled with increasing production is respectively mixed with a conventional bacterial fermentation medium at a volume ratio of 1:200-800 for scaled-up fermentation production, the temperature being 30-37°C, the tank pressure being 0.03-0.06 MPa, the initial rotation speed being 150-300 r / min, the ventilation ratio being 0.8-1.5 V / V∙m, and the fermentation being carried out for more than 15 hours to obtain batch fermentation liquid of each strain of the combined ARC microbial agent.

5. The fermentation production method according to claim 1, characterized in that: The bacterial powder is prepared by using fermentation liquid powder spraying and drying equipment to spray batch fermentation liquids of various strains of the combined ARC microbial agent for controlling toxicity, fixing nitrogen and increasing production respectively by conventional powder spraying method to prepare bacterial powders of four strains of ARC microbial agent.

6. The fermentation production method according to claim 1, characterized in that: The microbial agent combination is: the batch fermentation liquid or bacterial powder of each strain of the combined toxic control and nitrogen fixation coupled with production increase ARC microbial agent is mixed uniformly in a certain proportion to form a microbial composition containing the above strains, namely the toxic control and nitrogen fixation coupled with production increase ARC microbial agent, which is a liquid composition or a powdered composition.

7. The fermentation production method according to claim 6, characterized in that: The certain proportion means that the proportion of viable bacteria count of any one of the various strains of the combined ARC microbial agent for controlling toxicity and nitrogen fixation and increasing production is not less than 1%.

8. The fermentation production method according to claim 6, characterized in that: The invention also includes preparation of dosage forms, which is prepared by adding a conventional protective agent to a liquid composition containing the various strains of the combined ARC microbial inoculant for controlling toxicity and nitrogen fixation coupled with increasing production, i.e., a microbial composition obtained by combining the batch fermentation liquid of the various strains of the combined ARC microbial inoculant, to prepare a liquid dosage form of the ARC microbial inoculant; or adding a conventional protective agent to a microbial composition obtained by combining the powdered composition containing the various strains of the combined ARC microbial inoculant, i.e., a microbial composition obtained by combining the powder prepared from the batch fermentation liquid of the various strains of the combined ARC microbial inoculant, to prepare a powdered ARC microbial inoculant; or mixing the above-mentioned liquid composition or powdered composition with a conventional carrier to prepare a granular ARC microbial inoculant for controlling toxicity and nitrogen fixation coupled with increasing production.

9. The fermentation production method according to claim 1, characterized in that: The ARC microbial agent for controlling poison and fixing nitrogen coupled with increasing production has an inhibitory effect on aflatoxin / toxin.

10. The fermentation production method according to claim 1, characterized in that: The ARC microbial agent has an inhibitory effect on one or more pathogens / toxins of Aspergillus other than Penicillium and Aspergillus flavus, Fusarium, Sclerotium sclerotiorum, Pseudomonas solanacearum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia soil-borne plant pathogens.

11. The fermentation production method according to claim 1, characterized in that: The DNA sequence genes shown in SEQ ID No. 1-4 may have a certain degree of variation in different strains. When the degree of variation is small, not exceeding 10% of the base variation and having the corresponding biological activity function, they constitute the functional equivalents of the DNA sequences shown in SEQ ID No. 1-4. The microbial composition contains all the gene sequences shown in SEQ ID NO. 1~4 or their functional equivalents, and has a coupled effect of toxic control and nitrogen fixation, and has the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules of leguminous crops, constituting the ARC microbial agent described in claim 1.

12. The fermentation production method according to claim 1, characterized in that: The ARC microbial agent for controlling toxicity and fixing nitrogen coupled with increasing production is a composition of the following four microorganisms: Bacillus laterosporus with a preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595. Or it is a combination of one or more microorganisms selected from Bacillus laterosporus with a preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595, and other microorganisms, so that the combined microbial agent meets the requirements of containing all gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 4 or their functional equivalents, has a coupled effect of toxic control and nitrogen fixation, and has the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, thereby constituting the ARC microbial agent described in claim 1.