Use of arc microbial agent for nodulation, nitrogen fixation, quality improvement and yield increase in leguminous grain and oil crop

Abstract

The present invention belongs to the field of microorganisms. Disclosed is the use of an ARC microbial agent for nodulation, nitrogen fixation, quality improvement and yield increase in a leguminous grain and oil crop. The leguminous grain and oil crop is peanut, soybean or a leguminous coarse cereal crop. The ARC microbial agent is a microbial composition, has a coupled effect of toxicity control and nitrogen fixation, and has the effects of regulating and improving the abundance of rhizobia in the rhizosphere of a leguminous crop and increasing the number of nodules in the leguminous crop. The ARC microbial agent contains DNA sequences as shown in SEQ ID NOs: 1-4. The ARC microbial agent in the present invention can be used for nodulation, nitrogen fixation, quality improvement and yield increase in a leguminous grain and oil crop, is simple to use, has significant social and ecological benefits, and is easy to popularize and apply.

Description

Application of ARC microbial agent in nodulation, nitrogen fixation, quality improvement and yield increase of leguminous grain and oil crops Technical Field

[0001] The present invention belongs to the field of microorganisms, and in particular relates to the use of ARC microbial agent for nodulation, nitrogen fixation, quality improvement and yield increase of leguminous grain and oil crops. Background Art

[0002] Peanuts are an important food, oil, and feed crop in my country. Improving peanut quality and safety and reducing losses caused by aflatoxin contamination are of vital practical significance for ensuring food safety and promoting high-quality development of the peanut industry. Increasing peanut yield and total biomass is also crucial.

[0003] Soybeans are an important food, oil, and feed crop in my country, and a fundamental, critical, and strategic industry crucial to national economy and people's livelihood. Improving soybean quality and reducing the risk of aflatoxin contamination are crucial for the high-quality development of the soybean industry. Increasing soybean yields and increasing production capacity are crucial for ensuring soybean supply.

[0004] Adzuki beans (also known as red adzuki beans, red beans), mung beans, kidney beans, and lentils are important legume grains rich in nutrients. Increasing the yield of legume grains is crucial for meeting people's needs for a better and healthier life.

[0005] Although leguminous grains have the habit of symbiotically forming nodules and fixing nitrogen with soil rhizobia, the number of nodules is small under natural conditions, the nitrogen fixation time is short, and the efficiency is low.

[0006] The development of the peanut and soybean industries faces two major global challenges. First, peanuts and soybeans are susceptible to contamination with highly toxic and carcinogenic aflatoxins, which not only reduces quality and reduces production, but also seriously threatens people's lives and health. Take aflatoxin B1 as an example. Its toxicity is 10 times that of potassium cyanide. It is classified as a Class I carcinogen by the WHO International Agency for Research on Cancer and causes 28.2% of human liver cancer worldwide. In recent years, aflatoxin contamination of peanuts has generally shown an increasing trend and has become a major risk factor for industries such as peanuts. Aflatoxin contamination of peanuts often leads to huge food losses. Existing methods for controlling aflatoxin in peanuts mainly rely on temperature and humidity control in the storage, transportation, and processing stages, which are energy-intensive and difficult to control. Aflatoxin contamination prevention and control has always been a global problem.

[0007] Secondly, legume crops like peanuts and soybeans, while symbiotically forming nodules and fixing nitrogen with soil rhizobia, naturally have few nodules, a short nitrogen fixation period (it's generally believed that no nodules and 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. However, these approaches are limited in scope and have long been constrained by the AON theory. Improvements in nodulation and nitrogen fixation efficiency are limited, with the best results being around 30%. Achieving a doubling of nodulation and nitrogen fixation efficiency while also significantly increasing growth (a challenge that defies the AON theory) is elusive. Improving the nodulation and nitrogen fixation efficiency of legumes like peanuts remains a hotly debated and challenging issue internationally.

[0008] 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 ARC microbial agent has been successfully developed. This agent effectively controls 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 fixations (nitrogen and carbon fixation), three increases (increased yield, efficiency, and safety), and five reductions (reduced toxicity, damage, weight, costs, and carbon emissions). This agent has demonstrated and validated its potential in field trials across major soybean, peanut, and pea production regions nationwide, demonstrating its significant potential for boosting peanut yields, increasing production capacity, and promoting green, low-carbon, and efficient production.

[0009] The applicant's research team further discovered that the application of ARC microbial agents also significantly improved the quality and quality safety level of peanuts and reduced the losses caused by aflatoxin contamination, thus providing a new solution for improving the quality and quality safety level of peanut production and reducing the losses of aflatoxin-contaminated peanuts. Summary of the Invention

[0010] In view of the shortcomings of the existing technology, the present invention provides an ARC microbial agent for the nodulation and nitrogen fixation of leguminous grain and oil crops and the improvement of quality and yield. The ARC microbial agent is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0012] The present invention first 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.

[0013] 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.

[0014] 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.

[0015] According to the above scheme, preferably, the ARC microbial agent is a combination of three or more microorganisms.

[0016] According to the above scheme, the ARC microbial agent has an inhibitory effect on aflatoxin / its toxins.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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%.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Based on the above research, the present invention further studies and provides the use of ARC microbial agent for nodulation and nitrogen fixation and quality improvement and yield increase of leguminous grain and oil crops. The leguminous grain and oil crops are peanuts, soybeans or leguminous grain crops. The use of ARC microbial agent for nodulation and nitrogen fixation and quality improvement and yield increase of leguminous grain and oil crops is: used to improve the quality and quality safety of peanuts and reduce losses, or promote peanut nodulation and nitrogen fixation to achieve green and low-carbon production, or used to increase peanut yield level and total biomass; or used to improve soybean quality and reduce the risk of aflatoxin contamination, or promote soybean nodulation and nitrogen fixation to achieve green and low-carbon production, or increase soybean yield level and total biomass; or promote leguminous grain crops Nodulation and nitrogen fixation and increase yield level.

[0032] The details are as follows:

[0033] Provided is the use of ARC microbial agent for improving peanut quality and safety and reducing peanut losses.

[0034] A method for improving peanut quality and safety and reducing losses, comprising applying an ARC microbial agent to peanut production.

[0035] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0036] According to the above scheme, the application method is one or more of broadcasting, spraying, drip irrigation, etc., and the application stage is when peanuts are sown and / or from the emergence of peanuts to the flowering and needle-setting period.

[0037] According to the above plan, the above-mentioned ARC microbial agent is mixed with the peanut sowing base fertilizer, and applied evenly into the field by one or more of the following methods: manual, seeding machine, drone, etc. The application rate of the agent is 80 billion to 100 billion live bacteria per mu. After sowing, drip irrigation can be adopted for fields with conditions to avoid severe dryness in the field, uneven emergence, etc., and conventional field management is adopted for other fields.

[0038] The ARC microbial agent can also be applied to peanut production as follows: After normal peanut sowing and emergence, the ARC microbial agent is evenly applied to the field through one or more topdressing methods, such as broadcasting, spraying, and drip irrigation, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Conventional field management is used for all other aspects.

[0039] Provided is the use of ARC microbial agent for promoting peanut nodulation and nitrogen fixation to achieve green and low-carbon production.

[0040] Furthermore, the present invention also provides a method for promoting peanut nodulation and nitrogen fixation to achieve green and low-carbon production, which comprises applying ARC microbial agent to peanut production.

[0041] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0042] According to the above scheme, the application method is one or a combination of broadcasting, spraying, drip irrigation, etc., and the application stage is when peanuts are sown and / or from the emergence of peanuts to the flowering and needle-setting period.

[0043] According to the above plan, the application method of ARC microbial agent in peanut production can be as follows: mix the above ARC microbial agent with peanut sowing base fertilizer, and apply it evenly to the field by manual means, seed drill or drone, etc. The application rate of the agent is 80 billion to 100 billion live bacteria per mu. After sowing, drip irrigation can be adopted for fields with conditions to avoid severe dryness in the field, uneven emergence, etc., and conventional field management is adopted for other purposes.

[0044] According to the above scheme, the application method of ARC microbial agent in peanut production can also be as follows: After the peanuts are sown and seedlings have emerged normally, and until the flowering and needle setting stage, the above-mentioned microbial agent should be evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods, with the application rate of the agent accumulating 80 billion to 100 billion viable bacteria per mu. All other field management methods are the same.

[0045] The application of the above-mentioned ARC microbial agent can significantly promote peanut nodulation and nitrogen fixation, reduce carbon dioxide emissions, and achieve green and low-carbon production.

[0046] Provided is the use of an ARC microbial agent for increasing the yield per unit area and the total biomass of peanuts.

[0047] A method for increasing peanut yield and total biomass, the method comprising applying an ARC microbial inoculant to peanut production.

[0048] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0049] According to the above scheme, the application method is one or more of broadcasting, spraying, drip irrigation, etc., and the application stage is when peanuts are sown and / or from the emergence of peanuts to the flowering and needle-setting period.

[0050] According to the above scheme, the ARC microbial agent can be applied to peanut production by mixing it with a base fertilizer for peanut sowing and evenly applying it to the field using one or a combination of methods, such as manual application, seed drills, or drones, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be used where possible to minimize dryness and uneven seedling emergence. Conventional field management is followed in all other areas.

[0051] The ARC microbial agent can also be applied to peanut production as follows: After normal peanut sowing and emergence, the ARC microbial agent is evenly applied to the field through one or more topdressing methods, such as broadcasting, spraying, and drip irrigation, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Conventional field management is used for all other aspects.

[0052] Provided is the use of ARC microbial agent for improving soybean quality and reducing the risk of aflatoxin contamination.

[0053] A method for improving soybean quality and reducing the risk of aflatoxin contamination, comprising applying an ARC microbial agent to soybean crops.

[0054] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0055] According to the above scheme, the application method is one or a combination of broadcasting, spraying, drip irrigation, etc., and the application stage is when soybeans are sown and / or from soybean emergence to flowering.

[0056] According to the above plan, the above-mentioned ARC microbial agent is mixed with the soybean sowing base fertilizer, and applied evenly into the field by one or more of the following methods: manual, seeding machine, drone, drip irrigation pipe, etc. The application rate of the agent is 80 billion to 100 billion live bacteria per mu. After sowing, drip irrigation can be adopted for fields with conditions to avoid severe dryness in the field, uneven emergence, etc., and conventional field management is adopted for other fields.

[0057] The ARC microbial agent can also be applied to soybean production as follows: After soybeans have been sown and seeded normally, until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods, or a combination thereof, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is followed for all other aspects.

[0058] Provided is the use of ARC microbial agent for promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production.

[0059] The present invention further provides a method for promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production, which comprises applying ARC microbial agent to soybean production.

[0060] According to the above scheme, the method includes applying the ARC microbial agent to the soybean crop.

[0061] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0062] According to the above scheme, the application method is broadcasting, spraying, drip irrigation, etc., and the application stage is at soybean sowing and / or after seedling emergence until the flowering and podding stage. The application method of the above-mentioned ARC microbial agent in soybean production can be as follows: the above-mentioned ARC microbial agent is mixed with soybean sowing base fertilizer and evenly applied to the field by one or more methods such as manual application, seed drill, drone, drip irrigation pipe, etc., with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be used if conditions permit to minimize severe field dryness and uneven seedling emergence. Otherwise, conventional field management is used.

[0063] The ARC microbial agent can also be applied to soybean production as follows: After soybeans have been sown and seeded normally, until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods, or a combination thereof, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is followed for all other aspects.

[0064] Provided is the use of an ARC microbial agent for increasing soybean yield and total biomass.

[0065] The present invention further provides a method for increasing soybean yield and total biomass, the method comprising applying the ARC microbial inoculant to soybean production.

[0066] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0067] According to the above scheme, the application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is when soybeans are sown and / or from soybean emergence to flowering.

[0068] The application method of the above-mentioned ARC microbial agent in soybean production can be as follows: mix the above-mentioned ARC microbial agent with soybean sowing base fertilizer, and evenly apply it to the field through one or more combinations of manual methods, seed drills, drones, drip irrigation pipes, etc. The application rate of the agent is 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be adopted for fields where conditions permit, and try to avoid severe dryness in the field, uneven emergence of seedlings, etc., and conventional field management is adopted for other purposes.

[0069] The ARC microbial agent can also be applied to soybean production as follows: After soybeans have been sown and seeded normally, until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods, or a combination thereof, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is followed for all other aspects.

[0070] Provided is the use of ARC microbial agent for promoting nodulation and nitrogen fixation in leguminous grain crops and increasing yield.

[0071] The present invention also provides a method for promoting nodulation and nitrogen fixation of leguminous grain crops and increasing yield, which comprises applying an ARC microbial agent to the leguminous grain crops.

[0072] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0073] According to the above scheme, the application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is when leguminous grain crops are sown and / or from the emergence of peanuts to the flowering stage.

[0074] The specific application method of the above-mentioned ARC microbial agent in the production of leguminous grain crops can be as follows: mix the above-mentioned ARC microbial agent with the base fertilizer for sowing of leguminous grain crops, and apply it evenly to the field by manual means, seeding machines or drones, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be adopted for fields where conditions permit, and try to avoid severe dryness in the field, uneven emergence of seedlings, etc., and conventional field management is adopted for other purposes.

[0075] The specific application method of the ARC microbial agent in the production of leguminous grain crops can also be as follows: after the leguminous grain crops have been sown and seeded normally, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods until the flowering stage, with the application rate of the agent reaching 80 billion to 100 billion viable bacteria per mu. All other conventional field management methods are followed.

[0076] According to the above scheme, the above-mentioned leguminous grains include but are not limited to red beans, green beans, flower beans, lentils, etc.

[0077] The beneficial effects of the present invention are:

[0078] 1. ARC microbial agent can be used for nodulation and nitrogen fixation of leguminous grain and oil crops, and to improve quality and yield. It can improve the quality and safety of peanuts and reduce peanut losses caused by aflatoxin contamination; significantly promote peanut nodulation and nitrogen fixation to achieve green and low-carbon production; can be used to increase peanut yield and total biomass; can be used to improve soybean quality and reduce the risk of aflatoxin contamination; can be used to promote soybean nodulation and nitrogen fixation to achieve green and low-carbon production; can be used to increase soybean yield and total biomass; can be used to promote nodulation and nitrogen fixation of leguminous grain crops and increase their yield. 2. It is easy to use, low-cost, and highly effective. 3. It is of great significance to ensuring peanut food safety and promoting the high-quality development of peanut production in my country; it is of great significance to promoting the increase of peanut yield and production capacity in my country; it is of great significance to promoting the high-quality development of my country's soybean industry and ensuring consumer safety; it is of great significance to promoting the increase of production capacity of leguminous grains and the green, low-carbon, and efficient production. Modes for Carrying Out the Invention

[0079] Part I ARC Microbial Agents

[0080] Example 1 Preparation of ARC microbial agent

[0081] Whole plants of peanut, soybean, pea, broad bean, cowpea, 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.

[0082] Table 1. Some of the strains isolated and identified from a mixture of major legume crops such as peanuts and soybeans are shown below:

[0083] 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

[0084] The 10 microbial strains in Table 1 were amplified one by one by conventional bacterial culture medium amplification method to prepare batches of fermentation broth or bacterial powder of the 10 strains.

[0085] The above-mentioned Bacillus laterosporus with a deposit number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC NO: M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC NO: M 20231595 are mixed with fermentation broths or bacterial powders of the four strains; and the combination information of the microbial agents formed by mixing some strains of Bacillus laterosporus with a deposit number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC NO: M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC NO: M 20231595 and other strains, as well as some other strains are mixed to form a microbial agent. The proportion of the viable count of any one strain in each microbial combination is greater than or equal to 1%.

[0086] Table 2. Microbial composition and bacterial agent information

[0087] Microbial agent number Microbial agent composition and proportion of viable bacteria count of constituent strains (%) Microbial agent number Microbial agent composition and proportion of viable bacteria count of constituent strains (%) Composition of agent 1: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 Ratio: 33 / 33 / 1 / 33 Composition of agent 7: Strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 33 / 1 / 33 / 33 Composition of agent 2: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 ratio: 1 / 33 / 33 / 33 Bacterial agent 8 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC M 20231595 ratio: 1 / 33 / 33 / 33 Bacterial agent 3 composition: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 ratio: 33 / 33 / 33 / 1 Bacterial agent 9 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 ratio: 33 / 33 / 33 / 1 Bacterial agent 4 composition: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 Ratio: 33 / 1 / 33 / 33 Bacterial agent 10 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 Ratio: 33 / 1 / 33 / 33 Bacterial agent 5 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 30 / 10 / 30 / 30 Bacterial agent 11 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 Ratio: 33 / 33 / 1 / 33 Bacterial agent 6 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 10 / 30 / 30 / 30 Inoculum 12 Composition: strain 202308 / strain 202311 / Strain 202312 / Strain 202326 Ratio: 1 / 33 / 33 / 33

[0088] Example 2: Sequencing of ARC microbial agents

[0089] A sufficient number of samples were taken from the microbial agents in Table 2 of Example 1, and total DNA was extracted from these samples in sequence using a conventional DNA extraction method. The DNA sequences of these samples were then determined using a conventional DNA sequencing method. Finally, conventional analysis methods were used to compare the homology of the DNA sequences determined above with the gene sequences shown in SEQ ID No. 1-4 provided in this patent text.

[0090] The results of the above determination and homology analysis showed that the bacterial agents 1 to 4 in Table 2 contained all four genes in the DNA sequences 1 to 4 shown in SEQ ID No. 1 to 4, respectively, and the sequence homology was 100%; the bacterial agents 5 to 12 in Table 1 contained all four genes in the DNA sequences 1 to 4 shown in SEQ ID No. 1 to 4, respectively, but with certain variations, and the four gene sequences had 90.3-100% homology with the DNA sequences 1 to 4.

[0091] Example 3: Determination of the Toxic Control and Nitrogen Fixation Effect of Peanut ARC Microbial Agent

[0092] Taking peanuts as an example, the steps for determining the toxicity control and nitrogen fixation effects of the above-mentioned ARC microbial agent are described as follows.

[0093] On the one hand, the bacterial agents described in Table 2 of Example 1 were co-cultured with a toxin-producing Aspergillus flavus strain under the same conditions. The expression of PAB-01 was measured, and the inhibition rate of PAB-01 expression was calculated. The results are shown in Table 3. The method for determining the inhibition rate of Aspergillus flavus protein expression can be referred to the method 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.

[0094] On the other hand, microbial agents 1-12 from Table 2 of Example 1 were applied to the field along with the peanut sowing base fertilizer, or during the peanut growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 100 million). A plot without any of the microbial agents was set up as a control, while all other plots were managed using conventional field management. Root nodulation of peanuts in the seedling stage was investigated 7 to 12 days after emergence; root nodulation of peanuts in the mature stage was investigated 1 to 3 days before harvest; and the effectiveness of toxicity control was investigated from the flowering stage to around harvest. Specifically, the effectiveness of field disease control and the abundance of aflatoxin in peanuts after harvest were investigated. The results of the investigation are shown in Table 3.

[0095] Based on the above results, microbial agents 1 to 12 simultaneously possess the following characteristics: 1) The microbial agents contain 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 agents have a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, have an inhibitory effect on Aspergillus flavus, and have 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.

[0096] 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.

[0097] Table 3. Results of the microbial agent test on the control of toxicity and nitrogen fixation in peanuts

[0098] Microbial agent number Inhibition rate of PAB-01 (%) Whether it has an inhibitory effect on aflatoxin / toxin Whether it has an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium, Aspergillus other than aflatoxin, Fusarium, Pseudomonas solanacearum, etc. Promotion of nodulation and nitrogen fixation: Percent increase in rhizospheric rhizobium abundance (%) / fold increase in nodule number (fold) Whether it promotes early nodulation of peanut and prolongs the time of nodulation and nitrogen fixation Agent 199 Yes Yes 23 / 16.6 Yes Agent 297 Yes Yes 20 / 13.7 Yes Agent 398 Yes Yes 22 / 15.3 Yes Agent 499 Yes Yes 18 / 6.5 Yes Agent 597 Yes Yes 21 / 14.2 Yes Agent 698 Yes Yes 22 / 15.5 Yes Agent 797 Yes Yes 19 / 8.1 Yes Agent 899 Yes Yes 18 / 7.1 Yes Agent 997 Yes Yes 20 / 10.2 Yes Agent 61098 Yes Yes 26 / 22.0 Yes Agent 1199 Yes Yes 17 / 3.2 Yes Agent 1296 Yes Yes 19 / 7.3 Yes

[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] Example 4: Determination of the Toxic Control and Nitrogen Fixation Effect of Soybean Microbial Agents

[0101] 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.

[0102] On the one hand, the bacterial agent in Table 2 of Example 1 above was co-cultured with the toxigenic Aspergillus flavus strain under the same conditions, the expression level of PAB-01 was determined using the literature method, and the inhibition rate of the bacterial agent on PAB-01 expression was calculated. The results are shown in Table 3.

[0103] On the other hand, microbial agents 1-12 from Table 2 of Example 1 were applied to the field along with soybean sowing base fertilizer, or during the soybean growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 100 million). A control plot was established without any of the microbial agents. Conventional field management was used for all other plots. Root nodulation in soybean seedlings was investigated 7 to 12 days after emergence; root nodulation in peanuts was investigated 1 to 3 days before harvest; and toxicity control efficacy, i.e., effectiveness in preventing and controlling pest toxin-induced diseases, was investigated from the flowering stage to harvest. The results are shown in Table 4.

[0104] Based on the above results, agents 1-12 simultaneously possess the following characteristics: 1) ARC microbial agents contain all four gene sequences in DNA sequences 1-4, and these genes may have no more than 10% base variation in different strains; 2) the agents have a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, have an inhibitory effect on Aspergillus flavus, and have inhibitory effects on soil-borne plant pathogens other than Penicillium and Aspergillus, Fusarium, Sclerotium sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani; 3) although not rhizobia themselves, the agents can simultaneously regulate and increase the abundance of rhizobia in the soybean rhizosphere and the number of soybean root nodules; 4) the agents can promote early soybean nodulation and prolong the time of nodulation and nitrogen fixation.

[0105] 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.

[0106] Table 4. Results of microbial agents on the control of toxicity and nitrogen fixation in soybeans

[0107] Microbial agent number Inhibition rate of PAB-01 (%) Whether it has an inhibitory effect on Aspergillus flavus / toxins Whether it has an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus other than Aspergillus flavus, Fusarium, Sclerotinia sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani Percent increase in rhizospheric rhizobium abundance (%) / fold increase in nodule number (fold) Whether it promotes early nodulation of soybean and prolongs the time of nodulation and nitrogen fixation Bacterial agent 199 Yes Yes 22 / 15 Yes Bacterial agent 297 Yes Yes 21 / 12 Yes Bacterial agent 398 Yes Yes 22 / 16 Yes Bacterial agent 499 Yes Yes 19 / 8 Yes Bacterial agent 598 Yes Yes 20 / 13 Yes Bacterial agent 697 Yes Yes 23 / 15 Yes Bacterial agent 799 Yes Yes 18 / 6 Yes Bacterial agent 896 Yes Yes 19 / 7 Yes Bacterial agent 997 Yes Yes 20 / 11 Yes Bacterial agent 1098 Yes Yes 22 / 13 Yes Bacterial agent 1199 Yes Yes 17 / 3 Yes Bacterial agent 1296 Yes Yes 19 / 6 Yes

[0108] 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, Fusarium, Sclerotinia, Phytophthora, Sclerotinia, Pythium, and Rhizoctonia other than Aspergillus flavus can reach at least 30% or more, preferably more than 60%, and more preferably 80% or even more than 90%.

[0109] Example 5: Determination of the Effect of Microbial Agents on Toxic Control and Nitrogen Fixation in Other Leguminous Crops

[0110] The sampling method and steps similar to those in Examples 3 and 4 were used to determine the toxicity control and nitrogen fixation effects of ARC microbial agents 1 to 12 on other leguminous crops such as peas, broad beans, cowpeas, and alfalfa, and the results were similar to those in Tables 3 and 4.

[0111] Part 2 Application of ARC Microbial Agents

[0112] Example 6: Use of ARC microbial agent - used to promote peanut nodulation and nitrogen fixation to achieve green and low-carbon production.

[0113] ARC microbial agents 1-8 were mixed with peanut seeding fertilizer and applied to the field via seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Plots not treated with any of the agents served as controls, while all others were managed using conventional field management. Continuous investigations of peanut root nodulation began after seedling emergence. These findings revealed that during the flowering phase, the number of root nodules in the peanuts treated with agents 1-8 increased by more than threefold, and nitrogenase activity per plant increased by more than fivefold. Continuous monitoring of carbon dioxide concentrations in both treated and control plots using a carbon dioxide infrared monitor revealed a reduction of more than 9.9%. These results demonstrate that the application of ARC microbial agents significantly promoted peanut nodulation and nitrogen fixation, as well as green, low-carbon production, in field production.

[0114] The above-mentioned ARC microbial agents were applied in peanut production by manual spreading, drone spreading, etc. during peanut sowing, and similar significant effects were achieved in promoting peanut nodulation and nitrogen fixation to achieve green and low-carbon production.

[0115] Applying the above-mentioned ARC microbial agents from the time peanut seedlings emerge to the time when they flower and needle drop also achieved similar significant effects in promoting peanut nodulation and nitrogen fixation to achieve green and low-carbon production.

[0116] Example 7: Application of ARC Microbial Agents - Improving the Quality and Safety of Peanut Products and Reducing Losses

[0117] The above-mentioned ARC microbial agents 1 to 8 were mixed with peanut seeding base fertilizer and applied to the field through a seed drill. The application rate of the microbial agents was 80 billion to 100 billion viable bacteria per mu. At the same time, a plot without any of the above microbial agents was set up as a control. The other plots were managed in the conventional field. After the above-mentioned peanuts were harvested, peanut samples were collected using the national standard sampling method. (1) The abundance of aflatoxin-producing fungi in these samples was determined using the classic colony counting method. The reduction rate of the abundance of aflatoxin-producing fungi in peanuts was calculated, which is the control effect of aflatoxin-producing fungi; (2) The resveratrol, protein, and amino acid content of peanuts was determined using the standard method, and the quality improvement level was calculated; (3) After the samples were placed under the same conditions for 6 months, the aflatoxin contamination level was determined using the national standard liquid chromatography-mass spectrometry method, and the control effect of aflatoxin was calculated. These test results showed that: (1) the resveratrol, protein, glutamic acid and other quality contents of peanut samples in the fields treated with microbial agents 1 to 8 were significantly improved; (2) the control effect of microbial agents 1 to 8 on aflatoxin-producing fungi carried by field peanuts was above 61.5%, which greatly reduced the risk of aflatoxin contamination in post-harvest peanuts; (3) after the peanut samples in the fields treated with microbial agents 1 to 8 and the control were placed under the same conditions for 6 months, the aflatoxin content of peanuts in the treatment group decreased by more than 82% compared with the control group. Among them, the number of peanut samples in the control group with aflatoxin content exceeding the national limit standard accounted for more than 17%, while the peanut samples in the treatment group had no aflatoxin content exceeding the national limit standard, thereby reducing the peanut losses caused by aflatoxin contamination. The above results show that the application of ARC microbial agents significantly improved the quality and quality safety level of field peanuts and reduced peanut losses.

[0118] The above-mentioned ARC microbial agents were applied in peanut production by manual spreading, drone spreading, etc. during peanut sowing, and similar effects of improving quality and quality safety levels were achieved.

[0119] Applying the above-mentioned ARC microbial agents from the time peanuts emerge to the time they bloom and needle drop also achieved similar results in improving quality and quality safety levels.

[0120] Example 8: Use of ARC microbial agent - Use for increasing peanut yield

[0121] ARC microbial agents 1-8 were mixed with peanut seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). A control plot was established without any of the agents, while all other plots were managed using conventional methods. At harvest time, peanuts were harvested from one mu (approximately 1.5 acres) of fields treated with agents 1-8 and the control plots. Calculations showed that the yield increase in the fields treated with agents 1-8 was over 19%, achieving a significant yield increase. These results demonstrate that the application of ARC microbial agents significantly increased field peanut yields.

[0122] The above-mentioned ARC microbial agents were applied in peanut production by manual spreading, drone spreading, etc. during peanut sowing, and similar significant results were achieved in increasing peanut yield.

[0123] Applying the above-mentioned ARC microbial agents from the time peanut seedlings emerge to the time when they bloom and needle drop also achieved similar significant results in increasing peanut yield.

[0124] Example 9: Use of ARC microbial agent - Use for increasing the total biomass of peanuts

[0125] ARC microbial agents 1-8 were mixed with peanut seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). A control plot was established without any of the agents, while all other plots were managed using conventional methods. A survey was conducted at harvest time to determine the total biomass of the treated and control peanut plants. These survey results showed that the total biomass of the peanut plants treated with ARC microbial agents 1-8 increased by more than 16%. These results demonstrate that the application of ARC microbial agents significantly increases the total biomass of peanut plants.

[0126] The above-mentioned ARC microbial agents were applied to peanut production by manual spreading, drone spreading, etc. during peanut sowing, and similar results were achieved that significantly increased the total biomass of peanuts.

[0127] Applying the above-mentioned ARC microbial agents from the time peanuts emerge to the time they bloom and needle set also achieved similar results in significantly increasing the total biomass of peanuts.

[0128] Example 10: Use of ARC microbial agent - Use for improving soybean quality and reducing the risk of aflatoxin contamination

[0129] ARC microbial agents 1-8 were mixed with soybean seeding fertilizer and applied to the field via seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Plots not treated with any of the agents served as controls, while all other plots were managed using conventional field management. After the soybeans were harvested, soybean samples were collected using national standard sampling methods. The isoflavone, protein, and amino acid content of the soybean samples were determined using standard methods to calculate the quality improvement. The abundance of aflatoxin-carrying Aspergillus or aflatoxin-producing fungi in soybeans was determined using a published colony count method or the PAB-01 marker molecule assay. These results showed that the application of ARC microbial agents 1-8 significantly increased the isoflavone, protein, and glutamate content of soybeans grown in the field, and reduced the abundance of aflatoxin-carrying Aspergillus or aflatoxin-producing fungi in soybeans by more than 60%, thereby reducing the risk of aflatoxin contamination. These results demonstrate that the application of ARC microbial agents significantly improved soybean quality and reduced the risk of aflatoxin contamination in soybeans grown in the field.

[0130] The above-mentioned ARC microbial agents were applied in soybean production by manual spreading, drone spreading, etc. during soybean sowing, and similar results were achieved that significantly improved soybean quality.

[0131] Similar significant effects of improving soybean quality were achieved by applying the above-mentioned ARC microbial agents from the time soybean seedlings emerge to the time when they bloom and set pods.

[0132] Example 11: Use of ARC microbial agent - Use for promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production

[0133] ARC microbial agents 1-8 were mixed with soybean seeding fertilizer and applied to the field via seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Plots not treated with any of the agents served as controls, while all other plots were managed using conventional field management. Continuous investigations of soybean root nodulation were conducted after soybean seedlings emerged. These findings revealed that during the flowering phase, the number of root nodules in soybeans treated with agents 1-8 increased by over 3.35 times, and the nitrogenase activity per soybean plant increased by over 5.5 times. Continuous monitoring of carbon dioxide concentrations in both treated and control plots using a carbon dioxide infrared monitor revealed a reduction of over 9.3%. These results demonstrate that the application of ARC microbial agents significantly promoted soybean nodulation and nitrogen fixation, as well as green, low-carbon production, in the field.

[0134] The above-mentioned ARC microbial agents were applied in soybean production by manual spreading, drone spreading, etc. during soybean sowing, and similar significant effects were achieved in promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production.

[0135] Applying the above-mentioned ARC microbial agents from the time soybean seedlings emerge to the time when they bloom and set pods also achieved similar significant effects in promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production.

[0136] Example 12: Use of ARC microbial agent - Use for increasing soybean yield

[0137] ARC microbial agents 1-8 were mixed with soybean seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). A control plot was established without any of the agents, while all other plots were managed conventionally. At harvest time, soybeans were harvested from one mu (approximately 1.5 acres) of fields treated with ARC microbial agents and the control plot. Calculations showed that soybean yield increases in the fields treated with ARC microbial agents 1-8 exceeded 17%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly improved soybean yields per unit area in large fields.

[0138] The application of the above-mentioned ARC microbial agents in soybean production by manual spreading, drone spreading, etc. during soybean sowing has also achieved similar results in significantly improving soybean yield levels.

[0139] Applying the above-mentioned ARC microbial agents from the time soybeans emerge to the time they bloom and set pods also achieved similar results in significantly increasing soybean yields.

[0140] Example 13: Use of ARC microbial agent - Use for increasing total soybean biomass

[0141] ARC microbial agents 1-8 were mixed with soybean seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million hectares). A control plot was established without any of the agents, while all other plots were managed conventionally. A survey was conducted at soybean maturity, measuring the total biomass of soybean plants treated with ARC microbial agents and those in the control plots. These survey results showed that soybeans treated with ARC microbial agents 1-8 had a greater than 13% increase in total plant biomass. These results demonstrate that the application of ARC microbial agents significantly increases soybean plant biomass.

[0142] The above-mentioned ARC microbial agents were applied in soybean production by manual spreading, drone spreading, etc. during soybean sowing, and similar effects of significantly increasing the total soybean biomass were achieved.

[0143] Similar significant increases in soybean biomass were achieved by applying the above-mentioned ARC microbial agents from the time soybean seedlings emerge to the time when they bloom and set pods.

[0144] Example 14: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation of red beans and increasing yield

[0145] ARC microbial agents 1-8 were mixed with adzuki bean seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. A plot without any of the agents was set up as a control, while all other plots were managed using conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC microbial agents 1-8 increased both root nodule count and nitrogenase activity by more than 2-fold. After maturity, the adzuki beans were harvested, weighed, and yields were calculated. The yield increase rates for adzuki beans treated with ARC microbial agents 1-8 were all above 9.7%, achieving a significant yield increase. These results indicate that the application of ARC microbial agents significantly promoted adzuki bean nodulation and nitrogen fixation and increased adzuki bean yields per unit area.

[0146] The above-mentioned ARC microbial agents were applied to adzuki bean production by manual spreading, drone spreading, etc. during adzuki bean sowing, and similar significant effects were achieved in promoting adzuki bean nodulation and nitrogen fixation and increasing adzuki bean yield.

[0147] Applying the above-mentioned ARC microbial agents after the emergence of red beans also achieved similar results in significantly increasing red bean yields.

[0148] Example 15: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation of mung beans and increasing yield

[0149] ARC microbial agents 1-8 were mixed with mung bean seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. Plots not treated with any of the agents served as controls, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased the number of mung bean root nodules by more than 2.1 times and nitrogenase activity by more than 2.3 times. Mung beans were harvested and weighed after maturity, and yield was calculated. The yield increase for mung beans treated with ARC agents 1-8 was over 9.5%, demonstrating significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promoted green nodulation and nitrogen fixation and increased mung bean yield per unit area.

[0150] The above-mentioned ARC microbial agents were applied in mung bean production by manual spreading, drone spreading, etc. during mung bean sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing mung bean yield were achieved.

[0151] Applying the above-mentioned ARC microbial agents after mung bean seedlings emerged also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing mung bean yield.

[0152] Example 16: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation in peas and increasing yield

[0153] ARC microbial agents 1-8 were mixed with pea seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot was established without any of the agents, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased the number of pea root nodules by more than 2-fold and nitrogenase activity by more than 2.1-fold. Beans were harvested and weighed after maturity, and yield per unit area was calculated. The yield increase for peas treated with ARC agents 1-8 was over 7.3%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promoted green nodulation and nitrogen fixation and increased pea yield per unit area in the field.

[0154] The above-mentioned ARC microbial agents were applied to the production of mung beans by manual spreading, drone spreading, etc. during the sowing of mung beans, and similar significant effects of promoting nodulation and nitrogen fixation and increasing mung bean yield were achieved.

[0155] The application of the above-mentioned ARC microbial agents after the emergence of the mung bean seedlings also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing the yield of the mung bean.

[0156] Example 17: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation of lentil and increasing yield

[0157] ARC microbial agents 1-8 were mixed with lentil seeding fertilizer and applied to the field via seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. Plots not treated with any of the agents served as controls, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 2.3 times and nitrogenase activity by more than 2.8 times. Lentils were harvested and weighed after maturity, and yield per unit area was calculated. The yield increase for lentil treated with ARC agents 1-8 was over 10.9%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promoted green nodulation and nitrogen fixation and increased lentil yield per unit area.

[0158] The above-mentioned ARC microbial agents were applied to lentil production by manual spreading, drone spreading, etc. during lentil sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing lentil yield were achieved.

[0159] Applying the above-mentioned ARC microbial agents after the emergence of lentil seedlings also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing lentil yield.

Claims

1. The use of ARC microbial agent for nodulation, nitrogen fixation, and quality improvement and yield increase of leguminous grain and oil crops. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, 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. The ARC microbial agent contains the DNA sequences shown in SEQ ID No. 1-4. The leguminous grain and oil crops are peanuts, soybeans, or leguminous grain crops. The use of ARC microbial agent for nodulation, nitrogen fixation, and quality improvement and yield increase of leguminous grain and oil crops is: for improving the quality and quality safety of peanuts and reducing losses, or promoting nodulation and nitrogen fixation of peanuts to achieve green and low-carbon production, or for increasing the yield level and total biomass of peanuts; or for improving the quality of soybeans and reducing the risk of aflatoxin contamination, or promoting nodulation and nitrogen fixation of soybeans to achieve green and low-carbon production, or increasing the yield level and total biomass of soybeans; or promoting nodulation and nitrogen fixation of leguminous grain crops and increasing the yield level.

2. A method for improving peanut quality and safety and reducing losses, comprising applying an ARC microbial agent to peanut production. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, 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. The composition comprises the DNA sequences shown in SEQ ID Nos. 1-4.

3. The method according to claim 1, wherein: The method is as follows: ARC microbial agent is mixed with peanut sowing base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, drone, etc. The application rate of the microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: After the peanuts are sown and seedlings emerge normally until the flowering and needle-setting stage, apply the ARC microbial agent evenly into the field by broadcasting, spraying, drip irrigation or a combination of one or more of the following methods, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

4. A method for promoting peanut nodulation and nitrogen fixation to achieve green and low-carbon production, characterized by: The method includes applying an ARC microbial agent to peanut production. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and the number of nodules in leguminous crops, and contains the DNA sequences shown in SEQ ID No. 1-4.

5. The method according to claim 4, characterized in that: The method is as follows: ARC microbial agent is mixed with peanut seeding base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, drone, etc., with the cumulative number of viable bacteria per mu being 80 billion to 100 billion, or: After the peanuts are sown and seedlings emerge normally until the flowering and needle-setting stage, ARC microbial agent should be evenly applied to the field by broadcasting, spraying or drip irrigation. The application rate of the agent should be 80 billion to 100 billion live bacteria per mu.

6. A method for increasing peanut yield and total biomass, characterized by: ARC microbial agent is applied to peanut production. The ARC microbial agent is a microbial composition with a coupled effect of toxicity control and nitrogen fixation, which 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. It contains the DNA sequence shown in SEQ ID No. 1-4.

7. The method according to claim 2, wherein: The method is as follows: ARC microbial agent is mixed with peanut sowing base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine or drone, with a cumulative viable bacterial count of 80 billion to 100 billion per mu. Or: After the peanuts are sown and seedlings emerge normally until the flowering and needle-setting stage, apply the ARC microbial agent evenly into the field by broadcasting, spraying, drip irrigation or a combination of one or more of the following methods, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

8. The method according to claim 2, 4 or 6, characterized in that: The application amount of the ARC microbial agent is 80 billion to 100 billion live bacteria per mu.

9. The method according to claim 2, 4 or 6, characterized in that: The application method is one or more of broadcasting, spraying, drip irrigation, etc., and the application stage is when the peanuts are sown and / or from the emergence of peanut seedlings to the flowering and needle-setting period.

10. A method for improving soybean quality and reducing the risk of aflatoxin contamination, comprising applying an ARC microbial agent to soybean crops. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and the number of nodules in leguminous crops, and comprises the DNA sequences shown in SEQ ID Nos. 1-4.

11. The method according to claim 10, characterized in that: The method is as follows: ARC microbial agent is mixed with soybean seeding base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, drone, drip irrigation pipe, etc. The application amount of the microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: After the soybeans are normally sown and seedlings emerge until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or topdressing, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

12. A method for promoting soybean nodulation and nitrogen fixation to achieve green and low-carbon production, characterized by: The method includes applying an ARC microbial agent to soybean production. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, 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. The ARC microbial agent contains the DNA sequences shown in SEQ ID No. 1-4.

13. The method according to claim 12, wherein: The method is as follows: ARC microbial agent is mixed with soybean seeding base fertilizer, and applied to the field by one or more of the following methods: manual, seeding machine, drone, drip irrigation pipe, etc. The application amount of the microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: After the soybeans are normally sown and seedlings emerge until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or topdressing, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

14. A method for increasing soybean yield and total biomass, characterized by: The method comprises applying an ARC microbial agent to soybean production. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules of leguminous crops, and contains the DNA sequences shown in SEQ ID No. 1-4.

15. The method according to claim 14, characterized in that: The method is as follows: ARC microbial agent is mixed with soybean seeding base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, drone, drip irrigation pipe, etc. The application amount of the microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: After the soybeans are normally sown and seedlings emerge until the flowering and podding stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or topdressing, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

16. The method according to claim 10, 12 or 14, characterized in that: The application amount of the ARC microbial agent is 80 billion to 100 billion live bacteria per mu.

17. The method according to claim 10, 12 or 14, characterized in that: The application method is one or a combination of broadcasting, spraying, drip irrigation, etc., and the application stage is when soybeans are sown and / or from soybean emergence to flowering.

18. A method for promoting nodulation and nitrogen fixation in leguminous grain crops and increasing yield, characterized by: The method includes applying an ARC microbial agent to leguminous grain crops. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and the number of nodules in leguminous crops, and contains the DNA sequences shown in SEQ ID No. 1-4.

19. The method according to claim 18, wherein: The legume grains include but are not limited to red beans, green beans, flower beans, and lentils; the application amount of the ARC microbial agent is 80 billion to 100 billion live bacteria per mu.

20. The method according to claim 18, wherein: The application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is when leguminous grain crops are sown and / or from the emergence of peanuts to the flowering stage.

21. The method according to claim 18, wherein: The method comprises: mixing the ARC microbial agent with the base fertilizer for sowing legumes and grain crops, and evenly applying the agent to the field by one or more of the following methods: manual application, seeding machine, or drone, with a cumulative viable bacterial count of 80 billion to 100 billion per mu. Or: after the leguminous grain crops are sown and germinated normally until the flowering stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or topdressing, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

22. The use according to claim 1, characterized in that: In the ARC microbial agent, 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, not exceeding 10% base variation and having corresponding biological activity functions, they constitute 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 to 4 or their functional equivalents, and has a coupled effect of toxicity 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 an ARC microbial agent.

23. The use according to claim 1, characterized in that: The ARC microbial agent 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 the ARC microbial agent 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 toxicity 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.

24. The use according to claim 1, characterized in that: The proportion of viable bacteria count of any one strain in the ARC microbial agent is greater than or equal to 1%.

25. The use according to claim 1, characterized in that: The ARC microbial agent has an inhibitory effect on aflatoxin and / or its toxins.

26. The use 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.