Quality-improving and nitrogen-fixing coupled microbial agent, and use thereof

Abstract

Provided are a quality-improving and nitrogen-fixing coupled microbial agent and the use thereof. The quality-improving and nitrogen-fixing coupled microbial agent is a microbial composition, has coupled functions of improving quality and fixing nitrogen so as to improve the quality of products of leguminous crops, and has the functions of regulating and increasing the rhizobial abundance for the rhizosphere of leguminous crops and increasing the amount of nodules of leguminous crops. The microbial agent contains all gene sequences of nucleotide sequences shown as SEQ ID NO.1-12 or four or more gene sequences among same. When being applied to production of leguminous crops, such as soybeans, peanuts, peas, broad beans and cowpeas, the quality-improving and nitrogen-fixing coupled microbial agent has coupled functions of improving quality and fixing nitrogen and achieves quality improvement and nitrogen fixation and green yield increase. The microbial agent is easy to use, achieves remarkable social and ecological benefits, and facilitates popularization and use.

Description

Quality-improving and nitrogen-fixing coupled microbial agent and its use Technical Field

[0001] The present invention belongs to the field of microorganisms, and in particular relates to a quality-improving and nitrogen-fixing coupled microbial agent and its use. Background Art

[0002] Legume crops, including soybeans, peanuts, peas, broad beans, cowpeas, and alfalfa, are important sources of food and feed for humans. Global agriculture today faces numerous challenges in its pursuit of green and high-quality development. The development of the legume industry faces two common challenges: First, legumes like peanuts and soybeans are susceptible to contamination by highly toxic and carcinogenic aflatoxins, which not only reduce quality and yield, but also pose a serious threat to human 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 World Health Organization's International Agency for Research on Cancer. It contributes to 28.2% of liver cancer worldwide. Improving the quality, safety, and performance of legume products has long been a hot topic in the high-quality development of the legume agriculture sector.

[0003] Secondly, while legume crops like soybeans and peanuts form symbiotic nodules and fix nitrogen with soil rhizobia, these nodules are naturally few in number, their nitrogen fixation duration is short (typically, no nodules form and fix nitrogen in the first month of crop growth, and nodules begin to decay during the pod-filling or fruit-full stages), and their efficiency is low. 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 that defies the AON theory) is elusive. Improving the nodulation and nitrogen fixation efficiency of legumes like peanuts and soybeans remains a hotly debated and challenging issue internationally.

[0004] To address these challenges, the inventors' team, after over 20 years of continuous research, have successfully developed a microbial agent for quality improvement and nitrogen fixation. This agent improves the quality of legume crops like soybeans and peanuts while simultaneously inducing efficient nodulation and nitrogen fixation in soybeans and peanuts, doubling their yields. This agent is simple to use, low-cost, and highly effective. It boasts significant advantages: two fixes (nitrogen and carbon fixation), three increases (increased yield, efficiency, and safety), and five reductions (reduced toxicity, damage, weight, cost, and carbon emissions). This agent has enormous potential for application and has been demonstrated and validated in field trials across major soybean, peanut, and pea producing regions nationwide. 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

[0005] The present invention addresses the deficiencies in the prior art and provides a quality-improving and nitrogen-fixing coupled microbial agent, which is applied to the production of legume crops such as soybeans, peanuts, peas, broad beans, and cowpeas. It has a quality-improving and nitrogen-fixing coupled effect, achieving quality-improving and nitrogen-fixing and green yield increase. It is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A quality-improving and nitrogen-fixing coupled microbial agent is a microbial composition that has a quality-improving and nitrogen-fixing coupled effect, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops. It contains all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or four or more of them.

[0008] According to the above scheme, the quality-improving and nitrogen-fixing coupled microbial agent is preferably a composition of more than three kinds of microorganisms, containing 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or more gene sequences of the nucleotide sequences shown in SEQ ID NO.1~12.

[0009] According to the above scheme, the microbial source includes but is not limited to Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Brevibacillus laterosporus, Bacillus mucilaginosus, Bacillus velezensis, Bacillus siamensis, Paenibacillus polymyxa, Paenibacillus timonensis, Pseudomonas fluorescens, Pseudomonas mendocina, Enterobacter ludwigii, Microbacterium proteolyticum, Leclercia adcarboxglata, Serratia marcescens. marcescens, Empedobacters sp., Priestia priestiamegaterium, Stenotrophomonas maltophilia and other bacteria.

[0010] According to the above scheme, the gene sequences shown in SEQ ID NOs. 1 to 12 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, when the identity is greater than 90%, preferably greater than 95%, and more preferably greater than 99%, and when the corresponding biological activity is present, these are called functional equivalents of the DNA sequences shown in SEQ ID NOs. 1 to 12, and containing these sequences is equivalent to containing the corresponding sequences of DNA sequences 1 to 12. Microbial compositions containing all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or four or more of these gene sequences, or functional equivalents of these gene sequences, and having the coupled effects of improving quality and fixing nitrogen as described above, such as improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops, are all coupled microbial agents for improving quality and fixing nitrogen of the present invention.

[0011] According to the above scheme, the green, yield-increasing, and nitrogen-fixing microbial agent can improve the quality of legume crops by increasing the content of one or more nutritional functional components, such as protein, glutamic acid, peanut resveratrol, and soy isoflavones. According to the above scheme, the coupled nitrogen-fixing and quality-enhancing microbial agent can alleviate or control one or more soil-borne diseases, such as peanut fruit rot, soybean green disease, white rot, bacterial wilt, root rot, and sclerotinia rot.

[0012] According to the above scheme, the quality-enhancing and nitrogen-fixing coupled microbial agent can reduce the aflatoxin level of crop products and improve the quality and safety level of leguminous crop products.

[0013] According to the above scheme, the quality-enhancing and nitrogen-fixing coupled microbial agent can reduce the spots on the peanut fruit surface.

[0014] The quality-enhancing and nitrogen-fixing coupled microbial agent can promote early nodulation of leguminous crops such as peanuts and soybeans and prolong the nodulation and nitrogen-fixation time.

[0015] The above-mentioned DNA sequences 1-12 are specific sequences obtained after comparison with Genbank genome big data. They have a direct or indirect correlation with the function of the microbial agent of this patent. When containing four or more of the above-mentioned DNA sequences 1-12, they have the effect of coupled quality improvement and nitrogen fixation, thereby improving the quality of legume products. They are the quality improvement and nitrogen fixation coupled microbial agent of the present invention. Furthermore, they can also promote early nodulation of legumes, prolong the nodulation and nitrogen fixation time, and increase the yield level and total biomass of legume production. These genes may vary to a certain extent in different strains. When the degree of difference is small, such as no more than 10% base variation, and the corresponding biological activity function is maintained, containing them is equivalent to containing the corresponding DNA sequences shown in SEQ ID No. 1-12. These are called functional equivalents of the DNA sequences shown in SEQ ID No. 1-12. Containing them is equivalent to containing all or four or more of the gene sequences in the DNA sequences 1-12 shown in SEQ ID No. 1-12.

[0016] The quality-enhancing and nitrogen-fixing coupled microbial agent provided by the present invention can be, but is not limited to, a combination of three or more of the following microorganisms: Bacillus subtilis with a deposit number of CCTCC NO: M 20231597, Bacillus velezensis with a deposit number of CCTCC NO: M 20231600, Bacillus laterosporus with a deposit number of CCTCC NO: M 20231808, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231810, Enterobacter ludwigii with a deposit number of CCTCC NO: M 20231812, and Microbacterium with a deposit number of CCTCC NO: M 20231814. Specifically, the agent can be a combination of three, four, five, or six of the above strains.

[0017] The quality-improving and nitrogen-fixing coupled microbial agent provided by the present invention can also be a combination of three or more of the above-mentioned microorganisms 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 12 or four or more gene sequences therein or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, and regulates and increases the abundance of rhizobia in the rhizosphere of leguminous crops and the number of nodules of leguminous crops, thereby constituting the quality-improving and nitrogen-fixing coupled microbial agent of the present invention.

[0018] The quality-improving and nitrogen-fixing coupled microbial agent provided by the present invention can also be a combination of one or more strains of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812, and the following three strains: Bacillus subtilis of CCTCC NO: M 20231597, Bacillus velez of CCTCC NO: M 20231600, and Microbacterium of CCTCC NO: M 20231814. For example, the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812 and Bacillus subtilis of CCTCC NO: M 20231597, or the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812 and Bacillus velez of CCTCC NO: M 20231600, or the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231600. The combination of Enterobacter Ludwigii of CCTCC NO: M 20231812 and Microbacterium of CCTCC NO: M 20231814, 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 12 or 4 or more gene sequences therein or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules of legume crops, constitutes the quality-improving and nitrogen-fixing coupled microbial agent of the present invention.

[0019] The quality-improving and nitrogen-fixing coupled microbial agent provided by the present invention can also be a combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812, and other bacteria, so that the combined microbial agent satisfies the requirement of containing all gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or 4 or more gene sequences therein, or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, thereby improving the quality of legume crop products, 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 quality-improving and nitrogen-fixing coupled microbial agent of the present invention.

[0020] Bacillus subtilis, deposit date is September 4, 2023, deposit number is CCTCC NO: M 20231597CCTCC NO: M 20231597, classification name is: Bacillus subtilis AR1003, deposited in: China Center for Type Culture Collection, address is Wuhan University, Wuhan, China.

[0021] Bacillus velezensis, deposited on September 4, 2023, with a deposit number of CCTCC NO: M 20231600, a classification name of Bacillus velezensis AR1006, and a depository unit named China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0022] Bacillus laterosporus, deposited on September 27, 2023, with a deposit number of CCTCC NO: M 20231808, a classification name of Brevibacillus laterosporus strain D-CB0402, and a depository unit named China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0023] Bacillus amyloliquefaciens, deposited on September 27, 2023, with a deposit number of CCTCC NO: M 20231810, is classified as Bacillus amyloliquefaciens strain D-JDFN02, and is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0024] Enterobacter ludwigii, deposited on September 27, 2023, with the deposit number CCTCC NO: M 20231812, classified as: Enterobacter ludwigii strain D-LD1601, and deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0025] Microbacterium, deposited on September 27, 2023, with the deposit number CCTCC NO: M 20231814, classified as: Microbacterium proteolyticum strain D-WG1701, and deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0026] According to the above scheme, the proportion of the number of live bacteria of any one strain of the above microbial agents in the microbial agent, that is, the mixed microbial composition, is greater than or equal to 1%.

[0027] The quality-improving and nitrogen-fixing coupled microbial agent of the present invention is a microbial composition. Through the synergistic effect of each component microorganism in the microbial composition, the quality-improving and nitrogen-fixing effect is exerted. When used in crop production, the quality-improving and nitrogen-fixing coupled effect is exerted, and the agent has the functions of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops. Although the agent itself is not a rhizobium, it has the functions of improving and nitrogen-fixing coupled effect, thereby improving the quality of leguminous crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops, increasing the number of nodules in leguminous crops, and improving the nitrogenase activity of individual plants. It can promote early nodulation of leguminous crops such as peanuts and soybeans, prolong the nodulation and nitrogen fixation time, and improve the yield level and total biomass of leguminous crop production.

[0028] The above-mentioned quality-enhancing and nitrogen-fixing coupled microbial agent can be prepared by the following method, which is prepared by combining the microorganisms in the above-mentioned microbial agent and fermenting it. The above-mentioned fermentation route can adopt conventional fermentation routes of bacteria or fungi that are already disclosed in existing technologies, including existing literature.

[0029] The second aspect of the present invention provides that the above-mentioned quality-improving and nitrogen-fixing coupled microbial agent can be used in the production of leguminous crops as follows: for improving the quality and safety level of leguminous crop products; for promoting nodulation and nitrogen fixation of leguminous crops; for improving the yield level of leguminous crops; for recruiting indigenous rhizobia and increasing the abundance of rhizobia in the rhizosphere soil of leguminous crops; for promoting early nodulation of leguminous crops and prolonging the nodulation and nitrogen fixation time; for preventing leguminous crops from losing fertilizer and aging prematurely during maturity; for increasing the number of leguminous crop pods; for increasing the fullness of leguminous crop pods and reducing the pod shrinkage rate; for promoting early flowering and early pod setting of leguminous crops; for reducing the occurrence of peanut fruit rot; for reducing leguminous crops The occurrence of bacterial wilt in legumes; used to reduce the occurrence of powdery mildew in legumes; used to reduce the occurrence of leaf spot in legumes; used to reduce the occurrence of root nematode in legumes; used to reduce the occurrence of root rot in legumes; used to reduce the occurrence of root nematode in legumes; used to reduce the occurrence of sclerotinia in legumes; used to reduce the occurrence of downy mildew in legumes; used to reduce the occurrence of wilt in legumes; used to reduce the occurrence of white rot in legumes; used to reduce the incidence of soybean greening; used to promote carbon emission reduction in legumes, which is beneficial to soil improvement; used to promote the increase of total biomass of legumes; used to reduce the surface spots of peanuts and increase marketability; used to promote the increase of soybean yield in saline-alkali land;

[0030] The third aspect of the present invention provides the above-mentioned quality-improving and nitrogen-fixing coupled microbial agent for use in the preparation of quality-improving and nitrogen-fixing coupled microbial compound fertilizer, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production; used to prepare quality-improving and nitrogen-fixing coupled microbial organic fertilizer, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production; used to prepare quality-improving and nitrogen-fixing coupled microbial fertilizer, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production; used to prepare quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production; used to prepare quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production; used to prepare quality-improving and nitrogen-fixing coupled microbial-seed dressing agent / seed dressing agent / seed soaking agent, which can simultaneously play the role of quality-improving and nitrogen-fixing coupled microbial agent in production.

[0031] The present invention further provides a quality-improving nitrogen-fixing coupled microbial compound fertilizer, a quality-improving nitrogen-fixing coupled microbial organic fertilizer, a quality-improving nitrogen-fixing coupled microbial micro-fertilizer, a quality-improving nitrogen-fixing coupled microbial moisturizing fertilizer, a quality-improving nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, a quality-improving nitrogen-fixing coupled microbial-rhizobium fertilizer, or a quality-improving nitrogen-fixing coupled microbial-seed dressing agent / seed dressing agent / seed soaking agent obtained by configuring the above-mentioned quality-improving nitrogen-fixing coupled microbial agent.

[0032] A fourth aspect of the present invention provides a method for preparing a quality-improving nitrogen-fixing coupled microbial compound fertilizer, a quality-improving nitrogen-fixing coupled microbial organic fertilizer, a quality-improving nitrogen-fixing coupled microbial micro-fertilizer, a quality-improving nitrogen-fixing coupled microbial moisturizing fertilizer, a quality-improving nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, a quality-improving nitrogen-fixing coupled microbial-rhizobium fertilizer, or a quality-improving nitrogen-fixing coupled microbial-seed dressing agent / seed dressing agent / seed soaking agent using the above-mentioned quality-improving nitrogen-fixing coupled microbial agent:

[0033] Method for preparing quality-improving and nitrogen-fixing coupled microbial compound fertilizer: according to the conventional dosage of compound fertilizer and the dosage of quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria per mu of land, compound fertilizer and quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on compound fertilizer particles by physical methods to prepare quality-improving and nitrogen-fixing coupled microbial compound fertilizer;

[0034] Method for preparing quality-improving and nitrogen-fixing coupled microbial organic fertilizer: according to the conventional amount of organic fertilizer per mu of land and the amount of quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria, the organic fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the organic fertilizer particles by physical methods to prepare a quality-improving and nitrogen-fixing coupled microbial compound fertilizer;

[0035] Method for preparing quality-improving and nitrogen-fixing coupled microbial fertilizer: according to the conventional amount of trace element fertilizer per mu of land and the amount of quality-improving and nitrogen-fixing coupled microbial agent with a viable count of not less than 80 billion, the trace element fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are proportioned, and then mixed by physical methods, and the quality-improving and nitrogen-fixing coupled microbial agent is adsorbed and fixed on the trace element fertilizer particles to prepare the quality-improving and nitrogen-fixing coupled microbial fertilizer;

[0036] Method for preparing the quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer: according to the conventional dosage of the water-retaining agent and the dosage of the quality-improving and nitrogen-fixing coupled microbial agent with a viable count of not less than 80 billion per mu of land, the water-retaining agent and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then mixed by conventional physical methods / or the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the water-retaining agent to prepare the quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer;

[0037] A method for preparing a quality-improving and nitrogen-fixing coupled microbial rhizobium fertilizer: according to the conventional amount of rhizobium fertilizer and the amount of the quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria per mu of land, the rhizobium fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in a conventional physical method or the two agents are mixed and then fixed on carrier particles to prepare the quality-improving and nitrogen-fixing coupled microbial rhizobium fertilizer;

[0038] The method for preparing the quality-improving and nitrogen-fixing coupled microbial organic-inorganic compound fertilizer is as follows: according to the conventional dosage of compound fertilizer per mu of land and the dosage of the quality-improving and nitrogen-fixing coupled microbial agent of not less than 80 billion viable bacteria, the organic-inorganic compound fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the organic and inorganic particles through physical methods to prepare the quality-improving and nitrogen-fixing coupled microbial organic-inorganic compound fertilizer.

[0039] The method for preparing quality-improving and nitrogen-fixing coupled microorganisms-seed dressing agent / seed mixing agent / seed soaking agent: according to the amount of seed dressing agent or seed mixing agent or seed soaking agent per mu of land and the amount of quality-improving and nitrogen-fixing coupled microorganisms not less than 80 billion viable bacteria, the seed dressing agent or seed mixing agent or seed soaking agent and the quality-improving and nitrogen-fixing coupled microorganisms are proportioned, and then the quality-improving and nitrogen-fixing coupled microorganisms and the seed dressing agent or seed mixing agent or seed soaking agent are evenly mixed through conventional physical blending to prepare the quality-improving and nitrogen-fixing coupled microorganisms-seed dressing agent / seed mixing agent / seed soaking agent.

[0040] The fifth aspect of the present invention provides any of the following applications of the quality-improving and nitrogen-fixing coupled microbial compound fertilizer, quality-improving and nitrogen-fixing coupled microbial organic fertilizer, quality-improving and nitrogen-fixing coupled microbial micro-fertilizer, quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer, quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, or quality-improving and nitrogen-fixing coupled microbial-rhizobium fertilizer configured using the above-mentioned quality-improving and nitrogen-fixing coupled microbial agent in crop production: for improving the quality and safety level of leguminous crop products; for promoting nodulation and nitrogen fixation of leguminous crops; for improving the yield per unit area of ​​leguminous crops; for recruiting indigenous rhizobia and increasing the abundance of rhizobia in the rhizosphere soil of leguminous crops; for promoting early nodulation of leguminous crops and prolonging the time of nodulation and nitrogen fixation; for preventing premature aging of leguminous crops due to lack of fertilizer during maturity; for increasing the number of pods of leguminous crops; for increasing leguminous crops. It can improve the fullness of crop pods and reduce the pod shrinkage rate; promote early flowering and pod setting of leguminous crops; reduce the occurrence of peanut fruit rot; reduce the occurrence of bacterial wilt of leguminous crops; reduce the occurrence of powdery mildew of leguminous crops; reduce the occurrence of leaf spot of leguminous crops; reduce the occurrence of root nematode disease of leguminous crops; reduce the occurrence of root rot of leguminous crops; reduce the occurrence of root nematode disease of leguminous crops; reduce the occurrence of sclerotinia disease of leguminous crops; reduce the occurrence of downy mildew of leguminous crops; reduce the occurrence of wilt of leguminous crops; reduce the occurrence of white rot of leguminous crops; reduce the occurrence of soybean green disease; promote carbon emission reduction of leguminous crops, which is beneficial to soil improvement; promote the increase of total biomass of leguminous crops; reduce the surface spots of peanut fruits and increase marketability; promote the increase of soybean yield in saline-alkali land.

[0041] A sixth aspect of the present invention provides a method for producing legume crops, wherein a microbial agent is selected so that the microbial agent contains all or four or more gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12 upon analysis, and has a coupled effect of improving quality and fixing nitrogen, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops; the microbial agent is applied to crops to improve quality, fix nitrogen, and increase yield in a green manner.

[0042] According to the above scheme, the gene sequences shown in SEQ ID NOs. 1 to 12 may have no more than 10% base variation in different strains. If they have more than 90% identity with the corresponding DNA sequence and have the corresponding biological activity, they are equivalent to the corresponding gene sequences in the DNA sequences 1 to 12 shown in SEQ ID NOs. 1 to 12, and these are called functional equivalents of the DNA sequences shown in SEQ ID NOs. 1 to 12. A microbial agent containing all or four or more of the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or functional equivalents thereof, and having the coupled effects of quality improvement and nitrogen fixation of the above-mentioned microbial agent, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops, constitutes the quality improvement and nitrogen fixation coupled microbial agent of the present invention, and is applied to crops for improving quality, nitrogen fixation, and green yield increase.

[0043] According to the above scheme, the gene sequences shown in SEQ ID NOs. 1 to 12 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, when the identity is greater than 90%, preferably greater than 95%, and more preferably greater than 99%, and when the corresponding biological activity is present, these are called functional equivalents of the DNA sequences shown in SEQ ID NOs. 1 to 12, and containing these sequences is equivalent to containing the corresponding sequences of DNA sequences 1 to 12. Microbial compositions containing all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or four or more of these gene sequences, or functional equivalents of these gene sequences, and having the coupled effects of improving quality and fixing nitrogen as described above, such as improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops, are all coupled microbial agents for improving quality and fixing nitrogen of the present invention.

[0044] In the above scheme, the green, yield-increasing, and nitrogen-improving microbial agent can improve the quality of legume crops by increasing the content of one or more nutritional functional components, such as protein, glutamic acid, peanut resveratrol, and soy isoflavones. According to the above scheme, the coupled nitrogen-improving and nitrogen-fixing microbial agent can alleviate or control one or more soil-borne diseases, such as peanut fruit rot, soybean green disease, white rot, bacterial wilt, root rot, and sclerotinia.

[0045] In the above scheme, the quality-enhancing and nitrogen-fixing coupled microbial agent can reduce the aflatoxin level of crop products and improve the quality and safety level of leguminous crop products.

[0046] In the above scheme, the quality-improving and nitrogen-fixing coupled microbial agent can reduce the spots on the peanut fruit surface.

[0047] According to the above scheme, the quality-improving and nitrogen-fixing coupled microbial agent is preferably a composition of more than three kinds of microorganisms, containing 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or more gene sequences of the nucleotide sequences shown in SEQ ID NO.1~12.

[0048] In the above scheme, the quality-enhancing and nitrogen-fixing coupled microbial agent can be, but is not limited to, a combination of three or more of the following microorganisms: Bacillus subtilis with a deposit number of CCTCC NO: M 20231597, Bacillus velezensis with a deposit number of CCTCC NO: M 20231600, Bacillus laterosporus with a deposit number of CCTCC NO: M 20231808, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231810, Enterobacter ludwigii with a deposit number of CCTCC NO: M 20231812, and Microbacterium with a deposit number of CCTCC NO: M 20231814. Specifically, the agent can be a combination of three, four, five, or six of the above strains.

[0049] In the above scheme, the quality-improving and nitrogen-fixing coupled microbial agent can also be a combination of one or more strains of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812, and the following three strains: Bacillus subtilis of CCTCC NO: M 20231597, Bacillus velez of CCTCC NO: M 20231600, and Microbacterium of CCTCC NO: M 20231814. For example, the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812 and Bacillus subtilis of CCTCC NO: M 20231597, or the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812 and Bacillus velez of CCTCC NO: M 20231600, or the combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231600. The combination of Enterobacter Ludwigii of CCTCC NO: M 20231812 and Microbacterium of CCTCC NO: M 20231814, 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 12 or 4 or more gene sequences therein or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules of legume crops, constitutes the quality-improving and nitrogen-fixing coupled microbial agent of the present invention.

[0050] In the above scheme, the quality-improving and nitrogen-fixing coupled microbial agent can also be a combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812 and other bacteria, 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 12 or 4 or more gene sequences therein or their functional equivalents, 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 12 or 4 or more gene sequences therein or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules of legume crops, thereby constituting the quality-improving and nitrogen-fixing coupled microbial agent of the present invention.

[0051] In the above solution, the proportion of viable bacteria count of any one strain of the above microbial agents in the microbial agent, i.e., the mixed microbial composition, is greater than or equal to 1%.

[0052] The beneficial effects of the present invention are:

[0053] 1. Quality-enhancing and nitrogen-fixing microbial agents coupled with microbial inoculants can be used to increase the production of legumes such as peanuts and soybeans. This single technology improves the quality of legume products, further reduces legume crop diseases, / or improves the quality and safety of crop products, and / or reduces peanut fruit spotting, while simultaneously inducing efficient nodulation and nitrogen fixation in soybeans and peanuts, significantly increasing yields. 2. The application is simple, low-cost, and highly effective. 3. It is of great significance to promoting the expansion of soybean oilseed production capacity, high-quality development, and green, low-carbon, and efficient production in my country. Modes for Carrying Out the Invention

[0054] Example 1 Isolation and Identification of Microbial Strains (Series of Microbial Strains, Used in Combinations)

[0055] A series of microbial strains were obtained through the following isolation and identification steps.

[0056] (1) Take whole plants and rhizosphere soil samples of peanuts, soybeans, peas, broad beans, cowpeas, and alfalfa, grind them and mix them evenly, isolate the strains using conventional bacterial isolation methods, and then identify them using conventional 16s rDNA methods. Through the above operations, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Brevibacillus laterosporus, Bacillus mucilaginosus, Bacillus velezensis, Bacillus siamensis, Paenibacillus polymyxa, Paenibacillus timonensis, Pseudomonas fluorescens, Pseudomonas mendocina, Enterobacter ludwigii, Microbacterium proteolyticum, Leclercia adcarboxglata, and Serratia serratia were obtained. marcescens, Empedobacters sp., Priestia priestiamegaterium, Stenotrophomonas maltophilia, etc. The detailed information is shown in Table 1.

[0057] Table 1. Strains isolated and identified from mixtures of peanut, soybean and other major legume crops

[0058] Strain code or deposition number Strain name Strain code or deposition number Strain name CCTCC NO: M 20231597 Bacillus subtilis CCTCC NO: M 20231600 Bacillus velezensis CCTCC NO: M 20231808 Brevibacillus laterosporus CCTCC NO: M 20231810 Bacillus amyloliquefaciens CCTCC NO: M 20231812 Enterobacter ludwigii CCTCC NO: M 20231814 Microbacterium proteolyticum Strain 207 Paenibacillus timonensis Strain 208 Enterobacter ludwigii ludwigii strain 209 Stenotrophomonas maltophilia strain 210 Bacillus mucilaginosus strain 211 Bacillus siamensis strain 212 Bacillus amyloliquefaciens strain 213 Bacillus subtilis strain 214 Bacillus licheniformis strain 215 Brevibacillus laterosporus strain 216 Bacillus mucilaginosus strain 217 Paenibacillus polymyxa strain 218 Paenibacillus timonensis strain 219 Pseudomonas fluorescens strain 220 Pseudomonas mendocina mendocina strain 221 Leclercia adcarboxglata strain 222 Serratia marcescens strain 223 Empedobacterium brevisStrain 224 Priestia megaterium Strain 225 Stenotrophomonas maltophilia Strain 226 Enterobacter ludwigii Strain 227 Bacillus velezensis Strain 228 Brevibacillus laterosporus Strain 229 Bacillus amyloliquefaciens Strain 230 Microbacterium proteolyticum strain 231 is not a rhizobium, strain 232 is not a rhizobium of unidentified genus and species, strain 233 is not a rhizobium of unidentified genus and species, strain 234 is not a rhizobium of unidentified genus and species, strain 235 is not a rhizobium of unidentified genus and species, strain 236 is not a rhizobium of unidentified genus and species, strain 237 is not a rhizobium of unidentified genus and species, strain 238 is not a rhizobium of unidentified genus and species, strain 239 is not a rhizobium of unidentified genus and species, strain 240 is not a rhizobium of unidentified genus and species, strain 241 is not a rhizobium of unidentified genus and species, strain 242 is not a rhizobium of unidentified genus and species, and strain 237 is not a rhizobium of unidentified genus and species.

[0059] Example 2 Preparation of microorganisms and their compositions

[0060] The series of bacterial strains obtained above are amplified and cultured by conventional bacterial culture medium amplification culture method to prepare batches of the above strain fermentation liquid or bacterial powder.

[0061] The fermentation broths or bacterial powders of the above strains are mixed one by one or more than two to form a series of microbial compositions. The information of these microbial compositions is shown in Table 2, wherein the proportion of viable bacteria count of any one strain in each microbial combination is greater than or equal to 1%.

[0062] Table 2. Information on microorganisms or microbial compositions

[0063] Composition number Composition strains and proportions (%) Composition number Composition strains and proportions Bacterial agent 1 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Proportion: 30 / 30 / 30 / 10 Bacterial agent 2 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Proportion: 30 / 30 / 10 / 30 Bacterial agent 3 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 30 / 10 / 30 / 30 Bacterial agent 4 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 10 / 30 / 30 / 30 Bacterial agent 5 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 33 / 33 / 33 / 1 Bacterial agent 6 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 33 / 33 / 1 / 33 Bacterial agent 7 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 33 / 1 / 33 / 33 Bacterial agent 8 composition: CCTCC NO: M 20231597 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 1 / 33 / 33 / 33 Bacterial agent 9 composition: CCTCC NO: M 20231814 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 20 / 20 Inoculant 10 Composition: CCTCCNO: M 20231600 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 11 composition: CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 / strain 207 Ratio: 30 / 30 / 30 / 10 Bacterial agent 12 composition: strain 209 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 13 composition: strain 210 / CCTCC NO: M 20231808 / M 20231812 / CCTCC NO: M 20231810 ratio: 30 / 1 / 30 / 10 / 29 Agent 14 composition: strain 211 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 30 / 30 / 30 / 10 Agent 15 composition: strain 213 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 ratio: 40 / 40 / 20 Agent 16 composition: strain 214 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 17 composition: strain 217 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 18 composition: strain 218 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 19 composition: strain 219 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 20 composition: strain 219 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 / strain 220Ratio: 30 / 30 / 30 / 10 Bacterial agent 21 composition: strain 221 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 22 composition: strain 222 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 23 composition: strain 223 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 24 composition: strain 224 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 25 composition: strain 225 / CCTCC NO: M 20231808 / CCTCC NO: M 20231812 / CCTCC NO: M 20231810 Ratio: 30 / 30 / 30 / 10 Bacterial agent 26 composition: strain 208 / strain 210 / strain 212 / strain 214 Ratio: 30 / 30 / 30 / 10 Bacterial agent 27 composition: strain 215 / strain 216 / strain 226 Ratio: 30 / 30 / 30 / 10 Bacterial agent 28 composition: strain 226 / strain 227 / strain 228 / strain 229 Ratio: 30 / 30 / 30 / 10 Bacterial Agent 29 Composition: Strain 230 / Strain 231 / Strain 232 Ratio: 45 / 45 / 10 Bacterial Agent 30 Composition: Strain 233 / Strain 234 Ratio: 90 / 10 Bacterial Agent 31 Composition: Strain 235 / Strain 236 Ratio: 50 / 50 Bacterial Agent 32 Composition: Strain 217 Ratio: 100 Bacterial Agent 33 Composition: Strain 218 Ratio: 100 Bacterial Agent 34 Composition: Strain 219 Ratio: 100 Bacterial Agent 35 Composition: Strain 220 Ratio: 100 Bacterial Agent 36 Composition: Strain 221 Ratio: 100 Bacterial Agent 37 Composition: Strain 222 Ratio: 100 Bacterial Agent 38 Composition: Strain 223 Ratio: 100 Bacterial Agent 39 Composition: Strain 224 Ratio: 100 Bacterial Agent 40 Composition: Strain 225 Ratio: 100 Bacterial Agent 41 4 composition: strain 236 / strain 239 / strain 240 ratio: 30 / 30 / 40 agent 42 3 composition: strain 237 / strain 238 / strain 241 / strain 242 ratio: 25 / 25 / 25 / 25

[0064] Example 3: Sequencing of microbial agents

[0065] A sufficient number of samples were taken from the microorganisms or microbial compositions in Table 2 of Example 2, and total DNA was extracted from these samples in sequence using conventional DNA extraction methods. The DNA sequences of these samples were then determined using conventional DNA sequencing methods. Finally, conventional analysis methods were used to compare the homology of the DNA sequences determined above with the gene sequences provided in this patent text.

[0066] The results of the above homology analysis are shown in Table 3.

[0067] Table 3. Information on microorganisms or microbial compositions

[0068] The numbers of microorganisms or their compositions containing the 12 characteristic genes provided by this patent (only those with homology of more than 90% are counted), DNA sequence homology and proportions. The numbers of microorganisms or their compositions containing the 12 characteristic genes provided by this patent (only those with homology of more than 90% are counted), DNA sequence homology and proportions. Bacterial agent 1: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 2: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 3: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 4: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 5: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 6: 12 sequences: sequences 1 to 12, all with 100% homology and 100% proportion. Bacterial agent 7: 12 sequences: sequences 1 to 12, all with 100% homology. 812 bacterial agents (accounting for 100%): sequences 1-12, all with 100% homology 99 bacterial agents (accounting for 100%): sequences 1-3, 7-12, all with 100% homology 109 bacterial agents (accounting for 75%): sequences 1-3, 7-12, all with 100% homology 119 bacterial agents (accounting for 75%): sequences 1-3, 7-12, all with 100% homology 129 bacterial agents (accounting for 75%): sequences 1-3, 7-12, all with 100% homology 139 bacterial agents (accounting for 75%): sequences 1-3, 7-12, all with 100% homology 149 bacterial agents (accounting for 75%): sequences 1-3, 7-12, all with 100% homology 157 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-9; and 90.3%-93.9% for sequences 10 and 11. 169 bacterial agents, accounting for 67%, have 100% homology for sequences 1-3 and 7-12. 179 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-12. 189 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-12. 199 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-12. 209 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-12. 219 bacterial agents, accounting for 75%, have 100% homology for sequences 1-3 and 7-12. 75% of the bacterial agents (229): sequences 1-3, 7-12, all with 100% homology. 75% of the bacterial agents (239): sequences 1-3, 7-12, all with 100% homology. 75% of the bacterial agents (249): sequences 1-3, 7-12, all with 100% homology. 75% of the bacterial agents (259): sequences 1-3, 7-12, all with 100% homology. 75% of the bacterial agents (266): sequences 1-3, 7-9, with homologies of 90.1%-99.9%. 50% of the bacterial agents (275): sequences 4-6, 8, 9, with homologies of 92.5%-99.6% (42%) 285 bacterial agents: sequences 1-3, 11, 12, with 90%-99.9% homology (42%) 295 bacterial agents: sequences 1-4, 7, with 91.2%-99.9% homology (42%) 304 bacterial agents: sequences 1, 2, 7, 9, with 90%-100% homology (33%) 314 bacterial agents: sequences 3, 4, 6, 8, with 90%-100% homology 33% of the total number of bacterial agents: 320, 0, 330, 0, 340, 0, 350, 0, 360, 0, 370, 0, 380, 0, 390, 0, 400, 0, 414: sequences 4, 7, 10, 12, with a homology of 90-99%; 33% of the total number of bacterial agents: sequences 3, 5, 12, with a homology of 96-100%; 25% of the total number of bacterial agents:

[0069] Example 4: Determination of the effect of peanut microbial inoculant on quality improvement and nitrogen fixation

[0070] Taking peanuts as an example, the steps for determining the quality-enhancing and nitrogen-fixing effects of the above-mentioned microbial agents are as follows.

[0071] The microbial agents in Table 3 of Example 3 above were applied to the field along with the peanut sowing base fertilizer, or they could be applied to the field during the peanut growth period, with an application rate of greater than or equal to 80 billion viable bacteria per mu. A plot not applied with any of the above microbial agents was set up as a control, and conventional field management was used for the others. A continuous investigation was conducted after emergence; an investigation was conducted within the first 30 days after emergence to determine whether nodules had formed in advance; an investigation was conducted during the flowering and needle-setting period to determine the number, weight, and nitrogenase activity of nodules per plant; an investigation was conducted 1 to 15 days before harvest to determine whether root nodules had formed during the mature stage of peanuts, i.e., whether the nodulation time had been extended; an investigation was conducted on the effects of field disease reduction, i.e., disease prevention and control, from the seedling stage to the harvest period; and an investigation was conducted after harvest to determine whether the total plant biomass, peanut yield per unit area, peanut quality, including protein, glutamic acid, peanut resveratrol, and the abundance of aflatoxin, to determine whether the quality and safety of the peanuts had been improved.

[0072] The above survey results are shown in Table 4. Based on the results in Table 4, agents 1–31 and 41 simultaneously possess the following characteristics: 1) they contain at least four of the DNA sequences 1–12, and these genes may vary by no more than 10% between different strains; 2) they can improve the quality of legume crops by increasing the content of one or more nutritional components, such as protein, glutamic acid, and peanut resveratrol; 3) they can alleviate or control one or more soil-borne diseases, such as peanut fruit rot, soybean green disease, white rot, bacterial wilt, root rot, and sclerotinia, or reduce peanut fruit surface spotting or significantly reduce aflatoxin levels, thereby improving quality and safety; 4) although not rhizobia themselves, they can simultaneously regulate and increase the abundance of rhizobia in the rhizosphere of legume crops, increase the number of nodules, and enhance nitrogenase activity per plant; 5) they can promote early nodulation and prolong the duration of nodulation and nitrogen fixation in legumes, such as peanuts and soybeans; and 6) they can increase the yield and total biomass of legume crops. However, bacterial agents 32~40 and 42 could not meet the above six characteristics at the same time.

[0073] Table 4. Results of the determination of the effect of microbial agents on improving the quality of peanuts and nitrogen fixation

[0074] Microorganism or its combination number: whether it improves the quality; whether it has the effect of preventing and controlling peanut diseases such as fruit rot, white rot, and bacterial wilt; or reduces the spots on the peanut fruit surface; or significantly reduces the aflatoxin level; improves the quality and safety level; whether it doubles the promotion of nodulation and nitrogen fixation, early nodulation, extended nodulation and nitrogen fixation time, increased yield and total biomass; Microorganism or its combination number: whether it improves the quality or significantly improves the quality and safety level; whether it has the effect of preventing and controlling peanut diseases such as fruit rot, white rot, and bacterial wilt; or reduces the spots on the peanut fruit surface; or significantly reduces the aflatoxin level; improves the quality and safety level; whether it doubles the promotion of nodulation and nitrogen fixation, early nodulation, extended nodulation and nitrogen fixation time, increased yield and total biomass; Microbial agent 1: yes: yes; Microbial agent 2: yes: yes; Microbial agent 3: yes: yes; Microbial agent 4: yes: yes; Microbial agent 5: yes: yes Yes, yes ... Yes Yes Bacteria 25 Yes Yes Yes Bacteria 26 Yes Yes Yes Bacteria 27 Yes Yes Yes Bacteria 28 Yes Yes Yes Bacteria 29 Yes Yes Yes Bacteria 30 Yes Yes Yes Bacteria 31 Yes Yes Yes Bacteria 32 No No No Bacteria 33 No No No Bacteria 34 No No No Bacteria 35 No No No Bacteria 36 No No No Bacteria 37 No No No Bacteria 38 No No No Bacteria 39 No No No Bacteria 40 No No No Bacteria 41 Yes Yes Yes Bacteria 42 No No No

[0075] The above-mentioned fungal agent can improve the quality of peanut products of leguminous crops - increase the content of one or more nutritional functional ingredients in peanut products such as protein, glutamic acid, peanut resveratrol, etc.: Peanut quality is improved: the protein content is increased by more than 0.2%, and preferably can reach more than 3%, the total content of essential amino acids for the human body such as leucine is increased by 0.15 mg / kg, and preferably, can reach more than 0.2 mg / kg, and the peanut resveratrol content is increased by more than 0.2 mg / kg, and more preferably, can reach more than 0.4 mg / kg.

[0076] Example 5: Determination of the Effect of Soybean Microbial Agent on Quality Improvement and Nitrogen Fixation

[0077] Taking soybean as an example, the steps for determining the quality improvement and nitrogen fixation effects of the above-mentioned microbial agents are described as follows.

[0078] The microbial agents described in Table 3 of Example 3 above were applied to the field along with soybean sowing base fertilizer, or during the soybean growth period, at an application rate of greater than or equal to 80 billion viable bacteria per mu. A plot not treated with any of the microbial agents was set up as a control, and conventional field management was used for all other plots. Continued investigations were conducted after seedling emergence. Early nodulation was investigated within the first 30 days after emergence. Nodule number, nodule weight, and nitrogenase activity per plant were investigated during the flowering and podding phase. Root nodulation during the mature peanut phase was investigated 1 to 15 days before harvest to determine whether the nodulation time was extended. Field disease mitigation, i.e., disease control efficacy, was investigated from the seedling stage to the harvest phase. After harvest, total plant biomass, soybean yield per unit area, soybean quality, including protein, glutamic acid, soy isoflavones, and the abundance of aflatoxin were investigated to determine whether the soybeans improved quality and safety.

[0079] The above survey results are basically consistent with the results in Table 4. The above-mentioned microbial agents can improve the quality of soybean products of leguminous crops - increasing the content of one or more nutritional functional components such as protein, glutamic acid, and soy isoflavones in soybean products: the protein content is increased by more than 0.3%, and preferably can reach more than 3%; the total content of essential amino acids for the human body, such as leucine, is increased by more than 0.2 mg / kg, and preferably can reach more than 0.3 mg / kg; the soy isoflavone content is increased by more than 50 mg / kg, and preferably can reach more than 200 mg / kg.

[0080] Based on this data, agents 1-31 and 41 simultaneously possess the following characteristics: 1) they contain at least four of the DNA sequences 1-12, with no more than 10% base variation in these genes between strains; 2) they can improve the quality of legume crops by increasing the content of one or more nutritional components, such as protein, glutamic acid, and soy isoflavones; 3) they can mitigate or control one or more soil-borne diseases, such as peanut fruit rot, soybean green disease, white rot, bacterial wilt, root rot, and sclerotinia, or significantly reduce aflatoxin levels, thereby improving quality and safety; 4) while not themselves rhizobia, they can simultaneously regulate and increase the abundance of rhizobia in the rhizosphere of legumes, increase the number of nodules, and enhance nitrogenase activity per plant; 5) they can promote early nodulation and prolong the duration of nodulation and nitrogen fixation in legumes, such as peanuts and soybeans; and 6) they can increase the yield and total biomass of legumes. Agents 32-40 and 42, however, do not meet all six of these characteristics.

[0081] Example 6: Determination of the Effect of Microbial Agents on Quality Improvement and Nitrogen Fixation of Other Leguminous Crops

[0082] The same sampling method and steps as in Examples 4 and 5 were used to determine the effect of microbial agents on the quality improvement and nitrogen fixation of other legume crops such as peas, broad beans, cowpeas, and alfalfa, and similar results were obtained as for peanuts and soybeans mentioned above, and the conclusions were consistent with those in Table 4.

[0083] Selection of microbial agents for quality improvement and nitrogen fixation

[0084] According to the results in Tables 1-4 above, agents 1-31 and 41 simultaneously possess the following characteristics: 1) The agents contain at least four of the DNA sequences 1-12, and these genes may vary by no more than 10% in different strains; 2) The agents can improve the quality of legume products by increasing the content of one or more nutritional functional components such as protein, glutamic acid, peanut resveratrol, and soy isoflavones; 3) The agents have a mitigating effect, or control effect, on one or more soil-borne diseases such as peanut fruit rot, soybean green disease, white rot of legumes, bacterial wilt, root rot, and sclerotinia, or reduce peanut fruit surface spotting or significantly reduce aflatoxin levels, thereby improving quality and safety; 4) Although not rhizobia themselves, the agents can simultaneously regulate and increase the abundance of rhizobia in the rhizosphere of legumes, increase the number of nodules in legumes, and thus increase the nitrogenase activity per plant; 5) The agents can promote early nodulation and prolong the time of nodulation and nitrogen fixation in legumes such as peanuts and soybeans; and 6) The agents can increase the yield and total biomass of legume production.

[0085] The microbial agent of the present invention was tested by knocking out 1 to 12 DNA sequences. The results showed that when more than 4 gene sequences remained after the knockout, the microbial agent still had the coupling effect of improving the quality and fixing nitrogen of the present invention, improving the quality of legume crop products, and had the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, increasing the number of nodules in legume crops, and further promoting the early nodulation of legume crops and prolonging the time of nodulation and nitrogen fixation. When 3 or fewer gene sequences were knocked out, the microbial agent no longer had the coupling effect of improving the quality and fixing nitrogen, but had the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, increasing the number of nodules in legume crops, and further promoting the early nodulation of legume crops and prolonging the time of nodulation and nitrogen fixation.

[0086] Example 7: Application of quality improvement and nitrogen fixation coupled with microbial inoculants - Improving the quality and safety of leguminous crop products

[0087] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing and nitrogen-fixing coupled microbial agents to improve the quality and safety level of leguminous crop products is described.

[0088] 1. Application on peanuts: Mix the above-mentioned microbial agents 1, 7, and 25 with peanut seeding base fertilizer respectively, and apply them to the field through a seed drill. The application rate of microbial agents is 80 billion to 100 billion viable bacteria per mu. At the same time, set up a plot without any of the above-mentioned microbial agents as a control. The rest are managed by conventional field management. After the soybeans are harvested, peanut samples are collected using the national standard sampling method. (1) The abundance of aflatoxin-producing fungi in these samples is determined by the classic colony counting method, and the reduction rate of the abundance of aflatoxin-producing fungi in soybeans is calculated, that is, the control effect on aflatoxin-producing fungi; (2) The content of soybean isoflavones, protein, and glutamic acid is determined by the standard method, and the quality improvement level is calculated; (3) After the samples are placed under the same conditions for 6 months, the aflatoxin contamination level is determined by the national standard liquid chromatography-mass spectrometry method, and the control effect on aflatoxin is calculated. These test results showed that agents 1, 7, and 25 all achieved over 62% control effectiveness against aflatoxin-producing fungi in field peanuts and over 80% control effectiveness against aflatoxin in peanuts. They also increased protein content by over 0.2%, the combined content of essential amino acids such as leucine increased by 0.15 mg / kg, and resveratrol content in peanuts by over 0.2 mg / kg. These results indicate that the application of quality-enhancing and nitrogen-fixing microbial agents significantly improved the quality and safety of field peanuts.

[0089] 2. Application on Soybeans: Before soybean sowing, agents 1, 7, and 25 were applied to the fields via drone at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million saplings). Plots not treated with any of the agents served as controls, while all other plots were managed conventionally. After harvest, soybean samples were collected using national standard sampling methods and assayed for isoflavone, protein, and amino acid content using standard methods to calculate the quality improvement. These assays showed that the application of agents 1, 7, and 25 significantly increased the isoflavone, protein, and glutamic acid content of soybeans grown in the field. Protein content increased by over 0.3%, essential amino acids such as leucine increased by over 0.2 mg / kg, and isoflavone content increased by over 50 mg / kg. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents significantly improved soybean quality in the field.

[0090] 3. The application on other leguminous crops has achieved similar results as the above-mentioned application on soybeans.

[0091] Example 8: Application of quality improvement and nitrogen fixation coupled with microbial inoculants - promoting nodulation and nitrogen fixation in leguminous crops

[0092] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing nitrogen fixation coupled with microbial agents to promote nodulation and nitrogen fixation in leguminous crops is described.

[0093] 1. Application on Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed using conventional methods. After the peanuts emerged, continuous investigations were conducted on the root nodules. These results showed that during the flowering phase, the number of root nodules in peanuts treated with agents 1, 7, and 25 increased by more than 2.3 times, and the nitrogenase activity per plant increased by more than 7.6 times. These results demonstrate that the application of quality-enhancing nitrogen-fixing agents coupled with microbial agents significantly promoted nodulation and nitrogen fixation in peanuts in the field.

[0094] 2. Application on Soybeans: Before soybean sowing, agents 1, 7, and 25 were applied to the fields via drone 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 using conventional methods. Root nodulation was continuously monitored after soybean seedlings emerged. These surveys revealed that during the flowering phase, the number of root nodules in soybeans treated with agents 1, 7, and 25 increased by over 2.3 times, and the nitrogenase activity per soybean plant increased by over 6.9 times. These results demonstrate that the application of quality-enhancing nitrogen-fixing agents coupled with microbial agents significantly promoted soybean nodulation and nitrogen fixation in the field.

[0095] 3. When applied to other leguminous crops, it achieved similar effects in promoting nodulation and nitrogen fixation as in peanuts and soybeans.

[0096] Example 9: Application of quality improvement and nitrogen fixation coupled with microbial inoculants to improve the yield of leguminous crops

[0097] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-improving and nitrogen-fixing coupled microbial agents to increase the yield level of leguminous crops is described.

[0098] 1. Application on Peanuts: Mix the aforementioned agents 1, 7, and 25 with peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. 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 the agents and the control plots. Calculations showed that the yield increase in the fields treated with agents 1, 7, and 25 was over 12%, achieving significant yield increases. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents significantly increased field peanut yields.

[0099] 2. Application on Soybeans: Before soybean sowing, microbial agents 1, 7, and 25 were evenly applied to the fields via drone at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot was established without any of the microbial agents, while all other plots were managed conventionally. At harvest time, soybeans were harvested from one mu of land in each of the treated and control plots. Calculations showed that peanut yield increases in the plots treated with microbial agents 1, 7, and 25 were all above 7%, achieving significant yield increases. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents coupled with microbial agents significantly increased soybean yields per unit area.

[0100] 3. Application on Peas: Before sowing peas, agents 1, 7, and 25 were evenly applied to the field by hand broadcasting, with a cumulative application 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 using conventional methods. Yields were measured by weighing the pea seedlings and green pea pods at harvest. The yield increases for pea seedlings and pea pods treated with agents 1, 7, and 25 were all above 5%, achieving significant yield increases. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents coupled with these agents significantly increased the yield per unit area of ​​pea seedlings and pea pods in the field.

[0101] 4. Application on other legume crops: Application of agents 1, 7, and 25 to legume crops such as broad beans yielded similar results to those achieved on soybeans. Application of agents 1, 7, and 25 to kidney beans and cowpeas yielded similar results to those achieved on peas. Application of agents 1, 7, and 25 to alfalfa also yielded similar results to those achieved on peas.

[0102] Example 10: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation - Recruiting Indigenous Rhizobia to Increase Rhizobium Abundance in the Rhizosphere Soil of Leguminous Crops

[0103] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-improving and nitrogen-fixing coupled microbial agents to increase the yield level of leguminous crops is described.

[0104] 1. Application on Peanuts: Inoculants 1, 7, and 25 were mixed with peanut seeding base 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). Plots not treated with any of the above agents served as controls, while all other plots were managed using conventional field management. Peanut rhizosphere soil samples were collected during the fruiting period and before harvest, and metagenomic sequencing and big data analysis were performed. These findings revealed that the abundance of rhizobia in the rhizosphere soil of peanuts treated with agents 1, 7, and 25 was significantly higher than in the control group, increasing by 10.3% to 27.1%. These results demonstrate that the application of microbial agents for quality improvement and nitrogen fixation, coupled with the recruitment of indigenous rhizobia, significantly increases the abundance of rhizobia in the rhizosphere soil of leguminous crops.

[0105] 2. When applied to other legume crops such as soybeans, peas, broad beans, cowpeas, and alfalfa, it achieved similar effects as the above-mentioned application on peanuts, which was to recruit indigenous rhizobia and thus increase the abundance of rhizobia in the rhizosphere soil of legume crops.

[0106] Example 11: Use of quality improvement and nitrogen fixation coupled with microbial agents - promoting early nodulation of leguminous crops and extending the time of nodulation and nitrogen fixation

[0107] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the purpose of coupling quality improvement and nitrogen fixation with microbial agents to promote early nodulation of leguminous crops and prolong the nodulation and nitrogen fixation time is described.

[0108] 1. Application on Peanuts: Inoculants 1, 7, and 25 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. Plots not treated with any of the above agents served as controls, while all other plots were managed under conventional practices. Root nodulation was continuously monitored after emergence. These results showed that peanuts treated with agents 1, 7, and 25 developed root nodules approximately 7 to 9 days after emergence, more than 20 days earlier than the conventional estimate of 30 days. New nodules were still observed during the full-fruiting stage, with the presence of old, middle-aged, and young nodules, as well as infant nodules, coexisting in the field. Fresh, active nodules with nitrogenase activity were still observed at harvest, disproving the conventional wisdom that no new nodules form after the seed-filling stage and that all nodules are depleted by harvest. This significantly extends the duration of nodulation and nitrogen fixation. The above results show that the application of quality improvement and nitrogen fixation coupled with microbial agents can achieve early nodulation of peanut legume crops and extend the time of nodulation and nitrogen fixation.

[0109] 2. Application on Soybeans: Before soybean sowing, microbial agents 1, 7, and 25 were applied to the fields via drone 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 practices. Root nodulation was continuously monitored after soybean seedlings emerged. These results showed that soybeans treated with microbial agents 1, 7, and 25 developed root nodules approximately 6 to 8 days after seedling emergence, approximately 20 days earlier than the conventional wisdom of around 30 days. New nodules were still observed during the grain-filling stage, with nodules from older, middle-aged, and young plants coexisting in the field. Fresh, viable nodules with nitrogenase activity were still observed at harvest, disproving the conventional wisdom that soybeans no longer develop new nodules after the grain-filling stage and that all nodules are depleted by harvest. This significantly extends the duration of nodulation and nitrogen fixation. The above results show that the application of quality improvement and nitrogen fixation coupled with microbial agents can achieve early nodulation and prolong the nodulation and nitrogen fixation time.

[0110] 3. When applied to other leguminous crops, the above-mentioned microbial agents 1, 7, and 25 respectively achieved similar effects as those achieved in peanuts and soybeans, in that they promoted early nodulation of leguminous crops and prolonged the time of nodulation and nitrogen fixation.

[0111] Example 12: Application of quality improvement and nitrogen fixation coupled with microbial agents to prevent premature aging of leguminous crops during maturity

[0112] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to prevent premature aging of leguminous crops during the maturity period is described.

[0113] 1. Application on Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed using conventional methods. Once the peanuts reached maturity, continuous monitoring of leaf photosynthetic rates and senescence was conducted. These results showed that leaf photosynthetic rates in peanuts treated with agents 1, 7, and 25 increased by over 33% compared to the control, demonstrating a significant effect in preventing premature aging due to nutrient deficiency. These results demonstrate that the application of microbial agents, coupled with quality improvement and nitrogen fixation, can effectively prevent premature aging of leguminous crops during their maturity period.

[0114] 2. Application on Soybeans: Before soybean sowing, agents 1, 7, and 25 were applied to the fields via drone 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 conventionally. Root nodulation was continuously monitored after soybean seedlings emerged. These surveys revealed that soybeans treated with agents 1, 7, and 25 increased their leaf photosynthetic rates by over 23.5%, demonstrating a significant protective effect against premature aging due to nutrient depletion. These results demonstrate that the application of quality-enhancing nitrogen fixation coupled with microbial agents resulted in earlier nodulation and prolonged nodulation and nitrogen fixation.

[0115] 3. Application on other leguminous crops: The application of fungicides 1, 7, and 25 on other leguminous crops such as peas, broad beans, and cowpeas has achieved similar effects as their application on peanuts and soybeans in preventing premature aging during maturity.

[0116] Example 12: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation to Increase the Number of Leguminous Crops

[0117] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to increase the number of pods in leguminous crops is described.

[0118] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed using conventional methods. Peanut pod counts were surveyed at harvest time. These survey results showed that the number of viable pods in the peanuts treated with agents 1, 7, and 25 increased by over 16.3% compared to the control, significantly increasing pod number. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents combined with these agents can significantly increase peanut pod number.

[0119] 2. Application on Soybeans: Before soybean sowing, agents 1, 7, and 25 were applied to the fields via drone 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 conventionally. Pod counts were surveyed at harvest time. These survey results showed that soybean pod counts increased by over 13.5% in the soybeans treated with agents 1, 7, and 25, significantly increasing pod number. These results demonstrate that the application of microbial agents combined with quality-enhancing and nitrogen-fixing properties can significantly increase soybean pod number.

[0120] 3. When applied to other leguminous crops such as peas, broad beans, kidney beans, and cowpeas, it achieved a similar effect as the above-mentioned application on peanuts and soybeans, which significantly increased the number of pods.

[0121] Example 12: Application of quality improvement and nitrogen fixation coupled with microbial inoculants - increasing leguminous crop pod plumpness and reducing pod shrinkage rate

[0122] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the purpose of coupling the quality-improving and nitrogen-fixing microbial agents to increase the plumpness of leguminous crop pods and reduce the pod shrinkage rate is described.

[0123] 1. Application on Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative bacterial count of 80 to 100 billion per mu (approximately 1.5 to 2.5 billion) should be achieved. A control plot without any of the agents should be established, while all other plots should be managed conventionally. At harvest, investigate the condition of the peanut pods and calculate the pod shriveling rate. These results show that the application of agents 1, 7, and 25 significantly improved pod plumpness and reduced pod shriveling rate by over 7.5%. These results demonstrate that the application of microbial agents, combined with quality-enhancing and nitrogen-fixing agents, significantly increases peanut pod plumpness and reduces pod shriveling rate.

[0124] 2. Application on Soybeans: Before soybean sowing, microbial agents 1, 7, and 25 were applied to the fields via drone 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 using conventional methods. At harvest, soybean pods were surveyed and pod shriveling rates were calculated. These survey results showed that soybeans treated with agents 1, 7, and 25 showed significantly improved pod plumpness and a reduction in pod shriveling rates by over 5.7%. These results demonstrate that the application of microbial agents, coupled with quality-enhancing and nitrogen-fixing agents, significantly increases soybean pod plumpness and reduces pod shriveling rates.

[0125] 3. The application of microbial agents 1, 7, and 25 on other leguminous crops such as peas, broad beans, kidney beans, and cowpeas achieved similar effects as their application on peanuts and soybeans, increasing the plumpness of peanut pods and reducing the rate of shrunken pods.

[0126] Example 12: Use of quality improvement and nitrogen fixation coupled with microbial agents to promote early flowering and pod formation in leguminous crops

[0127] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-improving and nitrogen-fixing coupled microbial agents to promote early flowering and pod setting in leguminous crops is described.

[0128] 1. Application on Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed using conventional methods. After the peanuts emerged, continuous surveys were conducted to analyze the flowering and pod setting times of the treated and control peanuts. These survey results showed that peanuts treated with agents 1, 7, and 25 flowered and podded at least two days earlier. These results demonstrate that the application of quality-enhancing, nitrogen-fixing microbial agents coupled with these agents significantly promotes earlier flowering and pod setting in peanuts.

[0129] 2. Application on Soybeans: Before soybean sowing, microbial agents 1, 7, and 25 were applied to the fields via drone 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 conventionally. After soybean seedlings emerged, continuous surveys were conducted to analyze the flowering and pod setting times of the treated and control soybeans. These surveys showed that soybeans treated with agents 1, 7, and 25 flowered and pod set at least two days earlier. These results demonstrate that the application of microbial agents, combined with quality-enhancing and nitrogen-fixing properties, significantly promotes earlier flowering and pod setting in soybeans.

[0130] 3. When applied to other leguminous crops such as peas, broad beans, kidney beans, and cowpeas, similar effects of early flowering and early pod setting as those mentioned above were achieved in peanuts and soybeans.

[0131] Example 13: Use of Quality Improvement and Nitrogen Fixation Coupled with Microbial Agents - Alleviating Peanut Fruit Rot

[0132] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of peanut fruit rot is described.

[0133] Inoculants 1, 7, and 25 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. A control plot was established without any of the above agents, while all other plots were managed conventionally. At harvest, the incidence of peanut fruit rot was statistically analyzed compared to the control plots. These findings showed that the incidence of peanut fruit rot in the plots treated with agents 1, 7, and 25 was reduced by over 60% compared to the control group. These results demonstrate that the application of microbial agents, combined with quality-enhancing and nitrogen-fixing agents, significantly reduces peanut fruit rot.

[0134] Example 14: Use of Nitrogen Quality Improvement and Fixation Coupled with Microbial Agents to Reduce the Occurrence of Bacterial Wilt in Leguminous Crops

[0135] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of bacterial wilt in leguminous crops is described.

[0136] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed conventionally. After the peanuts emerged, ongoing surveys were conducted to statistically analyze the incidence of peanut bacterial wilt compared to the control plots. These survey results showed that the incidence of peanut bacterial wilt in the peanuts treated with agents 1, 7, and 25 was reduced by over 55% compared to the control plots. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces the incidence of peanut bacterial wilt.

[0137] 2. Application in other leguminous crops: Application in other leguminous crops such as soybeans, peas, broad beans, kidney beans, and cowpeas has achieved similar effects as the above-mentioned application in peanuts in reducing the incidence of root rot in leguminous crops.

[0138] Example 15: Use of a Microbial Agent for Quality Improvement and Nitrogen Fixation to Reduce Powdery Mildew in Leguminous Crops

[0139] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of powdery mildew in leguminous crops is described.

[0140] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative bacterial count of 80 to 100 billion per mu (approximately 100 million saplings) should be achieved. A control plot without any of the agents should be established, while all other plots should be managed conventionally. After the peanuts emerge, ongoing surveys should be conducted to statistically analyze the incidence of peanut fruit rot compared to the control plots. These survey results show that the incidence of powdery mildew in peanuts treated with agents 1, 7, and 25 was reduced by over 60% compared to the control group. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces the incidence of powdery mildew in peanuts.

[0141] 2. Application in other leguminous crops: Application in other leguminous crops such as soybeans, peas, broad beans, kidney beans, and cowpeas has achieved similar effects as the above-mentioned application in peanuts in reducing the incidence of powdery mildew in leguminous crops.

[0142] Example 16: Use of a Microbial Agent for Nitrogen Quality Improvement and Fixation to Reduce Leaf Spot Disease in Leguminous Crops

[0143] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing nitrogen-fixing coupled microbial agents to reduce the incidence of leaf spot disease in leguminous crops is described.

[0144] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed conventionally. After the peanuts emerged, ongoing surveys were conducted to statistically analyze the incidence of peanut leaf spot compared to the control plots. These survey results showed that the incidence of peanut leaf spot in the plots treated with agents 1, 7, and 25 was reduced by over 53% compared to the control plots. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces the incidence of peanut leaf spot.

[0145] 2. Application in other leguminous crops: Application in other leguminous crops such as soybeans, peas, broad beans, kidney beans, and cowpeas has achieved similar effects as the above-mentioned application in peanuts in reducing the incidence of leaf spot disease in leguminous crops.

[0146] Example 17: Use of a Microbial Agent for Nitrogen Quality Improvement and Fixation to Reduce the Occurrence of Root Nematode Disease in Leguminous Crops

[0147] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing nitrogen-fixing coupled microbial agents to reduce the incidence of root nematode disease in leguminous crops is described.

[0148] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed conventionally. After the peanuts emerged, ongoing surveys were conducted to assess the incidence of root nematodes in the peanuts compared to the control plots. These survey results showed that the incidence of root nematodes in the peanuts treated with agents 1, 7, and 25 was reduced by over 60% compared to the control group. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces root nematode disease in peanuts.

[0149] 2. Application in other leguminous crops: It has been applied to other leguminous crops such as soybeans, peas, broad beans, kidney beans, and cowpeas, and has achieved similar effects as the above-mentioned application in peanuts in reducing the incidence of root nematode diseases in leguminous crops.

[0150] Example 18: Use of Quality Improvement and Nitrogen Fixation Coupled with Microbial Agents to Reduce the Occurrence of Root Rot in Leguminous Crops

[0151] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of root rot in leguminous crops is described.

[0152] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative bacterial count of 80 to 100 billion per mu (approximately 100 million active bacteria) was applied. A control plot was established without any of the agents, while all other plots were managed using conventional methods. After the peanuts emerged, ongoing surveys were conducted, and statistical analysis was performed to compare the incidence of peanut root rot with the control plots. These survey results showed that the incidence of peanut root rot in the peanuts treated with agents 1, 7, and 25 was reduced by over 57.5% compared to the control group. These results demonstrate that the application of microbial agents, combined with quality improvement and nitrogen fixation, significantly reduces peanut root rot.

[0153] 2. Application on Soybeans: Inoculants 1, 7, and 25 were mixed with soybean seeding base fertilizer and applied to the field via drone at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot was established without any of the above agents, while all other plots were managed conventionally. After soybean seedlings emerged, continuous surveys were conducted to statistically analyze the incidence of soybean root rot compared to the control plots. These survey results showed that peanut root rot incidence in the plots treated with agents 1, 7, and 25 was reduced by over 55% compared to the control plots. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces soybean root rot.

[0154] 3. Application in other leguminous crops: Application in other leguminous crops such as peas, broad beans, kidney beans, cowpeas, etc. has achieved similar effects in reducing root rot as the above-mentioned application in peanuts and soybeans.

[0155] Example 19: Use of a Microbial Agent for Quality Improvement and Nitrogen Fixation to Reduce the Occurrence of Sclerotinia in Leguminous Crops

[0156] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of sclerotinia disease in leguminous crops is described.

[0157] 1. Application on Soybeans: Mix the aforementioned agents 1, 7, and 25 with soybean seeding base fertilizer and apply them to the field using a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres). Set up plots without any of the agents as controls, while all other plots receive conventional field management. After soybean seedlings emerge, conduct ongoing surveys to statistically analyze the incidence of sclerotinia rot in comparison to the control plots. These survey results show that soybeans treated with agents 1, 7, and 25 experienced a reduction of over 62% in phytophthora sclerotiorum compared to the control plots. These results demonstrate that the application of microbial agents for quality improvement and nitrogen fixation, coupled with other strategies, significantly reduces sclerotinia rot in soybeans.

[0158] 2. Application in other leguminous crops: Application in other leguminous crops such as peanuts, peas, broad beans, kidney beans, cowpeas, etc. has achieved similar effects as the above-mentioned application in soybeans in reducing the incidence of sclerotinia disease in leguminous crops.

[0159] Example 20: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation to Reduce Downy Mildew in Leguminous Crops

[0160] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of downy mildew in leguminous crops is described.

[0161] 1. Application on Soybeans: Mix the aforementioned agents 1, 7, and 25 with soybean seeding base fertilizer and apply them to the field using a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million sq ft). Set up plots without any of the agents as controls, while all other plots receive conventional field management. After soybean seedlings emerge, conduct ongoing surveys to statistically analyze the incidence of downy mildew in soybeans compared to the control. These survey results show that soybeans treated with agents 1, 7, and 25 experienced a reduction of over 58% in downy mildew compared to the control. These results demonstrate that the application of microbial agents, coupled with quality improvement and nitrogen fixation, significantly reduces soybean downy mildew.

[0162] 2. Application in other leguminous crops: Application in other leguminous crops such as peanuts, peas, broad beans, kidney beans, and cowpeas has achieved similar effects as the above-mentioned application in soybeans in reducing the incidence of downy mildew in leguminous crops.

[0163] Example 21: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation to Reduce the Occurrence of Fusarium Wilt in Leguminous Crops

[0164] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of wilt disease in leguminous crops is described.

[0165] 1. Application on Soybeans: Mix the aforementioned agents 1, 7, and 25 with soybean seeding base fertilizer and apply them to the field using a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million sq ft). Set up plots without any of the agents as controls, while all other plots receive conventional field management. After soybean seedlings emerge, conduct ongoing surveys to statistically analyze the incidence of soybean wilt compared to the control. These survey results show that soybeans treated with agents 1, 7, and 25 exhibited a nearly 60% reduction in wilt compared to the control group. These results demonstrate that the application of microbial agents, combined with quality improvement and nitrogen fixation, significantly reduces soybean wilt.

[0166] 2. Application in other leguminous crops: Application in other leguminous crops such as peanuts, peas, broad beans, kidney beans, cowpeas, etc. has achieved similar effects as the above-mentioned application in soybeans in reducing the incidence of wilt disease in leguminous crops.

[0167] Example 22: Use of Microbial Agents for Nitrogen Quality Improvement and Fixation - Reducing the Occurrence of Sclerotinia rot in Leguminous Crops

[0168] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing nitrogen-fixing coupled microbial agents to reduce the incidence of white rot in leguminous crops is described.

[0169] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. A control plot was established without any of the agents, while all other plots were managed conventionally. After the peanuts emerged, ongoing surveys were conducted to statistically analyze the incidence of white rot in the peanuts compared to the control plots. These survey results showed that the incidence of white rot in the peanuts treated with agents 1, 7, and 25 was reduced by over 60% compared to the control group. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces the incidence of white rot in peanuts.

[0170] 2. Application in other leguminous crops: It has been applied to other leguminous crops such as soybeans, peas, broad beans, kidney beans, and cowpeas, and has achieved similar effects as its application in peanuts in reducing the incidence of white rot in leguminous crops.

[0171] Example 23: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation to Reduce the Incidence of Soybean Greening

[0172] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce the incidence of soybean greening is described.

[0173] Inoculants 1, 7, and 25 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). Plots not treated with any of the above agents served as controls, while all other plots were managed using conventional field management. After soybean seedlings emerged, ongoing surveys were conducted to assess the incidence of soybean greening compared to the control. These survey results showed that soybean greening was reduced by over 40% in the plots treated with inoculants 1, 7, and 25 compared to the control. These results demonstrate that the application of microbial agents, coupled with quality improvement and nitrogen fixation, significantly reduces the incidence of soybean greening.

[0174] Example 24: Use of Microbial Agents for Quality Improvement and Nitrogen Fixation - Promoting Carbon Emission Reduction in Leguminous Crops and Benefiting Soil Improvement

[0175] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to promote carbon emission reduction in leguminous crops is described.

[0176] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with a base fertilizer for peanut sowing, using 20% ​​and 30% less nitrogen than the conventional control. Apply the agents to the field using a seed drill, at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot without any of the agents was established, while all others were managed using conventional methods. After the peanuts emerged, continuous surveys were conducted to assess the carbon reduction levels of the treated and control peanuts. These survey results showed that peanuts treated with agents 1, 7, and 25 reduced carbon dioxide emissions by 20 to 30%, while also significantly increasing peanut yields by approximately 10%. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly reduces carbon emissions during peanut production.

[0177] 2. Application on Soybeans: Before soybean sowing, microbial agents 1, 7, and 25 were applied to the fields via drone at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million saplings). Nitrogen fertilizer was reduced by 20% and 50% in the treatment groups compared to the control group's conventional fertilization level. Plots not treated with any of the agents served as controls, while all other plots were managed under conventional field management. After soybean seedlings emerged, continuous surveys were conducted to assess carbon emission reductions in the treated and control soybeans. These survey results showed that soybeans treated with microbial agents 1, 7, and 25 reduced carbon dioxide emissions by 20% to 50%, while also significantly increasing soybean yields by more than 5%. These results demonstrate that the application of microbial agents, combined with quality improvement and nitrogen fixation, significantly reduces carbon emissions in soybean production.

[0178] 3. Its application on other leguminous crops such as peas, broad beans, kidney beans, and cowpeas has achieved similar effects in promoting carbon emission reduction as its application on peanuts and soybeans.

[0179] Example 25: Use of quality improvement and nitrogen fixation coupled with microbial agents - promoting the increase of total biomass of leguminous crops

[0180] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to promote the increase of the total biomass of leguminous crops is described.

[0181] 1. Application to Peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field using a seed drill. A cumulative application rate of 80 to 100 billion viable bacteria per mu (approximately 1.5 to 2.5 acres) was achieved. 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 agents 1, 7, and 25 increased by more than 22%. These results demonstrate that the application of microbial agents for quality improvement and nitrogen fixation, coupled with other microbial agents, significantly increases the total biomass of peanut plants.

[0182] 2. Application on Soybeans: Before soybean sowing, agents 1, 7, and 25 were applied to the fields via drone 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 conventionally. A survey was conducted at soybean maturity, and the total biomass of the treated and control soybean plants was calculated. These survey results showed that soybeans treated with agents 1, 7, and 25 increased their total plant biomass by over 13%. These results demonstrate that the application of microbial agents, combined with quality improvement and nitrogen fixation, significantly increases soybean plant biomass.

[0183] 3. When applied to other leguminous crops such as peas, broad beans, kidney beans, and cowpeas, it achieved similar effects as the above-mentioned application on peanuts and soybeans in promoting the increase of the total biomass of leguminous crop plants.

[0184] Example 26: Application of a microbial agent for quality improvement and nitrogen fixation: Reducing the abundance of harmful organisms such as Aspergillus terreus and Fusarium in the rhizosphere of leguminous crops, which is beneficial for improving the soil microbial population structure

[0185] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing and nitrogen-fixing coupled microbial agents to reduce the abundance of harmful organisms such as Aspergillus terreus and Fusarium in the rhizosphere of leguminous crops is described.

[0186] 1. Application on Peanuts: Inoculants 1, 7, and 25 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 100 to 100 million active bacteria per acre). A control plot was established without any of the above agents, while all other plots were managed conventionally. At harvest, rhizosphere soil samples were collected and, using metagenomic and big data analysis, the abundance of pests such as Aspergillus and Fusarium in the rhizosphere soil of the peanuts compared to the control samples was statistically analyzed. These findings showed that the abundance of Aspergillus and Fusarium in the rhizosphere of peanuts treated with agents 1, 7, and 25 decreased by between 45% and 70%. These results demonstrate that the application of microbial agents for quality improvement and nitrogen fixation, coupled with other beneficial effects, significantly reduces the abundance of pests such as Aspergillus and Fusarium in the rhizosphere of leguminous crops.

[0187] 2. Application on Soybeans: Inoculants 1, 7, and 25 were mixed with soybean seeding fertilizer and applied to the field via drone at a cumulative rate of 80 to 100 billion viable bacteria per mu (approximately 100 million saplings). Plots not treated with any of the above agents served as controls, while all other plots were managed conventionally. Rhizosphere soil samples were collected at harvest time. Metagenomic and big data analysis was used to analyze the abundance of pests such as Aspergillus and Fusarium in the rhizosphere soil compared to the control soil samples. These results showed that the abundance of Aspergillus and Fusarium in the rhizosphere of soybeans treated with agents 1, 7, and 25 decreased by between 45% and 70%. These results demonstrate that the application of microbial agents for quality improvement and nitrogen fixation, coupled with other effective methods, significantly reduces the abundance of pests such as Aspergillus and Fusarium in the rhizosphere of legume crops.

[0188] 3. When applied to other legume crops such as peas, broad beans, kidney beans, and cowpeas, it achieved similar effects as the above-mentioned application on peanuts and soybeans, significantly reducing the abundance of pests such as Aspergillus terreus and Fusarium in the rhizosphere of legume crops.

[0189] Example 28: Application of quality improvement and nitrogen fixation coupled with microbial inoculants - reducing surface spots on peanuts and improving marketability

[0190] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to reduce spots on the surface of peanuts is described.

[0191] Application on peanuts: Mix the aforementioned agents 1, 7, and 25 with the peanut seeding base fertilizer and apply them to the field via a seed drill, with a cumulative application rate of 80 to 100 billion viable bacteria per mu. A control plot without any of the agents was established, while all others were managed conventionally. At harvest, surveys were conducted to determine the presence of black spots on the shells of the treated and control peanuts. These survey results showed that the presence of black spots on the shells of peanuts treated with agents 1, 7, and 25 was reduced by over 70%. These results demonstrate that the application of quality-enhancing and nitrogen-fixing microbial agents, coupled with other microbial agents, significantly reduces surface spots on peanuts, enhancing market value and competitiveness, and increasing profitability.

[0192] Example 29: Use of quality-enhancing and nitrogen-fixing coupled microbial agents to promote crop resistance to repeated cropping

[0193] Continuous cropping of crops for many years often leads to the accumulation of crop pathogens, resulting in weak seedlings, diseased seedlings, and missing seedlings. Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality-enhancing nitrogen-fixing coupled microbial agents to promote crop resistance to continuous cropping is described.

[0194] 1. Application in Peanuts: A trial demonstrating continuous cropping resistance was conducted in fields in Xingcheng, Liaoning Province, where peanuts had been planted continuously for many years. Agents 1, 7, and 25 were mixed with peanut seeding base fertilizer and applied via seed drill to the continuously cropped fields 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 using conventional field management. Regular and ongoing surveys were conducted after peanut sowing. These surveys showed that peanut seedlings treated with agents 1, 7, and 25 were more robust and had a significantly lower number of diseased plants compared to the control group. The disease rate in the control group was over five times that of the treated group. These results demonstrate that the application of microbial agents, coupled with nitrogen fixation and quality improvement, significantly enhances crop resistance to continuous cropping.

[0195] 2. Application in Soybeans: A trial demonstrating continuous cropping resistance was conducted in fields planted with soybeans for many years. Agents 1, 7, and 25 were mixed with soybean seeding base fertilizer and applied via drone 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 using conventional methods. Regular surveys were conducted after soybean sowing. These surveys showed that soybean seedlings treated with agents 1, 7, and 25 exhibited stronger seedlings and a significantly lower number of diseased plants compared to the control group. The disease rate in the control group was over five times that of the treated group. These results demonstrate that the application of microbial agents, combined with nitrogen fixation and quality improvement, significantly enhances crop resistance to continuous cropping.

[0196] 3. Its application on other crops such as peas, broad beans, kidney beans, cowpeas, potatoes, etc. has achieved a similar significant effect in promoting the resistance of crops to repeated cropping as its application on peanuts and soybeans.

[0197] Example 30: Application of Quality Improvement and Nitrogen Fixation Coupled with Microbial Agents to Promote Soybean Yield in Saline-Alkali Land

[0198] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of quality improvement and nitrogen fixation coupled with microbial agents to promote soybean production in saline-alkali soil is described.

[0199] The pilot demonstration was conducted in saline-alkali soil with a pH range of 8.2 to 9.2. Before soybean sowing, microbial agents 1, 7, and 25 were applied by drone to the fields, with a cumulative application 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 conventionally. Yield surveys were conducted after the soybean harvest. These surveys showed that soybean yields increased by over 10% per unit area in the areas treated with agents 1, 7, and 25. These results demonstrate that the application of microbial agents, combined with quality improvement and nitrogen fixation, significantly boosts soybean yields in saline-alkali soil.

[0200] Example 31: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial Compound Fertilizer

[0201] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents in the preparation of the quality-improving and nitrogen-fixing coupled microbial compound fertilizer is described.

[0202] Based on the amount of compound fertilizer used per mu of land and a quantity of the quality-enhancing and nitrogen-fixing coupled microbial agent containing no less than 100 billion viable bacteria, the compound fertilizer is mixed with quality-enhancing and nitrogen-fixing coupled microbial agents 1, 7, and 25, respectively. The quality-enhancing and nitrogen-fixing coupled microorganisms are then adsorbed and fixed to the compound fertilizer using conventional physical methods to create three types of quality-enhancing and nitrogen-fixing coupled microbial compound fertilizers, 1, 7, and 25. These compound fertilizers are inorganic compound fertilizers that can be purchased commercially or prepared using inorganic fertilizers such as nitrogen fertilizer, phosphate fertilizer, and potassium fertilizer according to common mixing ratios.

[0203] The three quality-enhancing, nitrogen-fixing coupled microbial compound fertilizers, 1, 7, and 25, were used as seed fertilizers in the production of leguminous crops such as peanuts and soybeans at the time of sowing. Fields treated with the same compound fertilizers served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial compound fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, increasing nodule number by 3.5 to 13 times, increasing nitrogenase activity per plant by more than 10 times, and increasing yield per unit area by more than 6.5%, achieving significant yield increases and demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial agents described in the above examples.

[0204] The above results indicate that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial compound fertilizer, and it has the effects of both compound fertilizer and quality-improving and nitrogen-fixing coupled microbial agent.

[0205] Example 32: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial-Organic Fertilizer

[0206] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents in the preparation of the quality-improving and nitrogen-fixing coupled microbial-bioorganic fertilizer is described.

[0207] According to the amount of bio-organic fertilizer used per mu of land and the amount of the quality-improving and nitrogen-fixing coupled microbial agent with a count of no less than 80 billion viable bacteria, the bio-organic fertilizer is mixed with the quality-improving and nitrogen-fixing coupled microbial agents 1, 7, and 25 respectively. Then, the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed to the bio-organic fertilizer using conventional physical methods to prepare three types of quality-improving and nitrogen-fixing coupled microorganisms-bio-organic fertilizers 1, 7, and 25. The above-mentioned bio-organic fertilizers can be purchased from the market.

[0208] The three quality-enhancing, nitrogen-fixing coupled microbial-bioorganic fertilizers, 1, 7, and 25, were used in the production of leguminous crops such as peanuts and soybeans as seed fertilizers at the time of sowing. Fields treated with the same bio-organic fertilizers served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial-bioorganic fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, with the number of nodules increasing by more than 2 times, the nitrogenase activity per plant increasing by more than 5 times, and the yield per unit area increasing by 6% to 40%, achieving a significant yield increase, demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial agents in the above examples.

[0209] The above results show that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial-bioorganic fertilizer, and has the effects of both bio-organic fertilizer and quality-improving and nitrogen-fixing coupled microbial agent.

[0210] Example 33: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial-Trace Element Fertilizers

[0211] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents in the preparation of quality-improving and nitrogen-fixing coupled microbial-trace element fertilizers is described.

[0212] According to the micronutrient fertilizer dosage per mu and the requirement for a quality-enhancing and nitrogen-fixing coupled microbial agent with a count of no less than 80 billion viable bacteria, mix the trace element fertilizer with quality-enhancing and nitrogen-fixing coupled microbial agents 1, 7, and 25, respectively. Mix the mixture using conventional physical methods to create three types of quality-enhancing and nitrogen-fixing coupled microbial-trace element fertilizers: 1, 7, and 25. These trace element fertilizers can be purchased commercially.

[0213] The three quality-enhancing, nitrogen-fixing coupled microbial-trace element fertilizers, 1, 7, and 25, were used as seed fertilizers in the production of leguminous crops such as peanuts and soybeans at the time of sowing. Fields treated with the same bio-organic fertilizers served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial-trace element fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, increasing nodule number by more than 2-fold, increasing nitrogenase activity per plant by more than 5-fold, and increasing yield per unit area by more than 6.5%, achieving significant yield increases and demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial inoculants described in the above examples.

[0214] The above results show that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial-trace element fertilizer, and it has the effects of both trace element fertilizer and quality-improving and nitrogen-fixing coupled microbial agent.

[0215] Example 34: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial Moisturizing Fertilizer

[0216] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents in the preparation of the quality-improving and nitrogen-fixing coupled microbial-water-retaining fertilizer is described.

[0217] Based on the amount of water-retaining agent used per mu of land and the amount of the quality-improving and nitrogen-fixing coupled microbial agent with a count of no less than 80 billion viable bacteria, the water-retaining agent was mixed with the quality-improving and nitrogen-fixing coupled microbial agents 1, 7, and 25, respectively. The water-retaining agent was then mixed using conventional physical methods, or the quality-improving and nitrogen-fixing coupled microbes were adsorbed and fixed onto the water-retaining agent to form three types of quality-improving and nitrogen-fixing coupled microbial fertilizers: 1, 7, and 25. The water-retaining agents can be purchased commercially.

[0218] The three quality-enhancing, nitrogen-fixing coupled microbial-water-retaining fertilizers, 1, 7, and 25, were used as seed fertilizers in leguminous crops such as peanuts and soybeans at sowing. Fields treated with the same water-retaining agent served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial-water-retaining fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, increasing nodule number by more than 2.5 times, increasing nitrogenase activity per plant by 6 times, and increasing yield per unit area by more than 7.5%, achieving significant yield increases and demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial agents described in the above examples.

[0219] The above results show that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial-water-retaining fertilizer, and has the effects of both water-retaining agent and quality-improving and nitrogen-fixing coupled microbial agent.

[0220] Example 35: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial Inorganic-Organic Compound Fertilizer

[0221] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents in the preparation of the quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer is described.

[0222] Based on the dosage of inorganic-organic compound fertilizer and a dosage of no less than 80 billion viable bacteria of the quality-enhancing and nitrogen-fixing coupled microbial agent per mu of land, the inorganic-organic compound fertilizer is mixed with quality-enhancing and nitrogen-fixing coupled microbial agents 1, 7, and 25, respectively. The quality-enhancing and nitrogen-fixing coupled microorganisms are then adsorbed and fixed to the inorganic-organic compound fertilizer using conventional physical methods to prepare three types of quality-enhancing and nitrogen-fixing coupled microbial inorganic-organic compound fertilizers, 1, 7, and 25. The above-mentioned inorganic-organic compound fertilizers can be purchased commercially or prepared by purchasing the inorganic compound fertilizer and bio-organic fertilizer separately and then blending them according to conventional methods.

[0223] The three quality-enhancing, nitrogen-fixing coupled microbial inorganic-organic compound fertilizers, 1, 7, and 25, were used as seed fertilizers in the production of leguminous crops such as peanuts and soybeans at the time of sowing. Fields treated with the same inorganic-organic compound fertilizers served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial inorganic-organic compound fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, increasing the number of nodules by more than 2.3 times, increasing the nitrogenase activity per plant by more than 5 times, and increasing the yield per unit area by more than 8%, achieving a significant yield increase, demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial inoculants described in the above examples.

[0224] The above results show that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, and it has the effects of both inorganic-organic compound fertilizer and quality-improving and nitrogen-fixing coupled microbial agent.

[0225] Example 36: Use of Quality-Improving and Nitrogen-Fixing Coupled Microbial Agents - Preparation of Quality-Improving and Nitrogen-Fixing Coupled Microbial-Rhizobium Fertilizer

[0226] Taking the application of the above-mentioned microbial agents 1, 7, and 25 as an example, the use of the quality-improving and nitrogen-fixing coupled microbial agents for the preparation of quality-improving and nitrogen-fixing coupled microbial-rhizobium fertilizers is described.

[0227] According to the dosage of rhizobium fertilizer and the dosage of the quality-enhancing and nitrogen-fixing coupled microbial agent with a viable count of no less than 80 billion per mu, the rhizobium fertilizer is mixed with the quality-enhancing and nitrogen-fixing coupled microbial agents 1, 7, and 25, respectively, and then mixed by conventional physical mixing to prepare three types of quality-enhancing and nitrogen-fixing coupled microbial-rhizobium fertilizers 1, 7, and 25. The above-mentioned rhizobium fertilizers, also known as rhizobium agents, can be purchased from the market or obtained by isolating and amplifying fresh leguminous nodules.

[0228] The three quality-enhancing, nitrogen-fixing coupled microbial-rhizobium fertilizers, 1, 7, and 25, were used as seed fertilizers in the production of leguminous crops such as peanuts and soybeans at the time of sowing. Fields treated with the same rhizobium fertilizers served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the three quality-enhancing, nitrogen-fixing coupled microbial inorganic-organic compound fertilizers, 1, 7, and 25, significantly promoted nodulation and nitrogen fixation, increasing the number of nodules by more than 2-fold, increasing the nitrogenase activity per plant by more than 3-fold, and increasing the yield per unit area by more than 6%, achieving a significant yield increase, demonstrating the key characteristics of the quality-enhancing, nitrogen-fixing coupled microbial agents described in the above examples.

[0229] The above results show that the quality-improving and nitrogen-fixing coupled microbial agent can be used to prepare the quality-improving and nitrogen-fixing coupled microbial-rhizobium fertilizer, and it has the effects of both rhizobium fertilizer and quality-improving and nitrogen-fixing coupled microbial agent.

Claims

1. Quality improvement and nitrogen fixation coupled with microbial inoculant, characterized by: It is a microbial composition that has the coupled effects of improving quality and fixing nitrogen, thereby improving the quality of legume crop products. It has the functions of regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops and increasing the number of nodules in legume crops. It contains all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or four or more of them.

2. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The quality-improving and nitrogen-fixing coupled microbial agent is a composition of more than three kinds of microorganisms, containing 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more gene sequences of the nucleotide sequences shown in SEQ ID NOs. 1 to 12.

3. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1 or 2, characterized in that: The microbial sources include but are not limited to Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Brevibacillus laterosporus, Bacillus mucilaginosus, Bacillus velezensis, Bacillus siamensis, Paenibacillus polymyxa, Paenibacillus timonensis, Pseudomonas fluorescens, Pseudomonas mendocina, Enterobacter ludwigii, Microbacterium proteolyticum, Leclercia adcarboxglata, Serratia marcescens. marcescens, Empedobacters sp., Priestia priestiamegaterium, Stenotrophomonas maltophilia bacteria.

4. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The gene sequences shown in SEQ ID NOs. 1 to 12 may vary to a certain extent in different strains. When the degree of variation is small, not exceeding 10% base variation, and the corresponding biological activity functions are present, they constitute functional equivalents of the DNA sequences shown in SEQ ID NOs. 1 to 12. The microbial composition contains all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, or 4 or more of the gene sequences, or functional equivalents of these gene sequences, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agent, thereby improving the quality of legume crop products, regulating and increasing the abundance of rhizobia in the rhizosphere of legume crops, and increasing the number of nodules in legume crops. When this occurs, it constitutes the quality-improving and nitrogen-fixing coupled microbial agent according to claim 1.

5. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The green, yield-increasing, and nitrogen-fixing microbial agent can improve the quality of leguminous crop products by increasing the content of one or more nutritional functional components such as protein, glutamic acid, peanut resveratrol, and soy isoflavones.

6. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The quality-enhancing and nitrogen-fixing coupled microbial agent has an alleviating effect on one or more soil-borne diseases such as peanut fruit rot, soybean green disease, white rot of leguminous crops, bacterial wilt, root rot, and sclerotinia rot; And / or reduce the aflatoxin level in crop products and improve the quality and safety level of leguminous crop products. and / or reduce peanut fruit surface spots.

7. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The quality-improving and nitrogen-fixing coupled microbial agent is a composition of three or more of the following microorganisms: Bacillus subtilis with a deposit number of CCTCC NO: M 20231597, Bacillus velez with a deposit number of CCTCC NO: M 20231600, Bacillus laterosporus with a deposit number of CCTCC NO: M 20231808, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231810, Enterobacter ludwigii with a deposit number of CCTCC NO: M 20231812, and Microbacterium tumefaciens with a deposit number of CCTCC NO: M 20231814.

8. The microbial agent for improving quality and fixing nitrogen according to claim 1, characterized in that: The quality-improving and nitrogen-fixing coupled microbial agent is a combination of one or more of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812, and the following three strains: Bacillus subtilis of CCTCC NO: M 20231597, Bacillus velez of CCTCC NO: M 20231600, and Microbacterium of CCTCC NO: M 20231814.

9. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1, characterized in that: The quality-improving and nitrogen-fixing coupled microbial agent is a combination of Bacillus laterosporus of CCTCC NO: M 20231808, Bacillus amyloliquefaciens of CCTCC NO: M 20231810, Enterobacter Ludwigii of CCTCC NO: M 20231812, and other bacteria, 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 12 or 4 or more gene sequences therein or their functional equivalents, and has the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agents, and regulates and increases the abundance of rhizobia in the rhizosphere of leguminous crops and increases the number of nodules of leguminous crops, thereby constituting the quality-improving and nitrogen-fixing coupled microbial agent of claim 1.

10. The microbial agent for improving quality and fixing nitrogen according to claim 1, characterized in that: The proportion of viable bacteria count of any one strain in the microbial agent is greater than or equal to 1%.

11. The use of the quality-enhancing and nitrogen-fixing coupled microbial agent according to claim 1 in the production of leguminous crops as described in any of the following: for improving the quality and safety level of leguminous crop products; for promoting nodulation and nitrogen fixation of leguminous crops; for improving the yield level of leguminous crops; for recruiting indigenous rhizobia and increasing the abundance of rhizobia in the rhizosphere soil of leguminous crops; for promoting early nodulation of leguminous crops and prolonging the nodulation and nitrogen fixation time; for preventing leguminous crops from premature aging due to lack of fertilizer during maturity; for increasing the number of leguminous crop pods; for increasing the plumpness of leguminous crop pods and reducing the pod shrinkage rate; for promoting early flowering and early pod setting of leguminous crops; for reducing the occurrence of peanut fruit rot; for reducing the incidence of peanut fruit rot It is used to reduce the occurrence of bacterial wilt in leguminous crops; to reduce the occurrence of powdery mildew in leguminous crops; to reduce the occurrence of leaf spot in leguminous crops; to reduce the occurrence of root nematode disease in leguminous crops; to reduce the occurrence of root rot in leguminous crops; to reduce the occurrence of root nematode disease in leguminous crops; to reduce the occurrence of sclerotinia disease in leguminous crops; to reduce the occurrence of downy mildew in leguminous crops; to reduce the occurrence of wilt in leguminous crops; to reduce the occurrence of white rot in leguminous crops; to reduce the incidence of soybean green disease; to promote carbon emission reduction in leguminous crops, which is beneficial to soil improvement; to promote the increase of total biomass of leguminous crops; to reduce the surface spots of peanut fruits and increase marketability; to promote the increase of soybean yield in saline-alkali land.

12. The quality-improving and nitrogen-fixing coupled microbial agent according to claim 1 is used to prepare a quality-improving and nitrogen-fixing coupled microbial compound fertilizer, a quality-improving and nitrogen-fixing coupled microbial organic fertilizer, a quality-improving and nitrogen-fixing coupled microbial micro-fertilizer, a quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer, a quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, a quality-improving and nitrogen-fixing coupled microbial-rhizobium fertilizer, or a quality-improving and nitrogen-fixing coupled microbial-seed dressing agent / seed dressing agent / seed soaking agent, and is used in production to simultaneously exert the effects of the quality-improving and nitrogen-fixing coupled microbial agent.

13. A quality-improving nitrogen-fixing coupled microbial compound fertilizer, a quality-improving nitrogen-fixing coupled microbial organic fertilizer, a quality-improving nitrogen-fixing coupled microbial micro-fertilizer, a quality-improving nitrogen-fixing coupled microbial moisturizing fertilizer, a quality-improving nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, a quality-improving nitrogen-fixing coupled microbial-rhizobium fertilizer, or a quality-improving nitrogen-fixing coupled microbial-seed dressing agent / seed dressing agent / seed soaking agent obtained by using the quality-improving nitrogen-fixing coupled microbial agent according to claim 1.

14. A method for preparing a quality-improving nitrogen-fixing coupled microbial compound fertilizer, a quality-improving nitrogen-fixing coupled microbial organic fertilizer, a quality-improving nitrogen-fixing coupled microbial micro-fertilizer, a quality-improving nitrogen-fixing coupled microbial moisturizing fertilizer, a quality-improving nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, or a quality-improving nitrogen-fixing coupled microbial-rhizobium fertilizer using the quality-improving nitrogen-fixing coupled microbial agent according to claim 1: Method for preparing quality-improving and nitrogen-fixing coupled microbial compound fertilizer: according to the conventional dosage of compound fertilizer and the dosage of quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria per mu of land, compound fertilizer and quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on compound fertilizer particles by physical methods to prepare quality-improving and nitrogen-fixing coupled microbial compound fertilizer; Method for preparing quality-improving and nitrogen-fixing coupled microbial organic fertilizer: according to the conventional amount of organic fertilizer per mu of land and the amount of quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria, the organic fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the organic fertilizer particles by physical methods to prepare a quality-improving and nitrogen-fixing coupled microbial compound fertilizer; Method for preparing quality-improving and nitrogen-fixing coupled microbial fertilizer: according to the conventional amount of trace element fertilizer per mu of land and the amount of quality-improving and nitrogen-fixing coupled microbial agent with a viable count of not less than 80 billion, the trace element fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are proportioned, and then mixed by physical methods, and the quality-improving and nitrogen-fixing coupled microbial agent is adsorbed and fixed on the trace element fertilizer particles to prepare the quality-improving and nitrogen-fixing coupled microbial fertilizer; Method for preparing the quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer: according to the conventional dosage of the water-retaining agent and the dosage of the quality-improving and nitrogen-fixing coupled microbial agent with a viable count of not less than 80 billion per mu of land, the water-retaining agent and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in proportion, and then mixed by conventional physical methods / or the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the water-retaining agent to prepare the quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer; A method for preparing a quality-improving and nitrogen-fixing coupled microbial rhizobium fertilizer: according to the conventional amount of rhizobium fertilizer and the amount of the quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria per mu of land, the rhizobium fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed in a conventional physical method or the two agents are mixed and then fixed on carrier particles to prepare the quality-improving and nitrogen-fixing coupled microbial rhizobium fertilizer; A method for preparing a quality-improving and nitrogen-fixing coupled microbial organic-inorganic compound fertilizer: according to the conventional dosage of compound fertilizer per mu of land and the dosage of the quality-improving and nitrogen-fixing coupled microbial agent with a count of not less than 80 billion viable bacteria, the organic-inorganic compound fertilizer and the quality-improving and nitrogen-fixing coupled microbial agent are mixed, and then the quality-improving and nitrogen-fixing coupled microorganisms are adsorbed and fixed on the organic and inorganic particles by physical methods to prepare the quality-improving and nitrogen-fixing coupled microbial organic-inorganic compound fertilizer; The method for preparing the quality-improving and nitrogen-fixing coupled microorganisms-seed dressing agent / seed mixing agent / seed soaking agent: according to the amount of seed dressing agent or seed mixing agent or seed soaking agent per mu of land and the amount of quality-improving and nitrogen-fixing coupled microorganisms not less than 80 billion viable bacteria, the seed dressing agent or seed mixing agent or seed soaking agent and the ARC microbial agent are proportioned, and then the quality-improving and nitrogen-fixing coupled microorganisms and the seed dressing agent or seed mixing agent or seed soaking agent are evenly mixed through conventional physical blending to prepare the quality-improving and nitrogen-fixing coupled microorganisms-seed dressing agent / seed mixing agent / seed soaking agent.

15. The use of the quality-improving and nitrogen-fixing coupled microbial compound fertilizer, quality-improving and nitrogen-fixing coupled microbial organic fertilizer, quality-improving and nitrogen-fixing coupled microbial micro-fertilizer, quality-improving and nitrogen-fixing coupled microbial moisturizing fertilizer, quality-improving and nitrogen-fixing coupled microbial inorganic-organic compound fertilizer, or quality-improving and nitrogen-fixing coupled microbial-rhizobium fertilizer configured according to claim 14 in crop production as described in any of the following: for improving the quality and safety level of leguminous crop products; for promoting nodulation and nitrogen fixation in leguminous crops; for improving the yield per unit area of leguminous crops; for recruiting indigenous rhizobia and increasing the abundance of rhizobia in the rhizosphere soil of leguminous crops; for promoting early nodulation of leguminous crops and prolonging the nodulation and nitrogen fixation time; for preventing premature aging of leguminous crops due to defertilization during maturity; for increasing the number of leguminous crop pods; for increasing the plumpness of leguminous crop pods and reducing the shrunken pod rate ; Used to promote early flowering and early pod setting of leguminous crops; used to reduce the occurrence of peanut fruit rot; used to reduce the occurrence of bacterial wilt of leguminous crops; used to reduce the occurrence of powdery mildew of leguminous crops; used to reduce the occurrence of leaf spot of leguminous crops; used to reduce the occurrence of root nematode disease of leguminous crops; used to reduce the occurrence of root rot of leguminous crops; used to reduce the occurrence of root nematode disease of leguminous crops; used to reduce the occurrence of sclerotinia disease of leguminous crops; used to reduce the occurrence of downy mildew of leguminous crops; used to reduce the occurrence of wilt of leguminous crops; used to reduce the occurrence of white rot of leguminous crops; used to reduce the incidence of soybean green disease; used to promote carbon emission reduction of leguminous crops, which is beneficial to soil improvement; used to promote the increase of total biomass of leguminous crops; used to reduce the surface spots of peanut fruits and increase commercial value; used to promote the increase of soybean yield in saline-alkali land.

16. A method for leguminous crop production, characterized in that: The microbial agent is selected so that, upon analysis and determination, the microbial agent contains all or four or more gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12, and has a coupled effect of improving nitrogen quality and fixing nitrogen, 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; the microbial agent is applied to crops to improve nitrogen quality and fix nitrogen, and to increase yield in a green way.

17. The method according to claim 16, characterized in that: The gene sequences shown in SEQ ID NOs. 1 to 12 may have no more than 10% base variation in different strains. When the identity between them and the corresponding DNA sequences is more than 90% and they have corresponding biological activity functions, they constitute functional equivalents of the DNA sequences shown in SEQ ID NOs. 1 to 12. When they contain all the gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 12 or 4 or more gene sequences therein or functional equivalents of these gene sequences, and have the quality-improving and nitrogen-fixing coupled effects of the above-mentioned microbial agent, and regulate and increase the abundance of rhizobia in the rhizosphere of leguminous crops and increase the number of nodules of leguminous crops, they constitute the quality-improving and nitrogen-fixing coupled microbial agent of claim 1, and are applied to crops for improving quality, fixing nitrogen and increasing green yields.