Use of arc microbial agent in promoting nodulation and nitrogen fixation of leguminous vegetable or leguminous forage crop and in increasing yield per unit area

Abstract

The present invention belongs to the field of microorganisms. Disclosed is the use of an ARC microbial agent in promoting nodulation and nitrogen fixation of a leguminous vegetable or a leguminous forage crop and in improving yield per unit area. The use of an ARC microbial agent in promoting nodulation and nitrogen fixation of a leguminous vegetable or a leguminous forage crop and in improving yield per unit area. The ARC microbial agent is a microbial composition, has a coupled effect of toxicity control and nitrogen fixation, and has the effects of regulating and improving the abundance of rhizobia in the rhizosphere of a leguminous crop and increasing the number of nodules in the leguminous crop. The ARC microbial agent contains DNA sequences as shown in SEQ ID NOs: 1-4. The ARC microbial agent can promote the nodulation and nitrogen fixation of a leguminous vegetable or a leguminous forage crop and increase the yield per unit area. In addition, the ARC microbial agent is simple to use, has significant social and ecological benefits, and is easy to popularize and apply.

Description

Use of ARC microbial agent for promoting nodulation and nitrogen fixation of leguminous vegetables or leguminous forage crops and increasing yield Technical Field

[0001] The present invention belongs to the field of microorganisms, and in particular relates to the use of an ARC microbial agent for promoting nodulation and nitrogen fixation of leguminous vegetables or leguminous forage crops and increasing yield. Background Art

[0002] Leguminous vegetables, including soybeans, peas, broad beans, cowpeas, green beans, sword beans, and black-eyed peas, play a crucial role in ensuring the supply of my country's vegetable basket. Therefore, increasing the yield per unit area of ​​leguminous vegetable production is of great significance.

[0003] Forage is the material foundation for the development of animal husbandry. American scientists have concluded that high-quality forage should account for at least 70% of a dairy cow's daily diet. Artificial forage cultivation, particularly using leguminous grasses such as alfalfa, Astragalus, Sainfoin, clover, and Chinese milk vetch to establish artificial pastures or improve natural pastures, can increase forage production and is of great significance to the development of animal husbandry.

[0004] Although leguminous vegetables and forages form symbiotic nodules with soil rhizobia, they naturally form few nodules, resulting in short nitrogen fixation times and low efficiency. Research on rhizobium-based nitrogen fixation has a history of over 100 years, establishing the classically recognized legume AON theory, which states 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 specialized rhizobia suitable for specific production environments. This approach is geographically limited and has long been 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 potential violation of the AON theory) is difficult. Improving the efficiency of nodulation and nitrogen fixation in leguminous crops is a hotly debated and challenging issue internationally.

[0005] To address these challenges, the inventors' team, after over 20 years of continuous research, successfully developed the ARC microbial agent in the past five years. This agent effectively controls aflatoxin at the source while simultaneously promoting efficient nodulation and nitrogen fixation in legume crops and significantly increasing yields. This microbial agent, known as ARC (Aspergillus flavus / Aflatoxins and Rhizobia Coupling), combines the effects of controlling aflatoxin contamination and promoting nodulation and nitrogen fixation. This agent is simple to use, low-cost, and highly effective. It boasts significant advantages: two fixations (nitrogen and carbon fixation), three increases (yield, efficiency, and safety), and five reductions (reducing toxicity, damage, weight, costs, and carbon emissions). This agent has demonstrated significant application potential and has been demonstrated and validated in field trials across multiple major legume-producing regions across China. It holds significant promise for increasing legume crop yields and productivity, as well as promoting green, low-carbon, and efficient production. On this basis, the research team further discovered the effect of ARC microbial agent on promoting nodulation and nitrogen fixation and improving yield levels of leguminous vegetables such as soybeans, peas, broad beans, cowpeas, green beans, sword beans, and mung beans, providing a new solution for increasing the yield of leguminous vegetables. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides an ARC microbial agent for promoting nodulation and nitrogen fixation in leguminous vegetables or leguminous forage crops and increasing yield per unit area. The ARC microbial agent can promote nodulation and nitrogen fixation in leguminous vegetables and increase yield per unit area, is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0025] The above-mentioned ARC microbial agent can be used in the production of leguminous crops as follows: to improve the quality and safety level of leguminous crop products; to promote nodulation and nitrogen fixation of leguminous crops; to increase the yield level of leguminous crops; to recruit indigenous rhizobia and increase the abundance of rhizobia in the rhizosphere soil of leguminous crops; to promote early nodulation of leguminous crops and prolong the nodulation and nitrogen fixation time; to prevent leguminous crops from premature aging due to lack of fertilizer during maturity; to increase the number of leguminous crop pods; to increase the fullness of leguminous crop pods and reduce the rate of shrunken pods; to promote early flowering and early pod formation of leguminous crops; to reduce the occurrence of peanut fruit rot; to reduce the occurrence of bacterial wilt of leguminous crops; to reduce the occurrence of powdery mildew of leguminous crops; to reduce the occurrence of leaf spot of leguminous crops; to reduce the occurrence of root nematode disease of leguminous crops; to reduce Used to reduce the occurrence of root rot in leguminous crops; used to reduce the occurrence of root nematode disease in leguminous crops; used to reduce the occurrence of blight in leguminous crops; used to reduce the occurrence of sclerotinia rot in leguminous crops; used to reduce the occurrence of downy mildew in leguminous crops; used to reduce the occurrence of wilt in leguminous crops; used to reduce the occurrence of white rot in leguminous crops; used to reduce the incidence of soybean green spondylosis; used to reduce the incidence of corn ear rot and Fusarium toxin; used to reduce the abundance of wheat fusarium and reduce the incidence of fusarium toxin; used to promote carbon emission reduction in leguminous crops, which is beneficial to soil improvement; used to promote the increase of total biomass of leguminous crops; used to reduce the abundance of pests such as Aspergillus terreus and Fusarium in the rhizosphere of leguminous crops, which is beneficial to improving the soil microbial population structure; used to reduce the surface spots of peanuts and increase commercial value; used to promote soybean production in saline-alkali land.

[0026] The ARC microbial agent can be prepared by the following method: the microorganisms in the above microbial agent are combined and fermented. The fermentation route can adopt conventional fermentation routes of bacteria disclosed in the prior art, including existing literature.

[0027] Based on the above research, the present invention further studies and provides the use of ARC microbial agent for promoting nodulation and nitrogen fixation of leguminous vegetables and increasing yield.

[0028] The present invention further provides a method for promoting nodulation and nitrogen fixation of leguminous vegetables and increasing yield, which comprises applying ARC microbial agent to leguminous vegetable production.

[0029] According to the above scheme, the application amount of the ARC microbial agent is no less than 80 billion viable bacteria per mu, for example, the application amount is 80 billion to 100 billion viable bacteria, or more than 80 billion to 100 billion viable bacteria.

[0030] According to the above scheme, the application method is one or more of broadcasting, spraying, and drip irrigation, and the application stage is at the time of sowing of leguminous vegetables and / or from emergence to flowering. The specific application method of the above-mentioned ARC microbial agent in leguminous vegetable production can be as follows: the above-mentioned ARC microbial agent is mixed with the base fertilizer for sowing leguminous vegetables, and evenly applied to the field by one or more of the following methods: manual, seed drill, or drone, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be used in fields where conditions permit, to minimize severe field dryness and uneven emergence. Conventional field management is used for other purposes.

[0031] The specific application method of the ARC microbial agent in leguminous vegetable production can also be as follows: after the leguminous vegetables have been sown and seeded normally, until the flowering stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation, or other topdressing methods, or a combination thereof, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is followed for all other aspects.

[0032] According to the above scheme, the leguminous vegetables include but are not limited to edible soybeans, peas, broad beans, cowpeas, green beans, sword beans, black-eyed peas, etc.

[0033] Based on the above research, the present invention further studies and provides the use of ARC microbial agent to promote nodulation and nitrogen fixation of leguminous forage crops and increase yield.

[0034] A method for promoting nodulation and nitrogen fixation of leguminous forage and increasing yield, comprising applying an ARC microbial agent to the leguminous forage.

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

[0036] According to the above scheme, the application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is when the leguminous forage grass is sown and / or after emergence and / or after the previous crop is harvested.

[0037] The application method of the above-mentioned ARC microbial agent in the production of leguminous forage can be as follows: the above-mentioned ARC microbial agent is mixed with the sowing base fertilizer of leguminous forage such as alfalfa, and is evenly applied to the field by one or more methods such as manual, seeding machine, drone, drip irrigation pipe, etc., with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be adopted for fields where conditions permit, and severe dryness of the field and uneven emergence of seedlings can be avoided as much as possible. Conventional field management is adopted for the rest.

[0038] The ARC microbial agent can also be used in leguminous forage production as follows: After alfalfa seeding or previous crop harvest, the ARC microbial agent is evenly applied to the alfalfa field by broadcasting, spraying, drip irrigation, or other topdressing methods, or a combination thereof, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is used for all other purposes.

[0039] According to the above scheme, the above-mentioned legume forage includes alfalfa, Astragalus membranaceus, Sainfoin, clover, Chinese milk vetch, etc.

[0040] The beneficial effects of the present invention are:

[0041] 1. The ARC microbial agent can be used to promote nodulation and nitrogen fixation in leguminous vegetables or leguminous forage crops and increase the yield of leguminous vegetables. 2. It is easy to use, low-cost, and highly effective. 3. It is of great significance to promote the increase in leguminous vegetable production capacity and green, low-carbon, and efficient production; it is of great significance to promote the increase in alfalfa production capacity and green, low-carbon, and efficient production. Modes for Carrying Out the Invention

[0042] Part I ARC Microbial Agents

[0043] Example 1 Preparation of ARC microbial agent

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

[0045] Table 1. Some of the strains isolated and identified from a mixture of major legume crops such as peanuts and soybeans are shown below:

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

[0047] The 10 microbial strains in Table 1 were amplified one by one by conventional bacterial culture medium amplification method to prepare batches of fermentation broth or bacterial powder of the 10 strains.

[0048] The above-mentioned Bacillus laterosporus with a deposit number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC NO: M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC NO: M 20231595 are mixed with fermentation broths or bacterial powders of the four strains; and the combination information of the microbial agents formed by mixing some strains of Bacillus laterosporus with a deposit number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a deposit number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a deposit number of CCTCC NO: M 20231817, and Enterobacter Ludwigii with a deposit number of CCTCC NO: M 20231595 and other strains, as well as some other strains are mixed to form a microbial agent. The proportion of the viable count of any one strain in each microbial combination is greater than or equal to 1%.

[0049] Table 2. Microbial composition and bacterial agent information

[0050] Microbial agent number Microbial agent composition and proportion of viable bacteria count of constituent strains (%) Microbial agent number Microbial agent composition and proportion of viable bacteria count of constituent strains (%) Composition of agent 1: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 Ratio: 33 / 33 / 1 / 33 Composition of agent 7: Strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 33 / 1 / 33 / 33 Composition of agent 2: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 ratio: 1 / 33 / 33 / 33 Bacterial agent 8 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC M 20231595 ratio: 1 / 33 / 33 / 33 Bacterial agent 3 composition: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 ratio: 33 / 33 / 33 / 1 Bacterial agent 9 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 ratio: 33 / 33 / 33 / 1 Bacterial agent 4 composition: CCTCC NO: M 20231815 / CCTCC NO: M 20231598 / CCTCC NO: M 20231817 / CCTCC NO: M 20231595 Ratio: 33 / 1 / 33 / 33 Bacterial agent 10 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 Ratio: 33 / 1 / 33 / 33 Bacterial agent 5 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 30 / 10 / 30 / 30 Bacterial agent 11 composition: strain 202308 / strain 202311 / strain 202312 / strain 202326 Ratio: 33 / 33 / 1 / 33 Bacterial agent 6 composition: strain 202330 / CCTCC NO: M 20231598 / strain 202334 / CCTCC NO: M 20231595 Ratio: 10 / 30 / 30 / 30 Inoculum 12 Composition: strain 202308 / strain 202311 / Strain 202312 / Strain 202326 Ratio: 1 / 33 / 33 / 33

[0051] Example 2: Sequencing of ARC microbial agents

[0052] A sufficient number of samples were taken from the microbial agents in Table 2 of Example 1, and total DNA was extracted from these samples in sequence using a conventional DNA extraction method. The DNA sequences of these samples were then determined using a conventional DNA sequencing method. Finally, conventional analysis methods were used to compare the homology of the DNA sequences determined above with the gene sequences shown in SEQ ID No. 1-4 provided in this patent text.

[0053] The results of the above determination and homology analysis showed that the bacterial agents 1 to 4 in Table 2 contained all four genes in the DNA sequences 1 to 4 shown in SEQ ID No. 1 to 4, respectively, and the sequence homology was 100%; the bacterial agents 5 to 12 in Table 1 contained all four genes in the DNA sequences 1 to 4 shown in SEQ ID No. 1 to 4, respectively, but with certain variations, and the four gene sequences had 90.3-100% homology with the DNA sequences 1 to 4.

[0054] Example 3: Determination of the Toxic Control and Nitrogen Fixation Effect of Peanut ARC Microbial Agent

[0055] Taking peanuts as an example, the steps for determining the toxicity control and nitrogen fixation effects of the above-mentioned ARC microbial agent are described as follows.

[0056] On the one hand, the bacterial agents described in Table 2 of Example 1 were co-cultured with a toxin-producing Aspergillus flavus strain under the same conditions. The expression of PAB-01 was measured, and the inhibition rate of PAB-01 expression was calculated. The results are shown in Table 3. The method for determining the inhibition rate of Aspergillus flavus protein expression can be referred to the method in the following literature: Protein biomarker for early diagnosis of microbial toxin contamination: Using Aspergillus flavus as an example, Food Frontiers. 2023, 4, 2013-2023, DOI: 10.1002 / fft2.295.

[0057] On the other hand, microbial agents 1-12 from Table 2 of Example 1 were applied to the field along with the peanut sowing base fertilizer, or during the peanut growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 100 million). A plot without any of the microbial agents was set up as a control, while all other plots were managed using conventional field management. Root nodulation of peanuts in the seedling stage was investigated 7 to 12 days after emergence; root nodulation of peanuts in the mature stage was investigated 1 to 3 days before harvest; and the effectiveness of toxicity control was investigated from the flowering stage to around harvest. Specifically, the effectiveness of field disease control and the abundance of aflatoxin in peanuts after harvest were investigated. The results of the investigation are shown in Table 3.

[0058] Based on the above results, microbial agents 1 to 12 simultaneously possess the following characteristics: 1) The microbial agents contain all four gene sequences in DNA sequences 1 to 4, and these genes may have no more than 10% base variation in different strains; 2) The microbial agents have a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, have an inhibitory effect on Aspergillus flavus, and have an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium and Aspergillus other than Aspergillus flavus, Fusarium, and Pseudomonas solanacearum; 3) Although not a rhizobium itself, the ARC microbial agent can simultaneously regulate and increase the abundance of rhizobia in the peanut rhizosphere and increase the number of peanut root nodules; 4) It can promote early nodulation of peanuts and prolong the time of nodulation and nitrogen fixation.

[0059] The above-mentioned soil-borne plant pathogens are the pathogenic microorganisms of the above-mentioned corresponding field diseases. Therefore, the field disease prevention and control effect is equivalent to the inhibitory effect of ARC microbial agent on the pathogenic factors / toxins of the above-mentioned soil-borne plant pathogens.

[0060] Table 3. Results of the microbial agent test on the effects of peanut poison control and nitrogen fixation

[0061] Microbial agent number Inhibition rate of PAB-01 (%) Whether it has an inhibitory effect on aflatoxin / toxin Whether it has an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Penicillium, Aspergillus other than aflatoxin, Fusarium, Pseudomonas solanacearum, etc. Promotion of nodulation and nitrogen fixation: Percent increase in rhizospheric rhizobium abundance (%) / fold increase in nodule number (fold) Whether it promotes early nodulation of peanut and prolongs the time of nodulation and nitrogen fixation Agent 199 Yes Yes 23 / 16.6 Yes Agent 297 Yes Yes 20 / 13.7 Yes Agent 398 Yes Yes 22 / 15.3 Yes Agent 499 Yes Yes 18 / 6.5 Yes Agent 597 Yes Yes 21 / 14.2 Yes Agent 698 Yes Yes 22 / 15.5 Yes Agent 797 Yes Yes 19 / 8.1 Yes Agent 899 Yes Yes 18 / 7.1 Yes Agent 997 Yes Yes 20 / 10.2 Yes Agent 61098 Yes Yes 26 / 22.0 Yes Agent 1199 Yes Yes 17 / 3.2 Yes Agent 1296 Yes Yes 19 / 7.3 Yes

[0062] The above-mentioned microbial agent has an inhibition rate of more than 60% against aflatoxin / toxin, and further more than 90%; the inhibition rate of one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus, Penicillium, Fusarium, and Pseudomonas solanacearum other than aflatoxin can reach at least 30% or more, preferably more than 60%, and even more preferably 80% or even more than 90%.

[0063] Example 4: Determination of the Toxic Control and Nitrogen Fixation Effect of Soybean Microbial Agents

[0064] Taking soybean as an example, the steps for determining the toxicity control and nitrogen fixation effects of the above-mentioned microbial agents are described as follows.

[0065] On the one hand, the bacterial agent in Table 2 of Example 1 above was co-cultured with the toxigenic Aspergillus flavus strain under the same conditions, the expression level of PAB-01 was determined using the literature method, and the inhibition rate of the bacterial agent on PAB-01 expression was calculated. The results are shown in Table 3.

[0066] On the other hand, microbial agents 1-12 from Table 2 of Example 1 were applied to the field along with soybean sowing base fertilizer, or during the soybean growing season, at a cumulative application rate of 80 billion viable bacteria per mu (approximately 100 million). A control plot was established without any of the microbial agents. Conventional field management was used for all other plots. Root nodulation in soybean seedlings was investigated 7 to 12 days after emergence; root nodulation in peanuts was investigated 1 to 3 days before harvest; and toxicity control efficacy, i.e., effectiveness in preventing and controlling pest toxin-induced diseases, was investigated from the flowering stage to harvest. The results are shown in Table 4.

[0067] Based on the above results, agents 1-12 simultaneously possess the following characteristics: 1) ARC microbial agents contain all four gene sequences in DNA sequences 1-4, and these genes may have no more than 10% base variation in different strains; 2) the agents have a significant inhibitory effect on the expression of Aspergillus flavus PAB-01 protein, have an inhibitory effect on Aspergillus flavus, and have inhibitory effects on soil-borne plant pathogens other than Penicillium and Aspergillus, Fusarium, Sclerotium sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani; 3) although not rhizobia themselves, the agents can simultaneously regulate and increase the abundance of rhizobia in the soybean rhizosphere and the number of soybean root nodules; 4) the agents can promote early soybean nodulation and prolong the time of nodulation and nitrogen fixation.

[0068] The above-mentioned soil-borne plant pathogens are the pathogenic microorganisms of the above-mentioned corresponding field diseases. Therefore, the field disease prevention and control effect is equivalent to the inhibitory effect of ARC microbial agent on the pathogenic factors / toxins of the above-mentioned soil-borne plant pathogens.

[0069] Table 4. Results of microbial agents on the control of toxicity and nitrogen fixation in soybeans

[0070] Microbial agent number Inhibition rate of PAB-01 (%) Whether it has an inhibitory effect on Aspergillus flavus / toxins Whether it has an inhibitory effect on one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus other than Aspergillus flavus, Fusarium, Sclerotinia sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani Percent increase in rhizospheric rhizobium abundance (%) / fold increase in nodule number (fold) Whether it promotes early nodulation of soybean and prolongs the time of nodulation and nitrogen fixation Bacterial agent 199 Yes Yes 22 / 15 Yes Bacterial agent 297 Yes Yes 21 / 12 Yes Bacterial agent 398 Yes Yes 22 / 16 Yes Bacterial agent 499 Yes Yes 19 / 8 Yes Bacterial agent 598 Yes Yes 20 / 13 Yes Bacterial agent 697 Yes Yes 23 / 15 Yes Bacterial agent 799 Yes Yes 18 / 6 Yes Bacterial agent 896 Yes Yes 19 / 7 Yes Bacterial agent 997 Yes Yes 20 / 11 Yes Bacterial agent 1098 Yes Yes 22 / 13 Yes Bacterial agent 1199 Yes Yes 17 / 3 Yes Bacterial agent 1296 Yes Yes 19 / 6 Yes

[0071] The above-mentioned microbial agent has an inhibition rate of more than 60% against aflatoxin / toxin, and further more than 90%; the inhibition rate of one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus, Fusarium, Sclerotinia, Phytophthora, Sclerotinia, Pythium, and Rhizoctonia other than Aspergillus flavus can reach at least 30% or more, preferably more than 60%, and more preferably 80% or even more than 90%.

[0072] Example 5: Determination of the Effect of Microbial Agents on Toxic Control and Nitrogen Fixation in Other Leguminous Crops

[0073] The sampling method and steps similar to those in Examples 3 and 4 were used to determine the toxicity control and nitrogen fixation effects of ARC microbial agents 1 to 12 on other legume crops such as peas, broad beans, cowpeas, and alfalfa, and the results were similar to those in Tables 3 and 4.

[0074] Part 2 Application of ARC Microbial Agents

[0075] (I) Application of ARC microbial agent: It is used to promote nodulation and nitrogen fixation and increase yield of leguminous vegetables such as soybeans, peas, broad beans, cowpeas, green beans, sword beans, and black-eyed peas

[0076] Example 6 Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation in cowpea and improving yield

[0077] ARC microbial agents 1-8 were mixed with cowpea 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 agents served as controls, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased the number of cowpea root nodules by more than three times and increased nitrogenase activity by more than 3.5 times. Fresh cowpea pods were weighed and yields were measured at each harvest. The yield per unit area of ​​fresh cowpea pods was calculated by accumulating the harvested pods. The yield increases for cowpeas treated with ARC agents 1-8 were all above 12.4%, achieving significant yield increases. These results indicate that the application of ARC microbial agents significantly promoted cowpea nodulation and nitrogen fixation and increased cowpea yield per unit area.

[0078] The above-mentioned ARC microbial agents were applied to cowpea production by manual spreading, drone spreading, etc. during cowpea sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing cowpea yield were achieved.

[0079] Applying the above-mentioned ARC microbial agents after cowpea seedlings emerge also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing cowpea yield.

[0080] Example 7 Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation of broad beans and improving yield

[0081] ARC microbial agents 1-8 were mixed with broad bean seeding fertilizer and applied to the field via seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot was established without any of the agents, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 3.3 times and nitrogenase activity by more than 3.6 times. Bean yields were measured by weighing at harvest, and the yield increases for those treated with ARC agents 1-8 were all above 13.5%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promoted broad bean nodulation and nitrogen fixation and increased broad bean yield per unit area.

[0082] The above-mentioned ARC microbial agent was applied to pea production by manual spreading, drone spreading, etc. during broad bean sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing broad bean yield were achieved.

[0083] Applying the above-mentioned ARC microbial agents after the emergence of broad beans also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing broad bean yield.

[0084] Example 8 Use of ARC microbial agent - Use for promoting pea nodulation and nitrogen fixation and improving yield

[0085] ARC microbial agents 1-8 were mixed with pea seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. A control plot was set up without any of the agents, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 3.4 times and nitrogenase activity by more than 4 times. Peas were harvested and weighed for yield measurement. The yield increases for peas treated with ARC agents 1-8 were all above 15%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promoted pea nodulation and nitrogen fixation and increased field pea yields.

[0086] The above-mentioned ARC microbial agent was applied to pea production by manual spreading, drone spreading, etc. during pea sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing pea yield were achieved.

[0087] Applying the ARC microbial agent after pea seedling emergence also achieved similar significant results in promoting nodulation and nitrogen fixation, as well as increasing pea yield. Furthermore, using the same method, applying the ARC microbial agent to other leguminous vegetables, such as soybeans, green beans, sword beans, and black-eyed peas, also achieved similar significant results in promoting nodulation and nitrogen fixation, as well as increasing yield per unit area, as seen with cowpeas, broad beans, and peas.

[0088] (II) Application of ARC microbial agent: used to promote nodulation and nitrogen fixation and increase the yield of leguminous forage grasses

[0089] Example 6: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation and increasing the yield of leguminous forage alfalfa

[0090] ARC microbial agents 1-8 were mixed with alfalfa seeding fertilizer and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. Plots not treated with any of the agents served as controls, while all other plots were managed under conventional field management. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 2.6 times and nitrogenase activity by more than 2.9 times in Astragalus adductus. After each alfalfa harvest, yield was measured by weighing and calculating the annual per-mu yield of all alfalfa harvested within a year. The yield increase for alfalfa treated with ARC agents 1-8 was over 10%, demonstrating significant yield increases. These results demonstrate that the application of ARC agents significantly promotes nodulation and nitrogen fixation and increases alfalfa yield in the field.

[0091] The above-mentioned ARC microbial agents were applied to alfalfa production by manual spreading, drone spreading, etc. during alfalfa sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing alfalfa yield were achieved.

[0092] Applying the above-mentioned ARC microbial agent after alfalfa seedlings emerge or after the previous crop is harvested also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing alfalfa yield.

[0093] Example 7: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation and increasing the yield of leguminous forage grass Astragalus membranaceus

[0094] ARC microbial agents 1-8 were mixed with the seeding base fertilizer for the legume forage grass Astragalus membranaceus and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. Plots without any of the agents were set up as controls, while all other plots were managed under conventional conditions. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 2.7 times and nitrogenase activity by more than 3 times. Yields of Astragalus membranaceus were measured after each harvest. The annual yield per mu of Astragalus membranaceus harvested within a year was calculated. The yield increase for Astragalus membranaceus treated with ARC agents 1-8 was over 13%, achieving significant yield increases. These results demonstrate that the application of ARC microbial agents significantly promotes nodulation and nitrogen fixation and increases the yield of Astragalus membranaceus in the field.

[0095] The above-mentioned ARC microbial agent was applied to the production of the legume forage grass Astragalus membranaceus by manual spreading, drone spreading, drip irrigation, etc. during sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing Astragalus membranaceus yield were achieved.

[0096] Applying the above-mentioned ARC microbial agent after the emergence of the leguminous forage grass Astragalus membranaceus or after the previous crop was harvested also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing Astragalus membranaceus yield.

[0097] Example 8: Use of ARC microbial agent - Use for promoting nodulation and nitrogen fixation and increasing the yield of leguminous forage grass Sainfoin

[0098] ARC microbial agents 1-8 were mixed with a seeding base fertilizer for the legume forage grass, Sainfoin, and applied to the field via a seed drill at a cumulative rate of 80 to 100 billion viable bacteria per mu. Plots without any of the agents were set up as controls, while all other plots were managed under conventional conditions. Ten days after application, root nodulation and nitrogen fixation were investigated. Results showed that ARC agents 1-8 increased root nodules by more than 2.5 times and nitrogenase activity by more than 2.7 times. Yields were measured after each harvest, and the annual yield per mu of Sainfoin was calculated based on the cumulative yield of all Sainfoin harvests within a year. The yield increases for Sainfoin treated with ARC agents 1-8 were all above 11%, demonstrating significant increases. These results demonstrate that the application of ARC microbial agents significantly promoted nodulation and nitrogen fixation and increased the yield of Sainfoin in the field.

[0099] The above-mentioned ARC microbial agent was applied to the production of the legume forage grass sainfoin by manual spreading, drone spreading, drip irrigation, etc. during sowing, and similar significant effects of promoting nodulation and nitrogen fixation and increasing sainfoin yield were achieved.

[0100] Applying the above-mentioned ARC microbial agent after the emergence of the leguminous forage grass Sainfoin or after the previous crop was harvested also achieved similar significant effects in promoting nodulation and nitrogen fixation and increasing the yield of Sainfoin.

[0101] In addition, the application of ARC microbial agents 1 to 8 in the production of other legume forages such as clover and astragalus has also achieved similar effects in promoting nodulation and nitrogen fixation and increasing yield levels as in the production of alfalfa, Astragalus and Sainfoin.

Claims

1. The ARC microbial agent is used to promote nodulation and nitrogen fixation in leguminous vegetables or leguminous forage crops and increase yield. The ARC microbial agent is a microbial composition having a coupled effect of toxicity control and nitrogen fixation, and has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops. It contains the DNA sequences shown in SEQ ID Nos. 1-4.

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

3. The method according to claim 2, wherein: The leguminous vegetables include but are not limited to edible soybeans, peas, broad beans, cowpeas, green beans, sword beans, and black-eyed peas; the application rate of the ARC microbial agent is 80 billion to 100 billion live bacteria per mu.

4. The method according to claim 2, wherein: The application method is broadcasting, spraying, drip irrigation or a combination thereof, and the application stage is when leguminous vegetables are sown and / or from seedling emergence to flowering.

5. The method according to claim 2, wherein: The method is as follows: ARC microbial agent is mixed with base fertilizer for leguminous vegetable sowing, and applied evenly to the field by one or more of the following methods: manual, seeding machine or drone, with a cumulative viable bacterial count of 80 billion to 100 billion per mu. Or: after the leguminous vegetables are sown and seedlings emerge normally until the flowering stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or topdressing, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

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

7. The method according to claim 6, characterized in that: The legume forage grasses include alfalfa, Astragalus membranaceus, Sainfoin, clover and Chinese milk vetch; the application amount of the ARC microbial agent is 80 billion to 100 billion live bacteria per mu.

8. The method according to claim 6, wherein: The application method is broadcasting, spraying, drip irrigation or a combination thereof, and the application stage is when the leguminous forage grass is sown and / or after the seedlings emerge and / or after the previous crop is harvested.

9. The method according to claim 6, wherein: The method is as follows: ARC microbial agent is mixed with alfalfa and other legume forage sowing base fertilizer, and is evenly applied to the field by one or more of the following methods: manual, seeding machine, drone, drip irrigation pipe, etc. The application amount of the microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: after the alfalfa is sown and germinated normally or after the previous crop is harvested, the ARC microbial agent is evenly applied to the alfalfa field by broadcasting, spraying, drip irrigation or a combination of one or more of the following methods, with the application rate of the agent being 80 billion to 100 billion live bacteria per mu.

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

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

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

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

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