Use of arc microbial agent for producing arc+ rhizobium fertilizer (AGENT)

Abstract

The present invention relates to the field of microorganisms. Disclosed is a use of an ARC microbial agent for producing an ARC+ rhizobium fertilizer (agent). The ARC microbial agent is a microbial composition, has a coupled effect of toxicity control and nitrogen fixation and the effects of performing regulation and control to enhance the abundance of rhizobia in the rhizosphere of a leguminous crop and increase the quantity of nodules in the leguminous crop, and contains DNA sequences as shown in SEQ ID Nos. 1-4. Further disclosed are a method for producing an ARC+ rhizobium fertilizer (agent) by compounding an ARC microbial agent with a rhizobium fertilizer (agent), the ARC+ rhizobium fertilizer (agent) prepared by the described solution, and a use thereof in crop production. The ARC+ rhizobium fertilizer (agent) can act as an ARC microbial agent and a rhizobium fertilizer (agent) simultaneously, is simple and convenient to apply, and has low costs, and high benefits.

Description

Use of ARC microbial agent in the production of ARC+ rhizobium fertilizer (agent) Technical Field

[0001] The present invention belongs to the field of microorganisms and also to the field of crop fertilizers, and particularly relates to the use of an ARC microbial agent in producing ARC+rhizobium fertilizer (agent). Background Art

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

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

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

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

[0006] Rhizobium agent, also known as rhizobium fertilizer, refers to a microbial preparation product made with rhizobia as the production strain. Rhizobium fertilizer (agent) has the habit of symbiotic nitrogen fixation with leguminous crops and has application potential. It converts nitrogen in the fixed air into nitrogen fertilizer that can be absorbed and utilized by the host plant, thereby achieving the goal of increasing production. The inventor team further discovered that after the ARC microbial agent and rhizobium fertilizer (agent) are compounded to prepare ARC+rhizobium fertilizer (agent), it can be applied to the production of leguminous crops such as soybeans and peanuts. ARC+rhizobium fertilizer (agent) still has the ARC microbial agent effect on soybeans, peanuts and other legumes. Therefore, the use of ARC microbial agent for the production of ARC+rhizobium fertilizer (agent) was invented, providing a new method for the promotion and application of ARC microbial agent and promoting the development of legume industries such as soybeans and peanuts. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the present invention provides an ARC microbial agent for controlling toxicity and fixing nitrogen and increasing yield. The ARC microbial agent is used to produce ARC+rhizobium fertilizer (agent), is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] 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.

[0010] 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.

[0011] 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.

[0012] According to the above scheme, preferably, the ARC microbial agent is a combination of three or more microorganisms.

[0013] According to the above scheme, the ARC microbial agent has an inhibitory effect on aflatoxin / its toxins.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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, deposit unit name is China Center for Type Culture Collection, address is Wuhan University, Wuhan, China.

[0020] 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.

[0021] Bacillus mucilaginosus JZ2013, deposited on September 27, 2023, with the deposit number CCTCC NO: M 20231817, classified as: Bacillus mucilaginosus strain JZ2013, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0022] Enterobacter ludwigii AR1001, deposit date is September 4, 2023, deposit number is CCTCC NO: M 20231595, classification name is: Enterobacter ludwigii AR1001, deposit unit name is China Center for Type Culture Collection, address is Wuhan University, Wuhan, China.

[0023] 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.

[0024] 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%.

[0025] 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.

[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] 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.

[0028] More specifically, it can be used for the following purposes:

[0029] Use of ARC microbial agent to improve peanut quality and safety and reduce losses

[0030] Use of ARC microbial agent to promote peanut nodulation and nitrogen fixation to achieve green and low-carbon production

[0031] Use of ARC microbial agent to increase peanut yield and total biomass

[0032] Application of ARC microbial agent in peanut to prolong the nodulation and nitrogen fixation time and prevent premature aging due to nutrient deficiency

[0033] ARC microbial agent is used to promote early nodulation, nitrogen fixation, flowering, needle formation and fruit setting in peanuts

[0034] Use of ARC microbial agent to improve peanut fruit filling rate and increase pod number

[0035] Use of ARC microbial agent to reduce fungal diseases such as peanut fruit rot, white rot, root rot and their damage

[0036] Use of ARC microbial agent in reducing bacterial diseases such as peanut wilt and their damage

[0037] Use of ARC microbial agent in reducing peanut root knot nematode disease and its damage

[0038] ARC microbial agent is used to reduce mildew spots and other spots on the surface of peanuts to improve marketability

[0039] Use of ARC microbial agent to improve soybean quality and reduce the risk of aflatoxin contamination

[0040] The use of ARC microbial agent to promote soybean nodulation and nitrogen fixation to achieve green and low-carbon production

[0041] Application of ARC microbial agent in increasing soybean yield and total biomass

[0042] Application of ARC microbial agent in soybean to prolong the nodulation and nitrogen fixation time and prevent premature aging due to nutrient deficiency

[0043] The use of ARC microbial agent for early nodulation, nitrogen fixation, early flowering and pod formation in soybeans

[0044] Use of ARC microbial agent to reduce soybean pod shrinkage rate and increase soybean pod plumpness

[0045] Use of ARC microbial agent in alleviating soybean greening and its hazards

[0046] The use of ARC microbial agent to reduce fungal diseases and damages such as soybean powdery mildew and downy mildew

[0047] Use of ARC microbial agent to reduce soybean root rot and its damage

[0048] Use of ARC microbial agent to shorten soybean pod spacing and increase pod number

[0049] Application of ARC microbial agent in increasing soybean yield in saline-alkali land

[0050] Application of ARC microbial agent in promoting nodulation and nitrogen fixation of leguminous vegetables and improving yield

[0051] Application of ARC microbial agent in promoting nodulation and nitrogen fixation of leguminous grain crops and improving yield

[0052] Application of ARC microbial agent in promoting nodulation and nitrogen fixation of leguminous forage grasses and improving yield

[0053] The use of ARC microbial agents to promote carbon emission reduction and improve arable land quality in the production of leguminous crops such as soybeans and peanuts

[0054] The use of ARC microbial agent to reduce the harm of continuous cropping of leguminous crops such as peanuts and soybeans

[0055] The use of ARC microbial agent in corn production to control bacteria and reduce toxicity, improve quality and safety, and reduce losses

[0056] ARC microbial agent is used in wheat production to control bacteria and reduce toxicity, improve quality and safety levels and reduce losses.

[0057] Based on further research on the ARC microbial agent, the present invention further provides the use of the ARC microbial agent in producing ARC+rhizobium fertilizer (agent).

[0058] The present invention also provides a method for producing ARC+rhizobium fertilizer (agent) by compounding ARC microbial agent and rhizobium fertilizer (agent).

[0059] The above-mentioned ARC microbial agent is used to produce ARC+ rhizobium fertilizer (agent), and its production method can be as follows: according to the amount of rhizobium fertilizer (agent) per mu of land and the amount of ARC microbial agent with a viable count of not less than 80 billion, the rhizobium fertilizer (agent) and the ARC microbial agent are proportioned, and then the ARC microbial agent and the rhizobium fertilizer (agent) are mixed by conventional physical blending to prepare the ARC+ rhizobium fertilizer (agent).

[0060] The above-mentioned ARC microbial agent is used to produce ARC+ rhizobium fertilizer (agent), and its production method can also be as follows: according to the amount of rhizobium fertilizer (agent) per mu of land and the amount of ARC microbial agent with a viable count of not less than 80 billion, the bacterial liquid / powder of the rhizobium fertilizer (agent) and the bacterial liquid / powder of the ARC microbial agent are proportioned, and then uniformly mixed by conventional physical method, and then physically adsorbed onto a carrier to prepare ARC+ rhizobium fertilizer (agent).

[0061] According to the above scheme, the above-mentioned rhizobium fertilizer (agent), rhizobium liquid, and rhizobium powder can be purchased from the market, or can be prepared by separating, identifying, screening, and fermenting from leguminous crop nodules through conventional methods.

[0062] The above physical method can use a spray gun to spray a mixed bacterial liquid consisting of a liquid ARC microbial agent and a bacterial liquid of a rhizobium fertilizer (agent) or a mixed bacterial powder consisting of a dry powder ARC microbial agent and a bacterial powder of a rhizobium fertilizer (agent) onto the carrier particles for adsorption.

[0063] According to the above scheme, the above carrier can be one or a mixture of two or more of carbon powder, charcoal powder, humus, zeolite powder, diatomaceous earth, vermiculite, bentonite, weathered coal, etc.

[0064] The present invention further provides an ARC+rhizobium fertilizer (agent) prepared using the above-mentioned ARC microbial agent.

[0065] The present invention further provides application of ARC+rhizobium fertilizer (agent) in crop production.

[0066] According to the above scheme, the crops include but are not limited to leguminous crops, corn, and wheat. The leguminous crops include peanuts, soybeans, and leguminous grains such as red beans and mung beans; leguminous vegetables such as peas, broad beans, cowpeas, French beans, and kidney beans; and leguminous forage crops such as alfalfa and milk vetch.

[0067] The above-mentioned ARC+rhizobium fertilizer (agent) can be used in the production of leguminous crops such as peanuts and soybeans and other crops, while exerting the effects of ARC microbial agent and rhizobium fertilizer (agent).

[0068] The beneficial effects of the present invention are:

[0069] 1. ARC microbial agent can be used to produce ARC+Rhizobium fertilizer (agent), which can simultaneously exert the functions of ARC microbial agent and Rhizobium fertilizer (agent), avoiding the problem of Rhizobium deficiency in the application field while also regulating Rhizobia and promoting Rhizobium nodulation and nitrogen fixation. 2. It is easy to use, low cost, and highly effective. 3. ARC+Rhizobium fertilizer (agent) can be used in leguminous crop production, which is of great significance for promoting the productivity of leguminous crops and promoting green, low-carbon, and efficient production. Modes for Carrying Out the Invention

[0070] Part I ARC Microbial Agents

[0071] Example 1 Preparation of ARC microbial agent

[0072] 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.

[0073] Table 1. Some of the strains isolated and identified from a mixture of major legume crops such as peanuts and soybeans are shown below:

[0074] 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

[0075] 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.

[0076] 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%.

[0077] Table 2. Microbial composition and bacterial agent information

[0078] 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

[0079] Example 2: Sequencing of ARC microbial agents

[0080] 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.

[0081] 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.

[0082] Example 3: Determination of the Toxic Control and Nitrogen Fixation Effect of Peanut ARC Microbial Agent

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] Table 3. Results of the microbial agent test on the control of toxicity and nitrogen fixation in peanuts

[0089] 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

[0090] 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%.

[0091] Example 4: Determination of the Toxic Control and Nitrogen Fixation Effect of Soybean Microbial Agents

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] The above-mentioned soil-borne plant pathogens are the pathogenic microorganisms of the above-mentioned corresponding field diseases. Therefore, the field disease prevention and control effect is equivalent to the inhibitory effect of ARC microbial agent on the pathogenic factors / toxins of the above-mentioned soil-borne plant pathogens.

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

[0098] 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

[0099] The above-mentioned microbial agent has an inhibition rate of more than 60% against aflatoxin / toxin, and further more than 90%; the inhibition rate of one or more pathogenic factors / toxins of soil-borne plant pathogens such as Aspergillus, 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%.

[0100] Example 5: Determination of the Effect of Microbial Agents on Toxic Control and Nitrogen Fixation in Other Leguminous Crops

[0101] The sampling method and steps similar to those in Examples 3 and 4 were used to determine the toxicity control and nitrogen fixation effects of ARC microbial agents 1 to 12 on other leguminous crops such as peas, broad beans, cowpeas, and alfalfa, and the results were similar to those in Tables 3 and 4.

[0102] Part 2 Application of ARC Microbial Agents

[0103] Example 6: Use of ARC microbial agent - Use for producing ARC + rhizobium fertilizer (agent) (I)

[0104] The ARC microbial agents 1-8 are mixed with the rhizobium fertilizer (agent) at a dosage of no less than 80 billion viable bacteria per mu of land, and the dosage of the rhizobium fertilizer (agent) is adjusted accordingly. The ARC microbial agents and the rhizobium fertilizer (agent) are then evenly blended using conventional physical methods to prepare eight types of ARC+rhizobium fertilizers (agents) 1-8. The rhizobium fertilizers (agents) can be purchased commercially or prepared by isolating, identifying, and fermenting leguminous crop nodules using conventional methods.

[0105] The eight ARC+Rhizobium fertilizers (agents) 1-8 were used as seed fertilizers in leguminous crops such as peanuts and soybeans at sowing. Fields treated with the same Rhizobium fertilizers (agents) served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the eight ARC+Rhizobium fertilizers (agents) 1-8 significantly promoted nodulation and nitrogen fixation, increasing nodule number by more than 3.5 times, nitrogenase activity per plant by more than 10.5 times, and yield per unit area by more than 15.0%, achieving significant yield increases and demonstrating the key characteristics of the ARC microbial agents described in the above examples.

[0106] The above results indicate that ARC microbial agent can be used to prepare ARC+rhizobium fertilizer (agent), and it has the effects of both rhizobium fertilizer (agent) and ARC microbial agent.

[0107] Example 7: Use of ARC microbial agent - Use for producing ARC + rhizobium fertilizer (agent) (II)

[0108] The above-mentioned ARC microbial agents 1 to 8 are mixed in proportion with the amount of rhizobium fertilizer (agent) per mu of land at a rate of not less than 80 billion viable bacteria per mu of land and ARC microbial agents 1 to 8 respectively. The bacterial liquid / powder of the rhizobium fertilizer (agent) and the bacterial liquid / powder of the ARC microbial agent are uniformly mixed by conventional physical mixing, and then physically adsorbed onto a carrier to prepare ARC+rhizobium fertilizer (agent).

[0109] The above-mentioned rhizobium fertilizer (agent), rhizobium liquid and rhizobium powder are directly purchased from the market, or can be prepared by separation, identification, screening and fermentation from nodules of leguminous crops through conventional methods.

[0110] The above physical method can use a spray gun to spray the ARC microbial agent in liquid state or the ARC microbial agent in dry powder state onto the carrier particles for adsorption.

[0111] The carrier can be a mixture of carbon powder and humus at a ratio of 9:1, or a mixture of one or two or more of carbon powder, carbon powder, humus, zeolite powder, diatomaceous earth, vermiculite, bentonite, weathered coal, etc.

[0112] The eight ARC+Rhizobium fertilizers (agents) 1-8 were used as seed fertilizers in leguminous crops such as peanuts and soybeans at sowing. Fields treated with the same Rhizobium fertilizers (agents) served as controls, and ongoing surveys were conducted after sowing. These survey results showed that the eight ARC+Rhizobium fertilizers (agents) 1-8 significantly promoted nodulation and nitrogen fixation, increasing nodule number by more than 4.1 times, nitrogenase activity per plant by more than 12.7 times, and yield per unit area by more than 16.0%, achieving significant yield increases and demonstrating the key characteristics of the ARC microbial agents described in the above examples.

[0113] The above results indicate that ARC microbial agent can be used to prepare ARC+rhizobium fertilizer (agent), and it has the effects of both rhizobium fertilizer (agent) and ARC microbial agent.

Claims

1. The use of an ARC microbial agent for producing ARC+rhizobium fertilizer (agent), wherein 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 Nos. 1-4.

2. A method for producing ARC+rhizobium fertilizer (agent) by compounding ARC microbial agent with rhizobium fertilizer (agent), characterized by: According to the conventional dosage of rhizobium fertilizer (agent) per mu of land and the dosage of ARC microbial agent with a viable count of not less than 80 billion, the rhizobium fertilizer (agent) and ARC microbial agent are proportioned, and then the ARC microbial agent and rhizobium fertilizer (agent) are mixed by conventional physical blending to prepare ARC + rhizobium fertilizer (agent); Alternatively, according to the conventional dosage of rhizobium fertilizer (agent) per mu and the dosage of ARC microbial agent with a viable count of not less than 80 billion, the bacterial liquid / powder of rhizobium fertilizer (agent) and the bacterial liquid / powder of ARC microbial agent are mixed evenly in a conventional manner, and then physically adsorbed onto a carrier to prepare ARC+rhizobium fertilizer (agent). 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, and contains the DNA sequences shown in SEQ ID No. 1-4.

3. The method according to claim 2, wherein: The physical method is to use a spray gun to spray a mixed bacterial solution consisting of ARC microbial agent liquid and rhizobium fertilizer (agent) bacterial solution or a mixed bacterial powder consisting of ARC microbial powder and rhizobium fertilizer (agent) powder onto the carrier particles for adsorption.

4. The ARC+Rhizobium fertilizer (agent) prepared according to the method of claim 2 or 3.

5. Use of the ARC+Rhizobium fertilizer (agent) according to claim 4 in crop production.

6. The use according to claim 1, characterized in that: In the ARC microbial agent, the DNA sequence genes shown in SEQ ID Nos. 1-4 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 function is present, the functional equivalents of the DNA sequences shown in SEQ ID Nos. 1-4 are constituted. The microbial composition contains all the gene sequences shown in SEQ ID NOs. 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.

7. 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 preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595, and other microorganisms, so that the combined microbial agent meets the requirements of containing all gene sequences in the nucleotide sequences shown in SEQ ID NOs. 1 to 4 or their functional equivalents, has a coupled effect of toxic control and nitrogen fixation, and has the effect of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, thereby constituting the ARC microbial agent of claim 1.

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

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

10. 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.