Use of arc microbial agent for controlling bacteria, reducing toxicity, improving quality and safety levels and reducing losses in production of maize or wheat

Abstract

The present invention belongs to the field of microorganisms. Disclosed is the use of an ARC microbial agent for controlling bacteria, reducing toxicity, improving quality and safety levels and reducing losses in the production of maize or wheat. The use of the ARC microbial agent for controlling bacteria, reducing toxicity, improving quality and safety levels and reducing losses in the production of maize or wheat. The ARC microbial agent is a microbial composition, which has a coupled effect of toxicity control and nitrogen fixation, has the effects of regulating and improving the abundance of rhizobia in the rhizosphere of a leguminous crop and increasing the number of nodules of the leguminous crop; and contains a DNA sequence as shown in SEQ ID NOs: 1-4. The ARC microbial agent is used for controlling bacteria, reducing toxicity, improving quality and safety levels and reducing losses in the production of maize or wheat, is simple to use, has significant social and ecological benefits, and is easy to popularize and apply.

Description

ARC microbial agent is used to control bacteria and reduce toxicity in corn or wheat production to improve quality and safety and reduce losses. Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to the use of an ARC microbial agent in controlling bacteria and reducing toxicity to improve quality and safety levels and reduce losses in corn or wheat production. Background Art

[0002] Corn mycotoxins are toxic secondary metabolites produced by Fusarium and / or aflatoxin-producing fungi found in corn. They primarily include aflatoxin B1, zearalenone, and deoxynivalenol. These toxins often have triple-causing effects: carcinogenic, teratogenic, and mutagenic. Corn is susceptible to infection by Fusarium and / or aflatoxin-producing fungi during production, and harbors toxin-producing fungi, which in turn makes corn mycotoxins highly likely to form. This leads to food losses and poses a serious threat to food safety and human health.

[0003] Wheat mycotoxins are toxic secondary metabolites produced by Fusarium fungi when they infect wheat and cause fusarium head blight. These include deoxynivalenol (DNO) and zearalenone (ZEA). Deoxynivalenol, also known as vomitoxin, has immunotoxic and gastrointestinal effects on humans and livestock, while ZEA has carcinogenic, reproductive, neurotoxic, and immunotoxic effects on humans and livestock. Wheat mycotoxin contamination is common and widespread worldwide. Wheat head blight is also widespread in my country, with major outbreaks occurring every three to five years. It is particularly prevalent in the Yangtze, Huai, and Yellow River basins. Excessive wheat mycotoxin contamination often leads to significant grain losses. Therefore, implementing fungal control and toxicity reduction measures during corn and wheat production—that is, preventing toxin contamination by inhibiting the growth of toxin-producing fungi—is crucial for ensuring food security, food safety, and human health, and remains a key research focus and challenge internationally. In recent years, research on green microbial control has become an important research direction in this field. However, chemical control methods are generally used in corn production or wheat production. There is still a lack of effective corn control and attenuation microbial agents or wheat control and attenuation microbial agents that can be promoted and applied in production.

[0004] After more than 20 years of continuous research, the inventors' team has discovered the spatiotemporal correlation between the abundance and toxicity of peanut aflatoxin-producing Aspergillus flavus, geography, climate, and 53 other factors, and the occurrence of aflatoxin. They have also developed highly sensitive aflatoxin detection and early warning technology, 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 in peanuts with nodulation and nitrogen fixation. By analyzing the microbial population structure within the peanut rhizosphere, isolating and identifying the probiotic strain library, and assembling a large number of combinations, they have completed laboratory and field screening and identification, opening up research and exploration into the coupling of soil-source control of aflatoxin and induced nodulation and nitrogen fixation. Over the past five years, the ARC microbial agent has been successfully developed. This agent, known as an ARC microbial agent or simply an ARC agent, effectively controls aflatoxin at the source while simultaneously promoting efficient nodulation and nitrogen fixation in peanuts and significantly increasing yields. ARC stands for Aspergillus flavus / Aflatoxins and Rhizobia Coupling, and represents a coupled effect in 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 fixes (nitrogen and carbon fixation), three increases (increased yield, efficiency, and safety), and five reductions (reduced toxicity, damage, weight, costs, and carbon emissions). This agent has demonstrated and validated its potential in field trials across multiple major peanut and other legume-producing regions nationwide, demonstrating its significant potential for increasing peanut yields, boosting production capacity, and promoting green, low-carbon, and efficient production.

[0005] In response to the difficulty in preventing and controlling fungal toxin contamination in corn, the inventor team further used ARC microbial agents to conduct research and found that ARC microbial agents can be used in corn or wheat production, and have the application effects of controlling bacteria and reducing toxicity, improving quality and safety levels, and reducing losses. Therefore, the inventor team invented the use of ARC microbial agents for controlling bacteria and reducing toxicity in corn or wheat production to improve quality and safety levels and reduce losses, providing an effective method for preventing and controlling major fungal toxin contamination in corn and wheat. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides an ARC microbial agent, which is used for controlling bacteria and reducing toxicity in corn or wheat production to improve quality and safety levels and reduce losses. It 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 the number of viable bacteria of any one strain in the above microbial agent, i.e. the mixed microbial composition, is greater than or equal to 1%.

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

[0025] The 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 in corn or wheat production to control bacteria and reduce toxicity, improve quality and safety levels and reduce losses.

[0028] The details are as follows: The invention provides the use of ARC microbial agent for controlling bacteria and reducing toxicity in corn production to improve quality and safety level and reduce losses.

[0029] A method for controlling bacteria and reducing toxicity, improving quality and safety, and reducing losses in corn production, comprising applying an ARC microbial agent to corn crops.

[0030] According to the above scheme, preferably, the application amount of the ARC microbial agent is 80 billion to 100 billion viable bacteria per mu.

[0031] 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 corn is sown and / or before the corn becomes diseased after seedlings emerge.

[0032] The application method of the above-mentioned ARC microbial agent in corn production can be as follows: mix the above-mentioned ARC microbial agent with corn sowing base fertilizer, and evenly apply it to the field by one or more methods such as manual, seeding machine or drone, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be adopted for fields where conditions permit, and try to avoid severe dryness in the field, uneven emergence of seedlings, etc., and conventional field management is adopted for other purposes.

[0033] The ARC microbial agent can also be applied to corn production as follows: After corn seedlings have emerged normally, and until the silking stage, before disease develops, the ARC microbial agent is evenly applied to the field via one or more topdressing methods, such as broadcasting, spraying, and drip irrigation, at a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is used for all other aspects.

[0034] Provided is the use of ARC microbial agent for controlling bacteria and reducing toxicity in wheat production to improve quality and safety levels and reduce losses.

[0035] According to the above scheme, the above-mentioned fungal control and toxin reduction refers to the prevention and control of wheat fusarium rust pathogenic fungi and the reduction and control of vomitoxin and other Fusarium fungal toxins.

[0036] A method for controlling bacteria and reducing toxicity, improving quality and safety, and reducing losses in wheat production, comprising applying an ARC microbial agent to wheat crops.

[0037] 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.

[0038] 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 wheat is sown and / or before the wheat seedlings emerge and the disease occurs.

[0039] The application method of the above-mentioned ARC microbial agent in wheat production can be as follows: mix the above-mentioned ARC microbial agent with wheat sowing base fertilizer, and evenly apply it to the field by one or more methods such as manual, seeding machine or drone, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. After sowing, drip irrigation can be adopted for fields where conditions permit, and try to avoid severe dryness in the field, uneven emergence of seedlings, etc., and conventional field management is adopted for other purposes.

[0040] The ARC microbial agent can also be applied to wheat production as follows: After wheat seedlings have been properly sown and emerged, and until the onset of disease at the heading and 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, with a cumulative application rate of 80 billion to 100 billion viable bacteria per mu. Conventional field management is used for all other purposes. The present invention has the following beneficial effects:

[0041] 1. ARC microbial agent can be used in corn or wheat production to control bacteria and reduce toxicity, improve quality and safety, and reduce losses. 2. It is easy to use, low-cost, and highly effective. 3. It is of great significance to the high-quality development of the corn and wheat industries and to ensure food safety. 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 leguminous 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] Example 6: Use of ARC microbial agent - Use in corn production to control bacteria and reduce toxicity, improve quality and safety, and reduce losses

[0076] The above-mentioned ARC microbial agents 1 to 8 were mixed with corn seeding base fertilizer and applied to the field through a seed drill. The application rate of the microbial agents was 80 billion to 100 billion viable bacteria per mu. At the same time, a plot without any of the above microbial agents was set up as a control. The other plots were managed under conventional field management. After the above-mentioned corn was harvested, representative corn samples were collected. (1) The abundance of toxin-producing fungi carrying aflatoxin and Fusarium toxin in these samples was determined by the classic colony counting method. The reduction rate of the abundance of toxin-producing fungi carried by corn was calculated, which is the control effect of toxin-producing fungi. (2) After the samples were placed under the same conditions for 6 months, the contamination levels of aflatoxin and Fusarium toxin were determined by the national standard method. The control effect of aflatoxin and Fusarium toxin was calculated. These test results showed that agents 1-8 were effective at controlling aflatoxins and Fusarium toxin-producing fungi in field corn kernels at rates exceeding 53%, 80% and 60% respectively against aflatoxins and Fusarium toxin-producing fungi in corn, significantly reducing grain losses due to excessive toxin contamination. These results demonstrate that the application of ARC microbial agents in corn production has significant potential for controlling bacteria and toxicity, improving corn quality and safety, and reducing losses.

[0077] The above-mentioned ARC microbial agents were applied in corn production by manual spreading, drone spreading, etc. during corn sowing, and similar effects of controlling bacteria and reducing toxicity, improving corn quality and safety, and reducing losses were achieved.

[0078] Applying the above-mentioned ARC microbial agents from the time corn seedlings emerge to the silking stage has also achieved similar effects in controlling bacteria and reducing toxicity, improving corn quality and safety, and reducing losses.

[0079] Example 7: Use of ARC microbial agent - Use in wheat production to control bacteria and reduce toxicity and improve quality and safety

[0080] The above-mentioned ARC microbial agents 1 to 8 were mixed with wheat seeding base fertilizer and applied to the field by a seed drill. The application rate of the microbial agents was 80 billion to 100 billion viable bacteria per mu. At the same time, plots without any of the above microbial agents were set up as controls, and the others were managed in the conventional field. After the wheat was harvested, representative wheat samples were collected. (1) The abundance of Fusarium carried by these samples was determined by the classic colony counting method, and the reduction rate of Fusarium abundance in wheat was calculated, that is, the control effect on toxin-producing fungi; (2) The contamination level of Fusarium toxins such as vomitoxin and zearalenone was determined by the national standard method, and the control effect on Fusarium toxins was calculated. These test results showed that the control effect of microbial agents 1 to 8 on Fusarium carried by wheat grains in the field was above 55%, and the control effect on Fusarium mycotoxins in wheat was above 65%, significantly reducing the wheat losses caused by excessive mycotoxin contamination. The above results show that the application of ARC microbial agent in wheat production has significant effects on controlling bacteria and reducing toxicity, improving wheat quality and safety, and reducing losses.

[0081] The above-mentioned ARC microbial agents were applied in wheat production by manual spreading, drone spreading, etc. during wheat sowing, and similar effects of controlling bacteria and reducing toxicity, improving wheat quality and safety, and reducing losses were achieved.

[0082] Applying the above-mentioned ARC microbial agents from the time wheat seedlings emerge to the time it heads and flowers has also achieved similar effects in controlling bacteria and reducing toxicity, improving wheat quality and safety, and reducing losses.

Claims

1. The ARC microbial agent is used for controlling bacteria and reducing toxicity in corn or wheat production to improve quality and safety and reduce losses. The ARC microbial agent is a microbial composition with a coupled effect of toxicity control and nitrogen fixation, which has the function of regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops. It contains the DNA sequences shown in SEQ ID Nos. 1-4.

2. A method for controlling bacteria and reducing toxicity, improving quality and safety, and reducing losses in corn production, comprising applying an ARC microbial agent to corn crops. The ARC microbial agent is a microbial composition having a coupled effect of controlling toxicity and fixing nitrogen, regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, and comprises the DNA sequences shown in SEQ ID Nos. 1-4.

3. The method according to claim 2, wherein: The application amount 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 corn is sown and / or before the corn becomes diseased after seedlings emerge.

5. The method according to claim 2, wherein: The method is as follows: ARC microbial agent is mixed with corn seeding base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, and drone. The application rate of ARC microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: after the corn is sown and seedlings emerge normally until the corn silking stage when the disease occurs, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or a combination of one or more of the following methods, with the application rate of the agent accumulating 80 billion to 100 billion live bacteria per mu.

6. A method for controlling bacteria and reducing toxicity, improving quality and safety, and reducing losses in wheat production, comprising applying an ARC microbial agent to a wheat crop. The ARC microbial agent is a microbial composition having a coupled effect of controlling toxicity and fixing nitrogen, regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops and increasing the number of nodules in leguminous crops, and comprises the DNA sequences shown in SEQ ID Nos. 1-4.

7. The method according to claim 6, characterized in that: 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 wheat is sown and / or before wheat becomes diseased after seedlings emerge.

9. The method according to claim 6, wherein: The method is as follows: ARC microbial agent is mixed with wheat sowing base fertilizer, and applied evenly to the field by one or more of the following methods: manual, seeding machine, and drone. The application rate of ARC microbial agent is 80 billion to 100 billion viable bacteria per mu. Or: after the wheat is normally sown and seedlings emerge until the disease occurs during the heading and flowering stage, the ARC microbial agent is evenly applied to the field by broadcasting, spraying, drip irrigation or a combination of one or more of the following methods, with a cumulative application rate of 80 billion to 100 billion live bacteria per mu.

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 the ARC microbial agent is a combination of one or more microorganisms selected from Bacillus laterosporus with a preservation number of CCTCC NO: M 20231815, Bacillus amyloliquefaciens with a preservation number of CCTCC NO: M 20231598, Bacillus mucilaginosus with a preservation number of CCTCC NO: M 20231817, and Enterobacter ludwigii with a preservation number of CCTCC NO: M 20231595, and other microorganisms, so that the combined microbial agent 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, 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.

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 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.