Use of arc microbial inoculant for shortening of internode distance and increasing pod number in soybean

Abstract

The application belongs to the field of microorganisms, and discloses the use of an ARC microbial inoculant for shortening the interval of soybean nodes and increasing the number of pods. The use of the ARC microbial inoculant for shortening the interval of soybean nodes and increasing the number of pods, wherein the ARC microbial inoculant is a microbial inoculant composition, has a detoxification and nitrogen fixation coupling effect, has the effect of regulating and increasing the abundance of rhizobium leguminosarum in the rhizosphere of legume crops and the number of nodulation of legume crops, and contains the DNA sequences shown as SEQ ID No. 1-4. The ARC microbial inoculant can increase the number of pods, improve the yield level of soybean, is simple to use, has significant social and ecological benefits, and is easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to the use of ARC microbial agents to shorten internode spacing and increase pod number in soybeans. Background Technology

[0002] The development of the soybean industry faces two major challenges: First, soybeans are susceptible to contamination by toxin-producing Aspergillus flavus, which produces highly toxic and carcinogenic aflatoxins. Current methods for controlling aflatoxin in soybeans mainly rely on temperature and humidity control during storage, transportation, and processing, which is energy-intensive and difficult to control. Aflatoxin contamination control has always been a global challenge.

[0003] Secondly, while leguminous crops like soybeans exhibit symbiotic nodulation and nitrogen fixation with soil rhizobia, the number of nodules is low under natural conditions, the nitrogen fixation period is short (it is generally believed that crops do not form nodules and fix nitrogen in the first month of growth after sowing, and no new nodules appear during the pod-filling or fruit-filling stage, with existing nodules beginning to decay), and the efficiency is low. Research on biological nitrogen fixation by rhizobia has a history of over 100 years, forming the classic and universally accepted AON theory system—plants self-regulate the number of nodules and growth, maintaining total energy conservation. Excessive nodulation inevitably comes at the cost of sacrificing plant growth. Current practices mainly involve screening and applying superior rhizobia suitable for specific growing environments, which has geographical limitations and is long constrained by the AON theory. The improvement in nodulation and nitrogen fixation efficiency is limited, with a relatively good increase of about 30%, making it difficult to achieve a doubling of nodulation and nitrogen fixation efficiency while simultaneously significantly increasing growth (breaking the AON theory system). How to improve the nodulation and nitrogen fixation efficiency of leguminous crops like soybeans is also a cutting-edge international challenge.

[0004] To address the aforementioned challenges, the inventors' team, after more than 20 years of continuous research, successfully developed the ARC microbial agent. This agent achieves green control of aflatoxin at its source while simultaneously inducing and promoting efficient nodulation and nitrogen fixation in soybeans, resulting in a significant increase in yield. Microbial agents with these functions are called ARC microbial agents or ARC agents. ARC stands for Aspergillus flavus / Aflatoxins and Rhizobia Coupling, meaning it couples the control of aflatoxin contamination with the promotion of nodulation and nitrogen fixation. This type of agent is easy to apply, low-cost, and highly effective, exhibiting significant characteristics and enormous application potential, including two fixes (nitrogen and carbon fixation), three increases (increased yield, efficiency, and safety), and five reductions (reduced toxicity, damage, fertilizer, cost, and carbon emissions). It has already undergone large-scale field trials and verification in multiple major soybean and other leguminous crop producing areas across China. This is of great significance for promoting the improvement of soybean oilseed production capacity and green, low-carbon, and efficient production in my country.

[0005] In soybean production, shortening the internode spacing and increasing the number of pods are important ways to improve soybean yield. Our team further discovered that the application of ARC microbial agents can also significantly shorten the internode spacing and increase the number of pods, thus providing a new solution for increasing soybean yield and production capacity. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing an ARC microbial agent for shortening internode spacing and increasing pod number in soybeans. It can increase pod number, improve soybean yield, is simple to use, has significant social and ecological benefits, and is easy to promote and apply.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: This invention first provides a microbial agent for controlling toxins and fixing nitrogen, coupled with yield-increasing ARC, which is a microbial composition that has a toxin-controlling nitrogen-fixing coupling effect. It 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 all the gene sequences in DNA sequences 1 to 4 as shown in SEQ ID No. 1-4.

[0008] According to the above scheme, the ARC microbial agent has an inhibitory effect on Aspergillus flavus and / or its toxins. Furthermore, the inhibition rate against Aspergillus flavus can reach over 60%, and the inhibition rate against aflatoxins can reach over 80%. The inhibition of Aspergillus flavus and / or its toxins can be detected using other conventional detection methods in the prior art, or it can be detected through inhibition analysis of marker molecules of Aspergillus flavus toxin-producing bacteria. Specifically, the inhibition of Aspergillus flavus and / or its toxins by the ARC microbial agent can be detected through inhibition analysis of marker molecules of Aspergillus flavus toxin-producing bacteria.

[0009] According to the above scheme, the ARC microbial agent of the present invention significantly inhibits the expression level of 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 antiviral 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 referred to the method in the following literature: Protein biomarker for early diagnosis of microbialtoxin contamination: Using Aspergillus flavus as an example, Food Frontiers. 2023, 4, 2013-2023, DOI: 10.1002 / fft2.295.

[0010] According to the above scheme, preferably, the ARC microbial agent is a combination of three or more microbial bacteria.

[0011] According to the above scheme, the ARC microbial agent has an inhibitory effect on Aspergillus flavus / its toxins.

[0012] According to the above scheme, the ARC microbial agent has an inhibitory effect on one or more of the soil-borne plant pathogens (pathogenic factors) / toxins, such as Penicillium, Aspergillus flavus, Fusarium, Sclerotium sclerotiorum, Ralstonia solanacearum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani.

[0013] According to the above scheme, the ARC microbial agent can promote early nodulation and prolong the nodulation and nitrogen fixation time of leguminous crops such as peanuts and soybeans.

[0014] The DNA sequences shown in SEQ ID Nos. 1-4 may vary to some 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, more preferably no more than 1% base variation (i.e., identity of 90% or more, preferably 95% or more, more preferably 99% or more), and the corresponding biological activity is present, these are called functional equivalents of the DNA sequences shown in SEQ ID Nos. 1-4. Containing these sequences is equivalent to containing the corresponding sequences of DNA sequences 1-4. Microbial compositions containing the DNA sequences shown in SEQ ID Nos. 1-4 or their functional equivalents, and possessing a nitrogen-fixing coupling effect, and having 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, are all ARC microbial agents of the present invention.

[0015] The aforementioned DNA sequences 1-4 are specific sequences obtained after comparison with GenBank genomic big data. They are directly or indirectly related to the function of the microbial agent of this patent. When the entire gene sequence of the aforementioned DNA sequences 1-4 is contained, it has a nitrogen fixation and toxin control coupling effect, which can regulate and increase the abundance of rhizobia in the rhizosphere of leguminous crops and increase the number of nodules in leguminous crops, thus constituting the ARC microbial agent of this invention. These genes may vary to a certain extent in different strains. When the degree of difference is small, such as no more than 10% base variation, and the corresponding biological activity function is maintained, containing them is equivalent to containing the gene sequences shown in SEQ ID No. 1-4.

[0016] 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 microorganisms: Bacillus lateralis with accession number CCTCC NO: M 20231815, Bacillus amyloliquefaciens with accession number CCTCC NO: M20231598, Bacillus mucilaginosus with accession number CCTCC NO: M 20231817, and Enterobacter ludwig's bacterium with accession number CCTCC NO: M 20231595.

[0017] Bacillus laterosporus H-CB4802, deposited on September 27, 2023, with accession number CCTCC NO: M20231815, is classified as follows: Brevibacillus laterosporus strain H-CB4802, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0018] Bacillus amyloliquefaciens AR1004, deposited on September 4, 2023, with accession number CCTCC NO: M20231598, and classified as follows: Amylolytic Bacillus AR1004 is deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0019] Bacillus mucilaginosus JZ2013, deposited on September 27, 2023, with accession number CCTCC NO: M20231817, classified and named as follows: Bacillus mucilaginosus strain JZ2013, the depository is China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0020] Enterobacter ludwig's bacterium AR1001, deposited on September 4, 2023, with accession number CCTCC NO: M20231595, and classified as follows: Enterobacter ludwigii AR1001, the depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0021] According to the above scheme, preferably, the above-mentioned ARC microbial agent can be a combination of one or more of the following microorganisms: Bacillus lateralis with accession number CCTCC NO: M20231815, Bacillus amyloliquefaciens with accession number CCTCC NO: M 20231598, Bacillus mucilaginosus with accession number CCTCC NO: M 20231817, and Enterobacter ludwigii with accession number CCTCC NO: M 20231595, and other microorganisms, so that the combined microbial agent contains all gene sequences or their functional equivalents in the nucleotide sequences shown in SEQ ID NO. 1~4, has a nitrogen fixation coupling 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, thus constituting the ARC microbial agent of the present invention.

[0022] According to the above scheme, in the above microbial agents, the proportion of any one strain of bacteria in the microbial agent, i.e., the microbial composition formed by mixing, is greater than or equal to 1%.

[0023] The ARC microbial agent of this invention is a microbial composition. Through the synergistic effect of the various microorganisms in the composition, a nitrogen-fixing and toxin-controlling coupling effect is generated, achieving a coupled effect of toxin control and nitrogen fixation. When used in crop production, it exhibits this coupling effect, playing a role in controlling aflatoxin contamination and promoting nodulation and nitrogen fixation. It can regulate and increase the abundance of rhizobia in the rhizosphere of leguminous crops and increase the number of nodules. While not itself a rhizobium, it can regulate and increase the abundance of rhizobia in the rhizosphere of leguminous crops, increase the number of nodules, and increase the nitrogenase activity of individual plants. It can increase the abundance of rhizobia in the rhizosphere of leguminous crops by at least 15%, and the number of nodules can increase by more than 2 times. It can also promote early nodulation and prolong the nodulation and nitrogen fixation time in peanuts and soybeans.

[0024] The aforementioned ARC microbial inoculants can be used in legume production for the following purposes: improving the quality and safety of legume products; promoting nodulation and nitrogen fixation in legumes; increasing the yield of legumes; recruiting native rhizobia and increasing the abundance of rhizobia in the rhizosphere soil of legumes; promoting early nodulation and extending the nodulation and nitrogen fixation time in legumes; preventing premature senescence due to nutrient deficiency at maturity in legumes; increasing the number of pods in legumes; increasing pod fullness and reducing the rate of empty pods in legumes; promoting early flowering and pod formation in legumes; reducing peanut pod rot; reducing bacterial wilt in legumes; reducing powdery mildew in legumes; reducing leaf spot in legumes; reducing root nematode disease in legumes; and reducing... It is used to reduce the occurrence of root rot in leguminous crops; to reduce the occurrence of root nematode disease in leguminous crops; to reduce the occurrence of Phytophthora blight in leguminous crops; to reduce the occurrence of sclerotinia rot in leguminous crops; to reduce the occurrence of downy mildew in leguminous crops; to reduce the occurrence of wilt disease in leguminous crops; to reduce the occurrence of white mold disease in leguminous crops; to reduce the severity of soybean scab; to reduce the severity of ear rot and Fusarium toxin in corn; to reduce the abundance of Fusarium head blight pathogens in wheat and reduce the severity of Fusarium toxin; to promote carbon emission reduction in leguminous crops and benefit soil improvement; to promote the increase of total biomass in leguminous crops; to reduce the abundance of harmful organisms such as Aspergillus terreus and Fusarium in the rhizosphere of leguminous crops and benefit the improvement of soil microbial community structure; to reduce surface spots on peanut pods and increase marketability; and to promote soybean yield increase in saline-alkali land.

[0025] The aforementioned ARC microbial inoculant can be prepared by the following method: using microorganisms from the aforementioned microbial inoculant, and preparing it via a fermentation pathway. The fermentation pathway can employ conventional bacterial fermentation pathways as described in existing technologies, including publicly available literature.

[0026] Based on the above research, this invention further explores and provides the use of ARC microbial inoculants for shortening internode spacing and increasing pod number in soybeans.

[0027] The present invention also provides a method for shortening internode spacing and increasing pod number in soybeans, the method comprising applying ARC microbial agent to soybean production.

[0028] According to the above scheme, the application rate of the ARC microbial agent is no less than 80 billion live bacteria per acre, for example, the application rate is 80 billion to 100 billion live bacteria, or more than 80 billion to 100 billion live bacteria.

[0029] According to the above scheme, the application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is during soybean sowing and / or after soybean emergence to flowering.

[0030] The application method of the above-mentioned ARC microbial agent in soybean production can be as follows: Mix the above-mentioned ARC microbial agent with the soybean sowing base fertilizer, and apply it evenly to the field through one or more of the following methods: manual, seeder, drone, drip irrigation pipeline, etc. The cumulative number of live bacteria per mu is 80 billion to 100 billion. After sowing, drip irrigation can be adopted in fields with suitable conditions to avoid severe dryness and uneven emergence. Other conventional field management is adopted.

[0031] The application method of the above-mentioned ARC microbial inoculant in soybean production can also be as follows: After soybeans have emerged from normal sowing and until the flowering and pod-setting stage, apply the above-mentioned ARC microbial inoculant evenly to the field through one or more topdressing methods such as broadcasting, spraying, and drip irrigation, with a cumulative application rate of 80 billion to 100 billion live bacteria per acre. Other conventional field management methods are adopted.

[0032] The beneficial effects of this invention are as follows: 1. ARC microbial inoculants can be used to shorten internode spacing in soybeans and increase the number of pods. 2. They are beneficial for increasing the number of pods and improving soybean yield per unit area. 3. They are easy to apply, low in cost, and highly efficient. 4. They are of great significance for promoting the improvement of soybean oilseed production capacity and green, low-carbon, and efficient production in my country. Detailed Implementation

[0033] Part 1: ARC Microbial Agents Example 1: Preparation of ARC microbial inoculant Whole plant samples and rhizosphere soil samples from peanuts, soybeans, peas, broad beans, cowpeas, and alfalfa were collected, ground, mixed, and strains were isolated using conventional bacterial isolation methods, followed by identification using conventional 16S rDNA methods. A total of *Bacillus amylase-lysing* strains were obtained through these procedures. Bacillus amyloliquefaciens Lateral spores of Bacillus brevis Brevibacillus laterite Bacillus mucilaginosus Mucilaginous Bacillus Ludwig's Enterobacter Enterobacter Ludwig The series of bacteria are detailed in Table 1.

[0034] Table 1. Some of the strains isolated and identified from mixtures of major leguminous crops such as peanuts and soybeans are shown below:

[0035] The 10 microbial strains in Table 1 above were amplified one by one using conventional bacterial culture medium amplification methods to prepare batches of fermentation broth or bacterial powder of the above 10 strains.

[0036] Table 2 shows the information on microbial inoculants formed by mixing fermentation broth or powder of four strains: Bacillus laterosporus brevis (CCTCC NO: M 20231815), Bacillus amyloliquefaciens (CCTCC NO: M20231598), Bacillus mucilaginosus (CCTCC NO: M 20231817), and Enterobacter ludwigii (CCTCC NO: M20231595). It also includes information on combinations of some strains from these strains with other strains, as well as combinations of other microbial inoculants. In each microbial combination, the proportion of viable bacteria of any single strain is greater than or equal to 1%.

[0037] Table 2. Information on Microbial Compositions (Initials)

[0038] Example 2: Sequencing of ARC microbial agents Take a sufficient number of samples from the microbial agents in Table 2 of Example 1, extract total DNA from these samples sequentially using conventional DNA extraction methods, determine the DNA sequence of these samples using conventional DNA sequencing methods, and finally compare the homology of the determined DNA sequence with the gene sequence shown in SEQ ID No. 1-4 provided in this patent text using conventional analysis methods.

[0039] The above determination and homology analysis results show that the bacterial agents 1-4 in Table 2 contain all four genes in DNA sequences 1-4 as shown in SEQ ID No. 1-4, and the sequence homology is 100%; the bacterial agents 5-12 in Table 1 contain all four genes in DNA sequences 1-4 as shown in SEQ ID No. 1-4, but there are some variations, and the homology of the four gene sequences with DNA sequences 1-4 is 90.3-100%.

[0040] Example 3: Determination of the nitrogen fixation effect of peanut ARC microbial inoculant. Taking peanuts as an example, the determination steps for the nitrogen fixation effect of the above-mentioned ARC microbial inoculant are described as follows.

[0041] On one hand, the bacterial agent from Table 2 of Example 1 above was co-cultured with the toxin-producing Aspergillus flavus strain under the same conditions, and the expression level of PAB-01 was measured. The inhibition rate of the bacterial agent on PAB-01 expression was calculated, and the results are shown in Table 3. The method for measuring the inhibition rate of Aspergillus flavus PAB-01 protein expression can be found 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.

[0042] On the other hand, the microbial agents 1-12 in Table 2 of Example 1 were applied to the field along with the basal fertilizer for peanut sowing, or they could be applied during the peanut growing season. The cumulative application rate was greater than or equal to 80 billion live bacteria per acre. A control plot was set up where none of the above microbial agents were applied, while conventional field management was used in all other plots. The root nodulation of peanut seedlings was investigated 7 to 12 days after emergence; the root nodulation of peanuts at maturity was investigated 1 to 3 days before harvest; and the virus control effect was investigated from flowering and pegging stage to around harvest, i.e., the field disease control effect and the field control effect based on the abundance of Aspergillus flavus in peanuts after harvest. The results are shown in Table 3.

[0043] Based on the above results, inoculants 1-12 simultaneously possess the following characteristics: 1) The inoculants contain all four gene sequences in DNA sequences 1-4, and these genes may have base variations of no more than 10% in different strains; 2) The inoculants have a significant inhibitory effect on the protein expression level of Aspergillus flavus PAB-01, an inhibitory effect on Aspergillus flavus, and an inhibitory effect on one or more pathogenic factors / toxins in soil-borne plant pathogens such as Penicillium, Aspergillus flavus, Fusarium, and Ralstonia solanacearum; 3) They are not rhizobia themselves, but ARC microbial inoculants can simultaneously regulate and increase the abundance of rhizobia in the peanut rhizosphere and increase the number of nodules on peanut roots; 4) They can promote early nodulation in peanuts and prolong the nodulation and nitrogen fixation time.

[0044] The aforementioned soil-borne plant pathogens are the pathogenic microorganisms of the corresponding field diseases. Therefore, the field disease control effect is equivalent to the inhibitory effect of ARC microbial agents on the pathogenic factors / toxins of the aforementioned soil-borne plant pathogens.

[0045] Table 3. Results of the determination of the effect of microbial inoculants on nitrogen fixation and virus control in peanuts.

[0046] The above-mentioned microbial agents have an inhibition rate of over 60% against Aspergillus flavus / toxins, and further over 90%; the inhibition rate against one or more pathogenic factors / toxins of soil-borne plant pathogens other than Aspergillus flavus, such as Penicillium, Fusarium, and Ralstonia solanacearum, can reach at least 30%, preferably over 60%, and even more preferably over 80% or 90%.

[0047] Example 4: Determination of the nitrogen-fixing effect of soybean microbial inoculants Taking soybeans as another example, the steps for determining the nitrogen-fixing effect of the above-mentioned microbial agents are described below.

[0048] On the one hand, the bacterial agent in Table 2 of Example 1 above was co-cultured with the toxin-producing Aspergillus flavus strain under the same conditions, and the expression level of PAB-01 was determined by the literature method, and the inhibition rate of the bacterial agent on the expression of PAB-01 was calculated. The results are shown in Table 3.

[0049] On the other hand, the microbial agents 1-12 in Table 2 of Example 1 were applied to the field along with the soybean sowing basal fertilizer, or they could be applied to the field during the soybean growth period, with a cumulative application rate of ≥80 billion live bacteria per acre. A control plot was set up where none of the above microbial agents were applied, while all other plots were managed using conventional field practices. Nodulation of soybean seedling roots was investigated 7-12 days after emergence; nodulation of soybean roots at maturity was investigated 1-3 days before harvest; and the control effect (i.e., the control effect against pathogenic toxins) was investigated before harvest. The results are shown in Table 4.

[0050] Based on the above results, inoculants 1-12 simultaneously possess the following characteristics: 1) ARC microbial inoculants contain all four gene sequences in DNA sequences 1-4, and these genes may have base variations of no more than 10% in different strains; 2) The inoculants have a significant inhibitory effect on the protein expression level of Aspergillus flavus PAB-01, inhibit Aspergillus flavus, and inhibit soil-borne plant pathogens such as Aspergillus, Fusarium, Sclerotium sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani, in addition to Penicillium and Aspergillus flavus; 3) They are not rhizobia themselves, but the inoculants can simultaneously regulate and increase the abundance of rhizobia in the soybean rhizosphere and increase the number of nodules on soybean roots; 4) The inoculants can promote early nodulation in soybeans and prolong the nodulation and nitrogen fixation time.

[0051] The aforementioned soil-borne plant pathogens are the pathogenic microorganisms of the corresponding field diseases. Therefore, the field disease control effect is equivalent to the inhibitory effect of ARC microbial agents on the pathogenic factors / toxins of the aforementioned soil-borne plant pathogens.

[0052] Table 4. Results of the determination of the effect of microbial inoculants on nitrogen fixation and toxin control in soybeans.

[0053] The above-mentioned microbial agents have an inhibition rate of over 60% against Aspergillus flavus / toxins, and further over 90%; the inhibition rate against one or more pathogenic factors / toxins of soil-borne plant pathogens other than Aspergillus flavus, such as Fusarium, Sclerotium sclerotiorum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani, can reach at least 30%, preferably over 60%, and even more preferably over 80% or 90%.

[0054] Example 5: Determination of the nitrogen fixation and toxin control effects of microbial inoculants on other leguminous crops The sampling and sampling procedures were the same as those in Examples 3 and 4 to determine the nitrogen fixation and toxin control effects of ARC microbial agents 1-8 on peas, broad beans, cowpeas, alfalfa, and other legumes. Similar results were obtained as shown in Tables 3 and 4.

[0055] Part Two: Uses of ARC Microbial Inoculants – For Shortening Internode Spacing and Increasing Pod Number in Soybeans The aforementioned ARC microbial inoculants 1-8 were mixed with the soybean basal fertilizer and applied to the field using a seeder. The cumulative application rate was 80-100 billion live bacteria per acre. A control plot was set up without any of the above inoculants applied, while other plots followed conventional field management. The number of soybean pods was assessed at harvest. These results showed that soybeans treated with inoculants 1-8 had an average internode spacing more than 4.2 mm shorter than the control, an average increase in the number of soybean pods of more than 9.5%, and a yield increase of more than 16.7%. These results indicate that the application of ARC microbial inoculants can significantly shorten the internode spacing of soybeans, increase the number of soybean pods, and thus greatly improve yield.

[0056] When soybeans are planted, the above-mentioned ARC microbial agents are applied in soybean production by means of manual broadcasting or drone broadcasting, and similar effects of significantly shortening the internode spacing and increasing the number of pods are achieved.

[0057] Applying the aforementioned ARC microbial inoculant during the period from soybean emergence to flowering and pod formation also yielded similar results, significantly shortening internode spacing and increasing the number of pods.

Claims

1. The ARC microbial agent is used to shorten the internode spacing of soybeans and increase the number of pods. The ARC microbial agent is a composition of the following four microorganisms: Bacillus lateralis (CCTCC NO: M 20231815), Bacillus amyloliquefaciens (CCTCC NO: M 20231598), Bacillus mucilaginosus (CCTCC NO: M 20231817), and Enterobacter ludwigii (CCTCC NO: M 20231595). The ARC microbial agent has a combined effect of controlling toxins and fixing nitrogen, and can regulate and increase the abundance of rhizobia in the rhizosphere of leguminous crops, increase the number of nodules in leguminous crops, and promote early nodulation and prolong the nodulation and nitrogen fixation time in leguminous crops.

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

3. The use according to claim 1, characterized in that: The ARC microbial agent described above has an inhibitory effect on Aspergillus flavus and / or its toxins.

4. The use according to claim 1, characterized in that: The ARC microbial agent described herein has an inhibitory effect on one or more pathogens selected from Penicillium, Fusarium, Sclerotinia sclerotiorum, Ralstonia solanacearum, Phytophthora, Sclerotinia sclerotiorum, Pythium, and Rhizoctonia solani and / or the toxins produced by the pathogens.

5. A method for shortening internode spacing and increasing pod number in soybeans, the method comprising applying an ARC microbial agent to soybean production, wherein the ARC microbial agent is a composition of four microorganisms: *Bacillus laterosporus* (accession number CCTCC NO: M 20231815), *Bacillus amyloliquefaciens* (accession number CCTCC NO: M 20231598), *Bacillus mucilaginosus* (accession number CCTCC NO: M 20231817), and *Enterobacter ludwigii* (accession number CCTCC NO: M 20231595), wherein the ARC microbial agent simultaneously exhibits a nitrogen fixation and toxin control coupling effect, thereby regulating and increasing the abundance of rhizobia in the rhizosphere of leguminous crops, increasing the number of nodules in leguminous crops, and promoting early nodulation and prolonging the nodulation and nitrogen fixation time in leguminous crops.

6. The method according to claim 5, characterized in that: The application rate of the ARC microbial agent is 80 billion to 100 billion live bacteria per acre.

7. The method according to claim 5, characterized in that: The application method is one or a combination of broadcasting, spraying, and drip irrigation, and the application stage is at soybean sowing and / or after soybean emergence to flowering.

8. The method according to claim 5, characterized in that: The method involves mixing the ARC microbial inoculant with the soybean sowing base fertilizer and applying it evenly to the field using one or more methods, such as manual labor, a seeder, a drone, or drip irrigation. The cumulative inoculant application rate is 80 billion to 100 billion live bacteria per acre. Alternatively, after soybeans have emerged from normal sowing and until the flowering and pod-setting stage, apply ARC microbial agents evenly to the field through one or more methods, such as broadcasting, spraying, or drip irrigation, with a cumulative application rate of 80 billion to 100 billion live bacteria per acre.

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