Complex microbial inoculant for promoting nodulation performance of peanuts as well as preparation method and application of complex microbial inoculant
By screening and combining bacteria such as Pantotheca dispersalis, Microbacterium chocolate, Agrobacterium radiodurans, and Slow-growing Rhizobium yunnanense, the problem of unstable effects of compound microbial agents in peanut cultivation has been solved, resulting in increased peanut nodulation rate and yield. This provides a high-efficiency and low-cost compound microbial agent solution.
Patent Information
- Application Number
- CN202511223345.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2026-01-27
AI Technical Summary
Existing compound microbial agents have problems with fluctuating effects and overlapping microbial functions in peanut cultivation, making it difficult to maintain stability and high efficiency in the soil, thus affecting peanut nodulation rate and yield.
By employing a combination of bacteria such as Pantotheca dispersalis, Microbacterium chocolate, Agrobacterium radiodurans, and Slow-growing Rhizobium yumingense, a highly efficient compound bacterial agent was developed through high-throughput sequencing and pure culture screening. This ensures that there is no antagonistic effect between the strains and that they can coexist stably, thereby promoting peanut nodulation.
It significantly improves the agronomic traits and active root nodule ratio of peanuts, enhances the efficiency and yield of peanut symbiotic nitrogen fixation, and has low operating costs and is easy to scale up for production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology and biofertilizers, specifically relating to a compound microbial agent that promotes peanut nodulation performance, its preparation method, and its application. Background Technology
[0002] Peanuts are a widely cultivated oilseed and cash crop worldwide, grown in over 100 countries, and hold significant agricultural and economic importance. Stable peanut production is crucial for increasing farmers' income and developing social trade. Peanuts are highly nutritious, rich in high-quality protein and fat, particularly known for their high content of unsaturated fatty acids, offering multiple health benefits including nutritional, medicinal, and health-promoting properties.
[0003] In the hilly regions of southern China, due to limited arable land resources and the promotion of intensive farming methods, peanuts have been continuously planted on the same plot of land for over 20 years. This continuous cropping leads to acidification of the red soil, causing imbalances in the soil microbiome, deterioration of soil properties, and loss of available nutrients, ultimately resulting in low peanut nodulation rates and reduced yields. Currently, agriculture often uses chemical fertilizers and pesticides to alleviate the problems caused by continuous cropping; however, these measures lead to significant environmental pollution and increased production costs.
[0004] Microbial preparations, represented by probiotics (such as plant growth promoters and biocontrol bacteria), have gradually become a research focus due to their environmental friendliness, high safety, and high efficacy. Currently, single-strain preparations face application limitations due to their strong functional specificity, making the development of multi-strain synergistic compound microbial agents a significant trend in the field of biofertilizers. However, existing compound microbial agents often employ simple strain stacking strategies, which are prone to fluctuations in efficacy and functional overlap among microbial communities. Although breakthroughs in high-throughput sequencing technology and synthetic microbial community construction methods have provided theoretical support and strain resource libraries for microbial agent design based on the principle of functional complementarity, ensuring the maximization of strain interaction efficacy and field stability in compound microbial preparations remains a core bottleneck restricting their industrial application. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a compound microbial agent for promoting peanut nodulation performance, its preparation method, and its application. It combines microbiome sequencing technology from 33 peanut-growing areas in China with pure culture isolation and screening of bacteria. The process is simplified using core microbial community screening criteria, selecting bacteria with functional advantages for combination to obtain a highly efficient and stable bacterial combination for promoting peanut nodulation performance. Finally, pot experiments are used to verify the actual growth-promoting and nodulation-promoting effects of this compound microbial agent.
[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:
[0007] A compound microbial agent for promoting peanut nodulation performance is composed of Pantoea dispersa (Pd), Microbacterium chocolatum (Mc), Agrobacterium radiobacter (Ar), and Bradyrhizobium yuanmingense (Br) in equal volume ratios; the compound microbial agent contains 1×10⁻⁶ effective viable bacteria. 7 ~10 9 CFU, of which Pantotheca dispersa was deposited at the China Center for Type Culture Collection (CCTCC) on May 20, 2025, with accession number CCTCC NO: M20251133 and taxonomic name Pantotheca adenophora Pd; Microbacterium chocolatum was deposited at the CCTCC on May 12, 2025, with accession number CCTCC NO: M20251018 and taxonomic name Microbacterium chocolatum Mc; Agrobacterium radiobacterium was deposited at the CCTCC on May 19, 2025, with accession number CCTCC NO: M20251016 and taxonomic name Agrobacterium radiobacterAr; and Slow-growing rhizobium yunnanensis was deposited at the CCTCC on May 19, 2025, with accession number CCTCC. NO: M20251017, taxonomically named Bradyrhizobiumyuanmingense Br.
[0008] As an improvement, the colonies of the dispersed pantothenic bacteria are round, with smooth, wavy edges, and are transparent.
[0009] As an improvement, the colonies of the chocolate-colored microbacterium are round, with neat and smooth edges, and are translucent.
[0010] As an improvement, the colonies of the *Rhizobium yunnanense* are round with smooth, regular edges and are translucent.
[0011] As an improvement, the colonies of the *Agrobacterium radioactivee* are round with smooth, neat edges, and the mucilaginous colonies have a distinct luster.
[0012] The preparation method of the above-mentioned compound bacterial agent promoting peanut nodulation performance is as follows: Pantoea dispersa (Pd), Microbacterium chocolatum (Mc), Agrobacterium radiobacter (Ar), and Bradyrhizobium yuanmingense (Br) are inoculated into R2A medium, shake-cultured until the absorbance at OD 600nm is 1.0, washed with PBS and resuspended, so that the OD of each bacterium is... 600 After maintaining the value at the 0.005–0.01 level, mix in equal volume ratios to achieve a viable count of 1 × 10⁻⁶. 7 ~10 9 CFU, that is, you get it.
[0013] The above-mentioned compound microbial agent for promoting peanut nodulation is applied to the promotion of peanut growth.
[0014] The above-mentioned compound microbial agent for promoting peanut nodulation is used to promote peanut root nodule activity (increase the proportion of active root nodules).
[0015] Preferably, the above-mentioned compound microbial agent that promotes peanut nodulation is applied to the roots of peanut seedlings.
[0016] Preferably, the procedure specifically includes the following steps:
[0017] Select peanut seedlings that have germinated for 3 days and have roots 3cm long, soak them in the peanut growth-promoting and disease-resistant compound microbial agent as described in claim 1 for 3-5 hours, transplant them into the soil, and irrigate the roots with the compound microbial agent at a rate of 10mL / plant. Irrigate the roots with the compound microbial agent again at 7 and 14 days later, at a rate of 15mL / plant.
[0018] Beneficial effects:
[0019] This invention discloses a compound microbial agent for promoting peanut nodulation, its preparation method, and its application. It develops a simplified method for screening and synthesizing functional compound microbial agents, simplifying in vitro strain functional screening and community synthesis based on large-scale high-throughput data. This overcomes the problems of low adaptability and functional redundancy in soil caused by simple compounding of functional strains in previous methods. The strains of this invention can significantly improve the agronomic traits and active nodule ratio of peanuts. The screened and simplified compound microbial agent has a significant effect on promoting nodule activity in peanuts, providing an effective compound microbial agent for improving peanut symbiotic nitrogen fixation efficiency and yield. Furthermore, this invention has low operating costs, is universally applicable, and is easy to scale up for production. Attached Figure Description
[0020] Figure 1This describes the screening process for each member of the functional synthetic community and the control synthetic community in Example 1;
[0021] Figure 2 The phylogenetic tree for each strain in Example 1, constructed based on the 16S gene sequence, is as follows: ASV1836 is Pantoea dispersa; ASV31124 is Microbacterium chocolatum; ASV5 is Agrobacterium radiobacter; and ASV1 is Bradyrhizobium yuanmingense.
[0022] Figure 3 This is a graph showing the nitrogen fixation, phosphorus solubilization, and enzyme production capabilities that promote root nodule activity of each strain in Example 2.
[0023] Figure 4 This is a diagram showing the antagonistic interactions between the strains in the R2A fermentation broth pairing experiment in Example 2;
[0024] Figure 5 The graph shows the effect of the functional synthetic community treatment on peanut growth compared with the control group in Example 3. In the graph, (a) shows the change in plant biomass, (b) shows the change in plant nitrogen content, and (c) shows the change in plant soluble sugar content.
[0025] Figure 6 The diagram shows the effect of the functional synthetic community treatment on peanut nodulation in Example 3 compared with the control group. (a) shows the expression of nodulation genes; (b) shows the change in root nodule biomass; (c) shows the proportion of active root nodules; (d) shows the change in root nodule volume; and (e) shows the change in plant hemoglobin content.
[0026] Figure 7 The figure shows the yield of peanuts per plant in each treatment group in Example 3. (a) is the yield of Tianfu 16, (b) is the yield of Sunonghua 1, and (c) is the yield of Qianyou 1. Detailed Implementation
[0027] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0028] Example 1: Isolation and Screening of Functional Bacteria
[0029] Analysis of high-throughput sequencing results of endophytic bacteria from peanut root nodules in 33 farmlands in China (sequencing work was handled by Shanghai Meiji Biotechnology Co., Ltd.) revealed four amplicon sequence variants (ASVs) based on three screening criteria: conserved presence in all agroecological zones, significant enrichment in highly active root nodules, and a core position in the co-occurrence network of root nodules endophytic bacteria.
[0030] The nucleic acid sequences of the four amplicon sequence variants are shown below:
[0031] ASV1 (Bradyrhizobium sp.):
[0032] GTAGTCCACGCCGTAAACGATGAATGCCAGCCGTTAGTGGGTTTACTCACTAGTGGCGCAGCTAACGCTTTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGCAGAACCTTACCAGCC CTTGACATGTCCAGGACCGGTCGCAGAGATGTGACCCTCTCTTCGGAGCCTGGAGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCGTCCTTAGTTGCTACCATTTAGTTGAGCACTCTAAGGAGACTGCCGGTGATAAGCCGCGA
[0033] ASV5 (Agrobacterium sp.):
[0034] GTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCTCTTGACATTCGGGGTTTGGGCAGTGGAGACATTGTCCTTCAGTTAGGCTGGCCCCAGAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGA
[0035] ASV1836(Pantoea sp.):
[0036] GTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGA
[0037] ASV31124(Microbacterium sp.):
[0038] GTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCTTTCCACGGATTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCA AGGCTTGACATACACGAGAACACCCCAGAAATGGGGGACTCTTTGGACACTCGTGAACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCACGTTATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGA
[0039] By combining pure cultures of root nodule endophytic bacteria, root nodule endophytic bacterial strains with 100% sequence similarity to the aforementioned four ASVs were successfully isolated. Simultaneously, three endophytic bacteria strains that did not significantly accumulate in highly active root nodules were isolated: *Rahnella aquatilis*, *Xanthomonas sacchari*, and *Micromonospora* sp. All three strains are commercially available products.
[0040] The R2A medium used for activating the bacterial strain comprises the following components: 0.5g yeast extract, 0.5g peptone, 0.5g acid-hydrolyzed casein, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, and distilled water to a final volume of 1000mL, pH=7.2. If preparing a solid medium, add 15-20g of agar.
[0041] 1. Bioinformatics analysis of high-throughput data to identify potential functional microorganisms
[0042] (1) Screening of conserved root nodule endophytic bacteria in seven agricultural ecoregions in China
[0043] The original sequencing data were divided into seven subsets according to the distribution map of agricultural regions in China (data sourced from the Geographic Data Platform of the College of Urban and Environmental Sciences, Peking University (http: / / geodata.pku.edu.cn)). The Venn Analysis toolkit of the cloud platform of Meiji Biotechnology Co., Ltd. was used to perform Venn analysis on the ASV abundance table after high-throughput sequencing grouping, and root nodule endophytic bacterial taxa that are conserved in the seven agricultural ecological regions of China were screened at the genus level.
[0044] (2) Screening of endophytic bacteria in active root nodules that are significantly enriched
[0045] The color of the root nodule cross-section was observed under a microscope. The CYMK (printing four-color) parameters of the root nodule cross-section were determined using ImageJ software, and the samples were divided into inactive root nodules (white) and highly active root nodules (dark red). The Kruskal-Wallis Htest was used to screen for ASV taxa with significantly increased abundance in active root nodules.
[0046] (3) Screening of core species of endophytic bacteria in root nodules
[0047] By using the "Zi-Pi Analysis" cloud platform of Meiji Biotechnology Co., Ltd., the Zi (inter-module connectivity) value and Pi (inter-module connectivity) value of each ASV were calculated. Nodes with Zi > 2.5 were identified as module hubs, while nodes with Pi > 0.62 were identified as connection points. The above module hubs and connection points were identified as the core species of the root nodule endophytic bacterial community.
[0048] (4) ASVs that meet all three screening criteria are identified as potential functional strains of root nodule endophytic bacteria. The strains obtained from pure culture are compared with the strain sequences to determine the final functional synthetic community (SynCom).
[0049] (5) Randomly select root nodule endophytic strains from the ASV set that did not meet the functional strain screening criteria as the control group synthetic community (SynCtrl).
[0050] 2. Identification of the strain
[0051] (1) Strains meeting and not meeting the screening criteria were molecularly identified, and their 16S genes were amplified by PCR. PCR products were bidirectionally sequenced by Beijing Qingke Biotechnology Co., Ltd. (Nanjing). The obtained sequences were compared with homologous sequences in GenBank using NCBI-Blast. 16S genes of closely related species were downloaded from GenBank for phylogenetic analysis. The downloaded Serratia sp. was considered an outgroup.
[0052] (2) Use MEGA 8.0 to construct a Bayesian phylogenetic tree.
[0053] 3. Results and Analysis
[0054] Based on bioinformatics analysis of the high-throughput sequencing results, a total of 4 functional bacteria that met the screening criteria (ASV1, ASV1836, ASV5, ASV31124) and 3 control bacteria that did not meet the screening criteria (ASV49941, ASV45227, ASV22899) were screened. Figure 1 As shown.
[0055] After sequence alignment with the bacteria in the pure culture experiment, the above 7 potential functional strains were obtained. Note that ASV1 is Bradyrhizobium yuanmingense. To ensure peanut nodulation, subsequent experiments were all pre-inoculated with Bradyrhizobium yuanmingense to ensure that peanuts could form nodules.
[0056] Phylogenetic analysis such as Figure 2 As shown, ASV1 was identified as Bradyrhizobium sp., ASV49941 was identified as Rahnella aquatilis, Rahnella sp. ASV1836 was identified as Pantoea dispersa, Pantoea sp. ASV45227 was identified as Xanthomonas sacchari, Xanthomonas sp. ASV5 was identified as Agrobacterium radiobacter, Agrobacterium sp. ASV22899 was identified as Micrococcus, Micromonospora sp. ASV31124 was identified as Microbacterium chocolatum, Microbacterium sp.
[0057] A compound microbial agent for promoting peanut nodulation, comprising an equal volume ratio of Pantoea dispersa (Pd), Microbacterium chocolatum (MC), Agrobacterium radiobacter (Ar), and Bradyrhizobium yuanmingense (Br); the compound microbial agent contains 1 × 10⁻⁶ effective viable bacteria. 7 ~10 9CFU, of which Pantoea dispersa Pd. was deposited at the China Center for Type Culture Collection (CCTCC) on May 20, 2025, with accession number CCTCC NO: M20251133 and taxonomic name Pantoea dispersa Pd.; Microbacterium chocolatum Mc. was deposited at the CCTCC on May 12, 2025, with accession number CCTCC NO: M20251018 and taxonomic name Microbacterium chocolatum Mc.; Agrobacterium radiobacterium Ar. was deposited at the CCTCC on May 19, 2025, with accession number CCTCC NO: M20251016 and taxonomic name Agrobacterium radiobacter Ar.; and Slow-growing rhizobium yumingensis was deposited at the CCTCC on May 19, 2025, with accession number CCTCC. NO: M20251017, taxonomically named Bradyrhizobium yuanmingense Br.
[0058] The dispersed pantothenic colonies are round, with smooth, wavy edges, and are transparent.
[0059] The colonies of the chocolate-colored microbacterium are round, with neat and smooth edges, and are translucent.
[0060] The colonies of the *Rhizobium glomeratum* are round, with neat and smooth edges, and are semi-transparent.
[0061] The colonies of *Agrobacterium radioactivee* are round with smooth, neat edges, and the mucilaginous colonies have a distinct luster.
[0062] Example 2: Construction of Synthetic Communities
[0063] 1. Determination of the tested strains' ability to dissolve organic and inorganic phosphorus and potassium, nitrogen fixation capacity, and siderophore production capacity.
[0064] (1) Prepare PKO inorganic phosphorus culture medium according to the following formula:
[0065] 10g glucose, 5g calcium phosphate, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 0.03g ferrous sulfate heptahydrate, 15-20g agar, distilled water to a final volume of 1000mL, pH=7.
[0066] (2) Prepare the organic phosphorus culture medium according to the following formula:
[0067] 10g glucose, 5g sodium phytate, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 0.03g ferrous sulfate heptahydrate, 15-20g agar, distilled water to a final volume of 1000mL, pH=7.0.
[0068] (3) Prepare silicate bacteria culture medium according to the following formula:
[0069] 5.0g sucrose, 0.5g magnesium sulfate, 0.1g calcium carbonate, 2.0g disodium hydrogen phosphate, 0.005g ferric chloride, 1.0g glass powder, 15-20g agar, distilled water to a final volume of 1000mL, pH=7.0.
[0070] (4) Prepare Ashby culture medium according to the following formula:
[0071] Potassium dihydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, calcium carbonate 5.0g, mannitol 10.0g, calcium sulfate 0.1g, agar 15-20g, distilled water to a final volume of 1000mL, pH=7.0.
[0072] (5) Prepare CAS culture medium according to the following formula:
[0073] Chromium azurite S 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, ferric chloride hexahydrate 2.6 mg, sodium dihydrogen phosphate dihydrate 295.3 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125.0 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 15-20 g, distilled water to a final volume of 1000 mL, pH = 7.0.
[0074] (6) Inoculate the test strain into R2A medium and culture in a shake flask until the logarithmic growth phase. Collect the bacterial cells by centrifugation, wash three times with sterile PBS and resuspend the bacterial cells until the bacterial suspension reaches OD500. 600 =1. Each bacterial strain was inoculated into the above five culture media at an inoculum of 1%, and cultured at 28°C for 7 days. The changes in the phosphate-solubilizing zone, potassium-solubilizing zone, and color of the CAS medium were observed to evaluate the bacterial phosphate-solubilizing, potassium-solubilizing, and siderophore-producing abilities. The colony diameter of bacteria on Ashby medium was observed to evaluate whether the bacteria had nitrogen-fixing ability.
[0075] 2. Determination of auxin, cytokinin, and ACC deaminase production by the tested strains.
[0076] (1) Detection of the strain's ability to produce auxin
[0077] The strain was cultured for 3 days at 28°C and 180 rpm in R2A medium supplemented with 0.1 g L 1 L tryptophan. Then, 1 mL of the supernatant (obtained by centrifugation at 10000 rpm for 5 minutes) was mixed with 2 mL of Salkowsky's reagent (150 mL concentrated sulfuric acid, 250 mL distilled water, and 7.5 mL 0.5 M ferric chloride hexahydrate) and incubated in the dark for 20 minutes. The auxin concentration was determined colorimetrically at 530 nm.
[0078] (2) Detection of the strain's ability to produce cytokinins
[0079] The bacterial strain was cultured in R2A medium at 28°C and 180 rpm for 3 days. The cultured solution after 3 days was centrifuged at 5000 rpm for 10 min, and the supernatant was retained as the crude extract. The ACC deaminase activity and cytokinin concentration in the crude extract were detected using the bacterial ACC deaminase activity assay kit and the bacterial CTK ELISA kit from Jiangsu Enzyme Immunosorbent Assay Biotechnology Co., Ltd., respectively.
[0080] 3. Evaluation of antagonistic relationships among bacterial strains
[0081] An important principle for constructing artificially synthesized microbial communities is that the selected strains do not exhibit significant antagonistic effects and can establish a stable coexistence relationship. Therefore, to verify whether the seven candidate strains selected through initial screening in Example 1 can coexist amicably, this example conducted a strain fermentation broth pairing experiment.
[0082] Seven experimental bacterial strains were inoculated into 20 ml of R2A liquid medium and allowed to grow to the logarithmic growth phase. The bacterial cultures were centrifuged, and the supernatant was filtered through a 0.22 μm sterile filter to obtain the fermentation broth for each strain. Each strain was adjusted to the same bacterial concentration (OD). 600 =0.1), and inoculated into fermentation broth (1%, v / v) for all strains. As a control, each strain was inoculated into fresh R2A liquid medium. After incubation, the OD of all bacteria was measured. 600 The results were obtained by analyzing the OD values of the strains in different fermentation broths. 600 Dividing by the OD of the control culture medium 600 This indicates that each treatment is repeated three times.
[0083] Interactions between microorganisms are measured by OD 600 The OD was determined using fermentation broth / fresh R2A, and a t-test was used to determine the OD of three independent experiments. 600 Is the ratio of fermentation broth to fresh R2A significantly greater or less than 1? If OD 600If the ratio of fermentation broth to fresh R2A is significantly greater than or less than 1 (P<0.05), the interaction between the two strains is considered positive (+) or negative (-), respectively. In other cases (P>0.05), the interaction is considered neutral (0).
[0084] 4. Results and Analysis
[0085] Figure 3 The growth-promoting properties of seven bacteria were evaluated. In the evaluation of growth-promoting properties, three functional bacteria identified by high-throughput sequencing had a wider range of plant growth-promoting functions than the control strain. Dispersible pantothecin, chocolate-colored microbacterium, and agrobacterium radioactivity all showed significant abilities to produce auxin, cytokinin, siderophores, and dissolve organic phosphorus. Chocolate-colored microbacterium and dispersible pantothecin showed significant abilities to dissolve inorganic phosphorus, while chocolate-colored microbacterium and agrobacterium radioactivity showed significant nitrogen-fixing abilities.
[0086] Figure 4 The pairing experiment of the bacterial strain fermentation broth showed no obvious antagonistic relationship, indicating that there was no obvious antagonistic effect between any two bacteria, and subsequent co-inoculation experiments can be carried out.
[0087] Example 3: Application of synthetic communities in peanut growth and nodulation
[0088] 1. Pot Experiment Design
[0089] A pot experiment was conducted using sterile vermiculite, with a total of 4 treatment groups, as detailed in Table 1 below.
[0090] Table 1. Grouping of treatment groups
[0091]
[0092]
[0093] The test strain was inoculated into R2A medium and cultured overnight. The bacterial culture that reached the logarithmic growth phase was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were washed 2-3 times with sterile PBS. The bacterial culture was then resuspended at OD200. 600 =0.005~0.01, to prepare single-cell suspensions. Mix the above single-cell suspensions in equal (volume) proportions to obtain an effective viable count of 1×10. 7 ~10 9 CFU was used to prepare a compound bacterial suspension.
[0094] Peanut seedlings that had germinated for 3-4 days and had roots approximately 5cm in length were selected and soaked in the inoculum solutions for 3-5 hours. They were then transplanted into the tested red soil, and the roots were irrigated with the inoculum solution at a rate of 10mL per plant. The compound microbial agent was applied twice, at 7 and 14 days later, at a rate of 15mL per plant. During the later management phase, the same watering rate was maintained, watering once every 2-3 days. Samples were taken 42 days after the initial inoculation treatment to analyze the growth-promoting effect and root nodule performance. This example used red soil from Jiangxi province (peanuts continuously cropped for over 10 years).
[0095] 2. Measurement of peanut growth traits and nutritional status
[0096] Nine peanut plants were randomly selected from each treatment group, and their above-ground and underground dry weight, nitrogen content, and soluble sugar content were measured.
[0097] 3. Peanut nodulation performance testing
[0098] To determine the effect on promoting nodulation, after peanut samples were harvested at 42 days, the expression of nodulation genes (primers: AhCyclops-F: 5'-GAGAGCCCTATTGGAATGCCAGT-3'; AhCyclops-R: 5'-TCGCCTGTTAAGTCCCATGCTT-3'), nodule biomass, nodule number, nodule volume, and nodule hemoglobin content were statistically analyzed. All of these testing methods are commonly used techniques in this field.
[0099] 4. Peanut yield determination in outdoor pot experiments
[0100] Three commercially available peanut varieties with different nodulation abilities were planted in the same soil and planting environment (Qianyou No. 1 had a weak nodulation ability, Sunonghua No. 1 had a moderate nodulation ability, and Tianfu No. 16 had a strong nodulation ability). Three treatments were designed for each peanut variety in pot experiments, as shown in Table 2. This example used red soil from Jiangxi province (peanuts continuously cropped for more than 10 years). The experimental procedures were the same as described above. After harvesting mature peanuts, the dry weight of peanut pods per plant was recorded.
[0101] Table 2 Grouping of Treatment Groups
[0102]
[0103]
[0104] 4. Results and Analysis
[0105] (1) The effects of synthetic communities on peanut growth and nutritional status
[0106] Figure 5To compare peanut growth across treatment groups, (a) shows the effect of different treatments on plant biomass, (b) shows the effect of different treatments on plant nitrogen content, and (c) shows the effect of different treatments on plant soluble sugar content. The figures show that the SynCom community significantly promoted peanut growth during flowering, with the levels of the SynCom treatments inoculated with *S. truncatula* and the control group showing the same effect. The SynCom community significantly increased the soluble sugar content in peanut leaves, while the SynCom treatments inoculated with *S. truncatula* and the control group had no promoting effect on the soluble sugar content in peanut leaves.
[0107] (2) The promoting effect of synthetic communities on peanut nodulation
[0108] Figure 6 The nodulation indicators of peanuts in different treatment groups were compared. As can be seen from the figure, the SynCom community can effectively improve the nodulation gene expression (a), root nodule biomass (b), proportion of active root nodules (c), root nodule volume (d), and hemoglobin content (e), thus improving the root nodulation status.
[0109] (3) The promoting effect of synthetic communities on peanut yield
[0110] Figure 7 The dry weight of peanut pods in each treatment group was compared. As can be seen from the figure, the SynCom community can increase peanut yield. (a) shows the effect of different treatments on the yield of Tianfu 16, (b) shows the effect of different treatments on the yield of Sunonghua 1, and (c) shows the effect of different treatments on the yield of Qianyou 1.
[0111] In summary, this invention provides a compound microbial agent for promoting peanut nodulation, its preparation method, and its application. It develops a simplified method for screening and synthesizing functional compound microbial agents, simplifying in vitro strain functional screening and community synthesis based on large-scale high-throughput data. This overcomes the problems of low adaptability and functional redundancy in soil caused by simple compounding of functional strains in previous methods. The strains of this invention can significantly improve the agronomic traits and active nodule ratio of peanuts. The screened and simplified compound microbial agent has a significant effect on promoting nodule activity in peanuts, providing an effective compound microbial agent for improving peanut symbiotic nitrogen fixation efficiency and peanut yield.
Claims
1. A compound microbial agent for promoting peanut nodulation, characterized in that, The compound microbial agent consists of dispersed pantothecin ( Pantoea dispersa Pd), chocolate-colored microbacteria ( Microbacterium chocolatum , Mc), Agrobacterium radioactivee ( Agrobacterium radiobacter Ar), Yuanming slow-growing root nodules ( Bradyrhizobium yuanmingense The compound microbial agent is composed of equal volume ratios of ,Br; the effective viable bacteria count in the compound microbial agent is 1×10⁻⁶. 7 ~10 9 CFU, of which *Ureaplasma dispersans* was deposited at the China Center for Type Culture Collection on May 20, 2025, with accession number CCTCC NO: M20251133, and its taxonomic name is... Pantoea dispersa Pd; The described *Microbacterium chocolateii* was deposited at the China Center for Type Culture Collection on May 12, 2025, with accession number CCTCC NO: M20251018, and its taxonomic name is Microbacterium chocolatum Mc; The *Agrobacterium radiophyllum* described was deposited at the China Center for Type Culture Collection on May 19, 2025, with accession number CCTCC NO: M20251016, and its taxonomic name is... Agrobacterium radiobacter Ar; The *Rhizobium yunnanense* was deposited at the China Center for Type Culture Collection on May 19, 2025, with accession number CCTCC NO: M20251017, and its taxonomic name is... Bradyrhizobium yuanmingense Br.
2. The compound microbial agent for promoting peanut nodulation and antibacterial properties according to claim 1, characterized in that, The colonies of the dispersed pantothecin are round, with smooth, wavy edges, and are transparent.
3. The compound microbial agent for promoting peanut nodulation and antibacterial properties according to claim 1, characterized in that, The colonies of the chocolate-colored microbacterium are round, with neat and smooth edges, and are translucent.
4. The compound microbial agent for promoting peanut nodulation and antibacterial properties according to claim 1, characterized in that, The colonies of the *Rhizobium yunnanense* are round, with smooth and regular edges, and are semi-transparent.
5. The compound microbial agent for promoting peanut nodulation and antibacterial properties according to claim 1, characterized in that, The colonies of *Agrobacterium radioanalysus* are round with smooth, regular edges, and the mucilaginous colonies have a distinct luster.
6. A method for preparing a compound microbial agent for promoting peanut nodulation performance according to claim 1, characterized in that, Dispersed pantothecin ( Pantoea dispersa Pd), chocolate-colored microbacteria ( Microbacterium chocolatum Mc), Agrobacterium radioactivee ( Agrobacterium radiobacter Ar), Yuanming slow-growing root nodules ( Bradyrhizobium yuanmingense (Br) were inoculated separately into R2A medium and cultured in shake flasks until the absorbance at OD 600 nm was 1.
0. The cells were then washed with PBS and resuspended to adjust the OD of each bacterial species. 600 After maintaining the value at the 0.005~0.01 level, mix in equal volume ratios to achieve a viable count of 1×10⁻⁶. 7 ~10 9 CFU, that is, you get it.
7. The application of the compound microbial agent for promoting peanut nodulation as described in claim 1 in promoting peanut growth.
8. The application of the compound microbial agent for promoting peanut nodulation as described in claim 1 in promoting peanut root nodule activity.
9. The application according to claim 7 or 8, characterized in that, The compound microbial agent for promoting peanut nodulation as described in claim 1 is applied to the roots of peanut seedlings.
10. The application according to claim 9, characterized in that, Specifically, the following steps are included: Select peanut seedlings that have germinated for 3 days and have roots 3cm long, soak them in the compound microbial agent for promoting peanut nodulation as described in claim 1 for 3-5 hours, transplant them into the soil, and apply the compound microbial agent by root irrigation at a rate of 10 mL / plant. Repeat the application of the compound microbial agent at 7 and 14 days later, at a rate of 15 mL / plant.