Compound microbial agent for promoting development of plant lateral roots and soil holding capacity of root system and application thereof

Abstract

The application discloses a kind of composite microbial inoculant for promoting plant lateral root development and application thereof.The composite microbial inoculant contains Bacillus atrophaeus ED10 and Bacillus safensis HD9.Taking Arabidopsis thaliana as a model, the number of lateral roots can be increased by 53% and the number of true leaves can be increased by 8% by using the composite microbial inoculant, and the effect is stable (lateral root CV=0.36).Taking perennial ryegrass as a model for soil fixation, the root surface area density can be increased by 85%, the aboveground dry weight can be increased by 167%, and the specific root length can be reduced by 50% by using the composite microbial inoculant, the root system is optimized in the direction of more robust and high density, and the soil fixation potential is significantly enhanced.The composite microbial inoculant can promote plant lateral root development and optimize root architecture across families, and is suitable for slope protection, soil and water loss control, degraded grassland restoration and agricultural production, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to a compound microbial inoculant and its application, particularly to a compound microbial inoculant that promotes lateral root development and root-based soil stabilization in plants and its application. This invention belongs to the field of microbial technology. Background Technology

[0002] Currently, promoting plant growth using microbial inoculants is a research hotspot in agriculture and ecological restoration. Existing technologies disclose various functional bacterial strains, such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and potassium-solubilizing bacteria. These primarily promote the elongation of above-ground parts (plant height, leaf area) and taproots by increasing soil nutrient availability or secreting plant growth hormones (such as IAA). For example, some Bacillus strains have been reported to significantly increase plant fresh weight and taproot length. The screening and application of these inoculants mainly focus on increasing crop yield. However, existing technologies have the following shortcomings:

[0003] 1. Limited Function: Most microbial agents focus on promoting taproot growth and aboveground biomass accumulation, while research on functional agents that can effectively increase the number of lateral roots and build a well-developed fibrous root system is limited. A well-developed lateral root network has a more direct effect on improving plant lodging resistance, soil stabilization, and preventing soil erosion.

[0004] 2. Unstable efficacy: The efficacy of many single microbial agents is greatly affected by environmental factors such as soil type and climate conditions, resulting in poor reproducibility of field application effects and a lack of stability.

[0005] 3. Incomplete evaluation system: Existing studies often focus only on a single growth indicator (such as plant height or fresh weight), lacking a comprehensive evaluation of root system architecture (number of lateral roots), aboveground development (number of true leaves) and data stability, making it difficult to accurately evaluate the comprehensive potential of microbial agents in specific application scenarios (such as ecological restoration).

[0006] Therefore, there is an urgent need to develop a compound microbial agent that can promote the development of lateral roots and the soil-fixing ability of plant roots. Summary of the Invention

[0007] The purpose of this invention is to provide a compound microbial agent that can promote the development of lateral roots and the ability of roots to stabilize soil in plants.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] This invention discloses a compound microbial agent for promoting lateral root development and soil-fixing ability of plants. The compound microbial agent contains Bacillus atrophicus ED10 and Bacillus salsa HD9. The Bacillus atrophicus ED10 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38149, and the Bacillus salsa HD9 is deposited at the same center with accession number CGMCC No. 38150.

[0010] The *Bacillus atrophus* ED10 mentioned above mainly contributes to the construction of a stable root network in this invention. Experimental data show that composite groups containing this strain (such as ED10+HD9, ED10+WN7) all exhibit a significant increase in the number of lateral roots.

[0011] The *Bacillus salsa* HD9 strain described herein primarily contributes to maintaining the normal development of the aboveground parts of the plant. Experimental data show that complexes containing this strain (such as ED10+HD9) promote lateral root growth while also increasing the number of true leaves, thus avoiding growth imbalances caused by drastic changes in the root system.

[0012] The combination of the two strains produced a significant synergistic effect. While single-strain treatments (such as ED10 or HD9) showed advantages in some indicators (e.g., HD9 had a high fresh weight score, but its fresh weight CV was as high as 2.79, indicating extreme instability), they suffered from incomplete effects or poor stability. The ED10+HD9 combination group, however, demonstrated superior performance in both lateral root number (+53% compared to the control) and true leaf number (+8% compared to the control). Furthermore, the coefficients of variation for all indicators (root length CV = 0.19, lateral root CV = 0.36, fresh weight CV = 0.54) were among the lowest of all high-scoring combination groups, indicating a stable and reliable growth-promoting effect. This synergistic effect of promoting root growth and stability is not found in single strains or other combinations.

[0013] Preferably, the ratio of viable Bacillus atrophus ED10 and Bacillus saffron HD9 in the compound microbial agent is (1-2):(1-2).

[0014] More preferably, the ratio of viable Bacillus atrophus ED10 and Bacillus salsa HD9 in the compound microbial agent is 1:1.

[0015] Preferably, the composite microbial agent is prepared according to the following method:

[0016] Bacillus atrophus ED10 and Bacillus salsa HD9 were activated and inoculated into LB liquid medium, and cultured at 28°C and 180 rpm until the logarithmic phase. The bacterial concentration was adjusted to OD600=0.1 with sterile water and mixed at a volume ratio of (1-2):(1-2) to obtain the compound microbial agent.

[0017] Furthermore, the present invention also proposes the application of the aforementioned compound microbial inoculant in promoting the development of plant lateral roots and root system soil stabilization.

[0018] Furthermore, the present invention also proposes a method for promoting the development of lateral roots and the soil-fixing ability of plant roots, including the step of irrigating plants with the aforementioned compound microbial agent.

[0019] Compared with the prior art, the present invention has the following significant advantages:

[0020] 1. Significantly promotes lateral root development, optimizes root system architecture, and enhances soil-fixing capacity.

[0021] Using Arabidopsis thaliana as a model plant, treatment with the compound microbial agent (ED10+HD9) of this invention significantly increased the number of lateral roots by 53% and the number of true leaves by 8% compared to the control group. To further verify the effect on fibrous-rooted plants, a pot experiment was conducted using perennial ryegrass, a key grass species for ecological restoration in northern grasslands, as a model. The results showed that the root surface area of ​​the treated ryegrass was significantly increased by 85% compared to the control group, the root dry weight was increased by 104%, while the specific root length was significantly reduced by 50%. This result indicates that the compound microbial agent of this invention not only promotes the development of lateral roots in taprooted plants but also optimizes the root system architecture of fibrous-rooted plants, causing the roots to become thicker and have a higher surface area density, thereby significantly enhancing the contact and entanglement ability between the roots and soil particles. The 85% increase in root surface area density (a core indicator of soil-fixing capacity) proves that this agent can significantly improve the soil-fixing capacity and slope protection potential of plant roots, and has extremely high application value in ecological restoration fields such as slope protection and soil erosion control.

[0022] 2. Significantly promotes above-ground growth, achieving efficient "root-crown synergy".

[0023] While promoting root development, the compound microbial agent of this invention did not inhibit aboveground growth; on the contrary, it produced a significant synergistic growth-promoting effect. Experimental data on ryegrass showed that the aboveground dry weight of the treatment group increased by 167%, aboveground fresh weight by 137%, plant height by 30%, and tiller number by 37% compared to the control group. Although the root-to-shoot ratio decreased slightly (-24%), this was a result of the aboveground biomass increase (+167%) far exceeding the root increase (+104%), precisely demonstrating that the agent, while promoting root absorption, allocated more photosynthetic products to the aboveground parts, achieving a synergistic growth model of "healthy roots and robust seedlings." This ensures that the plant can maintain vigorous photosynthesis, providing sufficient energy support for deep root development and soil stabilization.

[0024] 3. The growth-promoting effect is stable and reliable, and it has excellent potential for field application.

[0025] Through systematic evaluation and stability analysis of multiple candidate strain combinations, the ED10+HD9 combination screened in this invention exhibited excellent stability and reproducibility. In Arabidopsis experiments, the coefficient of variation (CV) of lateral root number (CV=0.36) and fresh weight (CV=0.54) of this combination were significantly lower than those of single strains (e.g., HD9's fresh weight CV was as high as 2.79). In ryegrass experiments, the aboveground dry weight CV (0.524) and root dry weight CV (0.502) of the treatment group were both lower than those of the control group (0.595 and 0.555, respectively). These results indicate that the growth-promoting effect of the compound microbial agent of this invention is highly consistent among different plant individuals, exhibiting good environmental adaptability and reproducibility, and is suitable for large-scale field application.

[0026] 4. Broad-spectrum effectiveness across families and genera, with a wide range of applications.

[0027] This invention verified the root-promoting and growth-promoting effects of the microbial agent on a cruciferous model plant (Arabidopsis thaliana) and a gramineous soil-stabilizing plant (ryegrass). The results consistently showed that the compound microbial agent significantly optimized root architecture and increased aboveground biomass. This demonstrates that the microbial agent of this invention has broad applicability to different plant families and genera, and its application scope can cover taproot crops (such as soybeans, cotton, and rapeseed) and fibrous root crops (such as wheat, corn, and rice), as well as various herbaceous plants used for ecological restoration, showing broad industrialization prospects in agricultural production and ecological restoration. The above results collectively confirm that the microbial agent of this invention can promote lateral root development and enhance the soil-stabilizing capacity of the root system in different plants.

[0028] 5. Scientific methods for strain screening and efficacy evaluation were established.

[0029] This invention innovatively employs a multi-index weighted scoring method (root length, lateral root number / root surface area, aboveground development indicators, and biomass) combined with stability assessment (coefficient of variation) to comprehensively compare and screen candidate single strains and their combinations. This evaluation system overcomes the limitations of traditional single-index screening, accurately identifying strain combinations with synergistic effects, and providing new technical ideas and standardized procedures for the precise development of functional microbial agents.

[0030] Information on strain preservation:

[0031] 1. Strain name: ED10

[0032] Classification and naming: Bacillus atrophaeus

[0033] Preservation Institution: China General Microbiological Culture Collection Center (CGMCC)

[0034] Collection Number: CGMCC No. 38149

[0035] Preservation period: March 25, 2026

[0036] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0037] 2. Strain name: HD9

[0038] Classification and nomenclature: Bacillus safensis

[0039] Preservation Institution: China General Microbiological Culture Collection Center (CGMCC)

[0040] Collection Number: CGMCC No. 38150

[0041] Preservation period: March 25, 2026

[0042] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Attached Figure Description

[0043] Figure 1 Phylogenetic trees were constructed for ED10 and the type strain;

[0044] Figure 2 Phylogenetic trees were constructed for HD9 and the type strain;

[0045] Figure 3 The morphological characteristics of ED10 and HD9;

[0046] Figure 4The promoting effect of ED10 and HD9 on the lateral roots of Arabidopsis thaliana;

[0047] Figure 5 The effects of ED10 and HD9 on the aboveground parts and roots of ryegrass.

[0048] Where a is plant height; b is number of tillers; c is above-ground fresh weight; and d is above-ground dry weight. Detailed Implementation

[0049] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become clearer as a result. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0050] Example 1: Isolation and Identification of Target Strains

[0051] Sample collection: Natural soil and degraded soil samples were collected from five locations in Inner Mongolia: Holingol, Xilinhot, Ergun, Hulunbuir and Ulanqab. The samples were placed in sterile sampling bags and stored at 4°C for later use.

[0052] Strain isolation: Weigh 10g of soil sample, add it to 90mL of sterile water, shake for 30min to prepare a soil suspension. Perform serial dilutions of the soil suspension (10T). -3 Up to 10 -5 The samples were spread onto LB solid medium plates and incubated at 28°C for 24-48 hours.

[0053] Purification and Preservation: Based on colony morphology, color, size, and other characteristics, single colonies were selected and purified by streaking three times on LB agar plates. The purified single colonies were inoculated into LB liquid medium and incubated overnight at 28°C with shaking at 180 rpm. The bacterial suspension was then mixed with 50% sterile glycerol at a 1:1 ratio and stored at -40°C.

[0054] Molecular identification: The preserved bacterial culture was sent to a sequencing company for sequencing. The sequencing results were then compared with the NCBI database using BLAST. Figure 1 A phylogenetic tree was constructed for ED10 and the type strain. Figure 2Phylogenetic trees were constructed for HD9 and the model strain. The results showed that ED10 was Bacillus atrophaeus, HD9 was Bacillus safensis, WN7 was Microbacterium sp., and XN8 was Patulibacteramericanus.

[0055] Example 2: Evaluation of the effect of compound microbial inoculants on Arabidopsis growth

[0056] Preparation of bacterial suspension: ED10, HD9, WN7 and XN8 strains stored at -40℃ were activated by streaking on LB plates. Figure 3 To determine the morphological characteristics of ED10 and HD9, single colonies were picked and inoculated into LB liquid medium, and cultured at 28°C with shaking at 180 rpm until the logarithmic growth phase (OD600≈0.8-1.0). The bacterial concentration was then adjusted to OD600=0.1 with sterile water for later use.

[0057] Plant materials and culture: Plump and uniform Arabidopsis thaliana (Col-0 ecotype) seeds were selected, sterilized with 75% ethanol for 2 min, then sterilized with 5% sodium hypochlorite for 4-5 min, rinsed 7-8 times with sterile water, and sown on 1 / 2 MS solid medium plates. After vernalization at 4℃ for 2 days, the seeds were transferred to a light-controlled culture rack (16h light / 8h dark, 25℃) for cultivation. Five days after germination, seedlings with uniform growth were selected and transplanted into new 1 / 2 MS solid medium square petri dishes, with 6 seedlings per dish.

[0058] Inoculum treatment: The following treatment groups were set up (at least 5 replicate dishes in each group): (1) Control group (CK), 10 μL of sterile water was added to the roots of each seedling; (2) Single strain treatment group (ED10, HD9, WN7, XN8), 10 μL of the corresponding OD600=0.1 bacterial suspension was added to each seedling; (3) Paired compound treatment group (such as ED10+HD9, ED10+WN7, etc.), after mixing the two bacterial suspensions in equal volumes, 10 μL of the mixture was added to each seedling; (4) Triple and quadruple compound treatment group, the mixing method was the same as above. The culture dishes were placed vertically on the culture rack and cultured for 7 days.

[0059] Data Measurement and Standardization: Seven days later, the Arabidopsis plants were carefully removed, and the root length (mm), number of lateral roots, number of true leaves, and fresh weight (g*100) of each plant were measured and recorded. The raw data are shown in Table 1 below.

[0060] Table 1. Root length, number of lateral roots, number of true leaves, and fresh weight of Arabidopsis thaliana in each treatment group.

[0061]

[0062] Comprehensive Evaluation and Screening: All indicators were standardized (normalized with the maximum value of each indicator as 100%). A weighted scoring method was used to comprehensively evaluate each treatment group. The weights of each indicator were set according to their importance to the soil consolidation target as follows: root length 30%, lateral root number 30%, true leaf number 25%, and fresh weight 15%. The comprehensive score was calculated as: (standardized root length score × 0.3) + (standardized lateral root score × 0.3) + (standardized true leaf score × 0.25) + (standardized fresh weight score × 0.15). Simultaneously, the coefficient of variation (CV = standard deviation / mean) of each indicator for each treatment group was calculated to assess its stability. The comprehensive score and stability analysis results are shown in Table 2. The results show that the ED10+HD9 compound group has outstanding effects in promoting lateral roots (12.35±4.47, standardized score 0.82) and increasing true leaves (7.59±2.83, standardized score 0.85), and the data are relatively stable (root length CV=0.19, lateral root CV=0.36, fresh weight CV=0.54). It has the best overall performance and was selected as the optimal target fungal agent.

[0063] Table 2. Overall Score and Stability Analysis Results

[0064]

[0065] The isolated strains ED10 and HD9 were deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 38149 and CGMCC No. 38150, respectively.

[0066] Example 3: Validation and stability analysis of the optimal compound microbial inoculant (ED10+HD9)

[0067] Experimental design: Repeat all the operations in Example 2 regarding the control group, ED10 single-strain group, HD9 single-strain group, and ED10+HD9 compound group, and increase the sample size (no less than 20 replicate plants in each group).

[0068] Results Analysis: The results are as follows Figure 4 As shown in the figure. The results of the verification experiment are consistent with the trend of the initial screening results. Compared with the control group, the number of lateral roots in the ED10+HD9 treatment group increased by an average of 53%, and the number of true leaves increased by an average of 8%. Although root length (-64%) and fresh weight (-48%) decreased, the significant increase in the number of lateral roots is the core beneficial effect under the goal of ecological restoration, indicating that the root system architecture is being reshaped in a direction conducive to soil stabilization. At the same time, the coefficients of variation of various indicators of this combination are relatively small (root length CV=0.232, lateral root CV=0.295, fresh weight CV=0.324), which is much lower than that of single strains (such as HD9 fresh weight CV exceeding 2.0), proving the reliability and stability of its effect.

[0069] Example 4: Effects of compound microbial inoculants on root development and soil-fixing potential of ryegrass

[0070] 1. Experimental materials

[0071] Test strains: Bacillus atrophus ED10 and Bacillus salsa HD9, isolated from Example 1, with accession numbers CGMCC No. 38149 and CGMCC No. 38150, respectively.

[0072] Test plant: Perennial ryegrass (Lolium perenne) seeds, commercially available, with a germination rate ≥90%.

[0073] 2. Preparation and treatment of microbial agents

[0074] ED10 and HD9 were activated separately and inoculated into LB liquid medium, and cultured at 28℃ and 180 rpm with shaking until the logarithmic growth phase (OD600≈1.0). The bacterial concentration was adjusted to OD600=0.1 with sterile water, and the inoculum was mixed in equal volumes to obtain a compound microbial agent. The first inoculum treatment was administered 14 days after germination (3-4 leaf stage) of ryegrass seeds, with each plant irrigated with 20 mL of the compound microbial agent (the control group was irrigated with an equal volume of sterile water). The second treatment was administered 7 days after the first treatment. Each treatment was replicated 10 times.

[0075] 3. Indicator Measurement

[0076] Harvest 14 days after the second treatment (35 days after sowing). Measure plant height, number of tillers, fresh weight, total root length, root surface area, and root volume; calculate root-to-shoot ratio, specific root length, root length density, and root surface area density.

[0077] 4. Data Processing and Statistical Analysis

[0078] All data are expressed as mean ± standard deviation. Independent samples t-tests were used to compare differences between the treatment and control groups, with a significance level set at p < 0.05. The coefficient of variation (CV) = standard deviation / mean was used to assess data stability.

[0079] 5. Experimental Results

[0080] The results are as follows Figure 5 As shown in Tables 3 and 4.

[0081] Table 3. Aboveground indicators of ryegrass (mean ± standard deviation)

[0082]

[0083] Compared with the control group, the ED10+HD9 treatment significantly increased ryegrass plant height by 29.8% (p<0.01), tiller number by 36.7% (p<0.05), aboveground fresh weight by 137% (p<0.01), and aboveground dry weight by 167% (p<0.01). The coefficient of variation (CV) for aboveground dry weight decreased from 0.595 to 0.524, indicating improved data stability.

[0084] Table 4. Ryegrass root system and soil stabilization related indicators

[0085]

[0086] Compared with the control group, the ED10+HD9 treatment group:

[0087] 1) The root surface area increased significantly by 85.1% (p<0.01), the root dry weight increased by 104% (p<0.05), and the root volume increased by 62.3% (p=0.082, marginally significant).

[0088] 2) The total root length increased by 6.3%, which was not significant (p=0.690), indicating that the fungicide mainly promoted the thickening and branching of the root system (increasing the surface area) rather than the elongation.

[0089] 3) The root length was significantly reduced by 49.5% (p<0.01), indicating that the root length produced by the unit of root dry matter input was reduced, that is, the root system was thicker and the tissue density was higher, which is conducive to enhancing the root system's ability to hold the soil.

[0090] 4) Root surface area density (a core indicator of soil stabilization capacity) increased by 85.1% (p<0.01), and root length density increased by 6.3% (no significant difference).

[0091] 5) The root-to-shoot ratio decreased from 1.839 to 1.402 (-23.8%), indicating that the increase in aboveground biomass was greater than that in the root system, which is consistent with the healthy growth model of "root-shoot synergy".

[0092] 6. Conclusion

[0093] Compared with the control group, treatment with the ED10+HD9 compound microbial inoculant increased the aboveground dry weight of ryegrass by 167%, root surface area by 85%, and root surface area density (a core indicator of soil-fixing potential) by 85%, while decreasing specific root length by 49.5%. The results indicate that this compound microbial inoculant significantly promoted the growth and root development of ryegrass, particularly optimizing root architecture (stronger roots and higher surface area density), thus enhancing its soil-fixing potential. Combined with results from Arabidopsis thaliana experiments, this demonstrates that the compound microbial inoculant of this invention has a broad-spectrum effect on different families and genera of plants (Arabidopsis thaliana of the Brassicaceae family and ryegrass of the Poaceae family), promoting lateral root development or optimizing root architecture, and has broad application prospects in ecological restoration and agricultural production.

[0094] Using perennial ryegrass as a model, it was confirmed that the ED10+HD9 compound microbial inoculant increased root surface area density by 85.1%, aboveground dry weight by 167%, and specific root length by 49.5%. Root surface area density is a key parameter for evaluating the soil-fixing capacity of plants, and its significant increase directly indicates that the inoculant can enhance the plant's soil-fixing effect. This result is corroborated by the effect of promoting lateral root development in Arabidopsis thaliana, proving that the compound microbial inoculant of this invention has a broad-spectrum root-promoting effect across families and genera, and has important industrialization value in the field of ecological restoration.

Claims

1. A compound microbial inoculant that promotes lateral root development and root system soil-fixing ability in plants, characterized in that, The compound microbial agent contains Bacillus atrophicus ED10 and Bacillus salsa HD9; wherein Bacillus atrophicus ED10 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38149; and Bacillus salsa HD9 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38150.

2. The compound microbial agent as described in claim 1, characterized in that, The ratio of viable Bacillus atrophus ED10 and Bacillus salsa HD9 in the compound microbial agent is (1-2):(1-2).

3. The compound microbial agent as described in claim 1, characterized in that, The ratio of viable Bacillus atrophus ED10 and Bacillus salsa HD9 in the compound microbial agent is 1:

1.

4. The compound microbial agent according to any one of claims 1-3, characterized in that, The aforementioned compound microbial agent is prepared according to the following method: Bacillus atrophus ED10 and Bacillus sabovella HD9 were activated and inoculated into LB liquid medium, respectively, and cultured at 28°C with shaking at 180 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to OD using sterile water. 600 =0.1, and the compound microbial agent is obtained by mixing at a volume ratio of (1-2):(1-2).

5. The application of the compound microbial agent according to any one of claims 1-4 in promoting the development of plant lateral roots and the ability of roots to stabilize soil.

6. A method for promoting the development of lateral roots and the soil-fixing capacity of plant roots, characterized in that, The step includes irrigating plants with the compound microbial agent as described in any one of claims 1-4.

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