A soybean rhizoctonia solani biocontrol agent with yield-increasing function, a bacterial agent, a preparation method and applications

Abstract

This invention belongs to the field of microbial control technology, specifically relating to a biocontrol bacterium for soybean root rot with growth-promoting and yield-increasing functions, its preparation method, and its application. The biocontrol bacterium is a halophilic Bacillus (Bacillus halophilus). Bacillus halotolerans A novel halophilic Bacillus strain, named GY6, was deposited at the China Center for Type Culture Collection (CCTCC) on November 12, 2025, with accession number CCTCC M 20252537. This strain exhibits antagonistic activity against soybean root rot and promotes growth through IAA production, phosphorus solubilization, and potassium release. By synergistically promoting disease control and healthy crop growth, strain GY6 can achieve a synergistic improvement in crop yield and quality, providing an effective approach to increasing soybean yield in a green manner. Inoculants prepared using GY6 have a positive role in reducing agricultural non-point source pollution, promoting soil health, and even building sustainable farmland ecosystems.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically relating to a biocontrol bacterium for soybean root rot with growth-promoting and yield-increasing functions, a bacterial agent, its preparation method, and its application. Background Technology

[0002] Soybeans Glycine max (L.) Merr. Soybean (Glycine max) is an annual herbaceous plant belonging to the genus *Glycine* in the legume family and is an important grain and oil crop. However, during the long-term cultivation of soybeans, the imbalance of the soil ecosystem caused by continuous cropping obstacles and unreasonable fertilization practices has become increasingly prominent, specifically manifested as the degradation of soil physical and chemical properties, dysbiosis of the microbial flora, and the continuous enrichment of soil-borne pathogens. Among these, soybean root rot, caused by the combined infection of multiple pathogenic fungi, has become one of the key diseases restricting the sustainable development of the industry. Its dominant pathogens include *Fusarium* species (…). Fusarium sp.) and Pythium ( Pythium sp.) etc.

[0003] As a typical soil-borne disease, soybean root rot is characterized by diverse pathogens, strong soil survival ability, and the ability to infect throughout the entire growth cycle, thus exhibiting a high degree of sudden onset and severe damage. With increasing years of continuous cropping, the relative abundance of pathogenic fungi such as Fusarium in the soil increases significantly, while the number of beneficial microorganisms decreases accordingly, further exacerbating the imbalance of the rhizosphere microecology. Once this disease occurs, it is extremely difficult to control, severely inhibiting root development, leading to a decline in plant absorption function, which in turn causes a reduction in the number of pods, the number of seeds, and the 100-seed weight, ultimately resulting in yield loss and quality deterioration, posing a serious economic threat to soybean production.

[0004] Currently, the control of soybean root rot still relies mainly on crop rotation and the application of chemical agents. A reasonable crop rotation pattern can effectively reduce the initial inoculum of pathogens in the soil; however, due to constraints such as farmland size, planting system, and economic benefits, it is difficult to implement sufficient long-term crop rotation in practice. For chemical control, applying metalaxyl granules or metalaxyl powder as a seed dressing at sowing, and spraying fungicides such as thiophanate-methyl, metalaxyl, and carbendazim in the field are the main methods for controlling root rot. However, long-term single application of chemical fungicides not only easily induces drug resistance in pathogens, leading to decreased control efficacy, but also causes secondary ecological problems such as soil acidification, nutrient imbalance, and soil compaction, threatening the health and sustainability of the farmland ecosystem.

[0005] Green strategies for disease control using antagonistic microorganisms and their active metabolites are gaining increasing attention due to their environmental friendliness and sustainability. For example, Bacillus spp. ( BacillusBiocontrol bacteria such as *Bacillus sp.* have been reported to have good inhibitory effects on soybean root rot pathogens. These strains can directly inhibit pathogen growth through nutrient competition, spatial site competition, and the secretion of antagonistic substances such as lipopeptide antibiotics (e.g., subtilisin) and chitinase. In addition, some Bacillus species also possess various plant growth-promoting functions, such as increasing the content of available nutrients in the soil by secreting organic acids to dissolve insoluble phosphorus and potassium minerals. However, in actual field applications, most microbial agents are difficult to establish stably in the rhizosphere and form a dominant population due to the influence of multiple factors such as soil environment, crop root exudates, and competition from indigenous microorganisms. This results in their biocontrol effects not being fully realized, becoming a key bottleneck restricting their large-scale application.

[0006] Screening functional strains with specific affinity for soybean roots and capable of efficient colonization, and developing efficient and stable specialized microbial agents based on these strains, and then systematically integrating them into a green integrated prevention and control system for soybean root rot, is the key to breaking through the current bottleneck in the prevention and control of soybean root rot. Summary of the Invention

[0007] To address the problems existing in the prior art, one of the objectives of this invention is to provide a salt-resistant Bacillus (Bacillus) Bacillus halotolerans The sample, named GY6, was deposited on November 12, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC M 20252537.

[0008] A second objective of this invention is to provide a biocontrol agent comprising the halophilic Bacillus GY6 as described above.

[0009] Preferably, the biocontrol agent is a liquid agent, which is fermented from halophilic Bacillus GY6. Compared with solid agents, the liquid system helps live bacterial cells to adsorb onto the root system more directly and quickly, and penetrate deep into the micropores and crevices of the root surface. At the same time, the evenly distributed bacterial cells can continuously secrete antibacterial substances and growth-promoting metabolites, constructing a highly efficient "micro-ecological disease-suppressing circle" at the root-soil interface, achieving multi-dimensional and continuous control of soybean root rot.

[0010] Preferably, the number of viable bacteria in the liquid bacterial agent is 1×10⁻⁶. 8 -9×10 8 CFU·mL -1 Within the range.

[0011] A third objective of this invention is to provide a method for preparing the biocontrol agent as described above, wherein the method for preparing the biocontrol agent is as follows:

[0012] S1. Activation of bacterial strain: Inoculate GY6 strain into LB liquid medium and incubate at 28-32℃ for 14-20 h to obtain activated bacterial solution;

[0013] S2. Fermentation culture: The activated bacterial solution from step S1 is inoculated into 180 mL of LB liquid medium at a volume ratio of 1-5%, and placed on a shaker at 28-32℃ and 160-200 r·min. -1 After shaking culture for 14-20 h, the biocontrol agent was obtained.

[0014] The fourth objective of this invention is to provide a microbial compound fertilizer, comprising the halophilic Bacillus GY6 as described above and excipients that are well compatible with the halophilic Bacillus GY6.

[0015] Preferably, the excipients include any one or more combinations of fermentation substrate, organic and / or inorganic fertilizers permissible in soil fertility science, and slow-release adsorption carriers.

[0016] The fifth objective of this invention is to provide the application of the biocontrol agent or the microbial compound fertilizer described above in the prevention and control of soybean root rot and / or the promotion of soybean growth and development.

[0017] The sixth objective of this invention is to provide a method for promoting soybean growth and / or preventing soybean root rot, wherein the method is to apply an effective amount of the biocontrol agent as described above to soybeans, or to apply the microbial compound fertilizer as described above to soybeans.

[0018] Preferably, the soybean roots are drenched with the biocontrol agent described above, at a dosage of 20-25 L per acre. This root-drenching method delivers a high concentration of functional bacterial solution directly to the core area of ​​disease occurrence—the soybean rhizosphere soil—achieving targeted delivery and effectively avoiding the problems of the agent remaining on the soil surface or insufficient contact with the roots. This ensures sufficient live bacterial cells directly contact the roots, thereby significantly improving the initial inoculation amount and colonization success rate of the functional bacterial strain.

[0019] The beneficial effects of this application are as follows:

[0020] 1. The halophilic Bacillus GY6 provided in this application exhibits a high specific affinity for soybean roots, enabling long-term stable colonization on the soybean root surface and in the rhizosphere soil, thereby continuously inhibiting the infection of root rot pathogens. Soybean roots can secrete a type of lectin, which is a protein or glycoprotein not derived from the immune system, capable of recognizing and binding to specific polysaccharide structures on the surface of bacterial cell walls. The GY6 antagonistic strain obtained in this application has a cell surface polysaccharide component that highly matches soybean lectins, enabling a specific agglutination reaction, thus achieving rapid recognition and preferential adsorption in the root microdomain. Based on this, the GY6 strain, through a lectin-mediated root recognition mechanism, significantly enhances its initial colonization efficiency and population stability in the soybean rhizosphere, continuously inhibiting the growth and infection of root rot pathogens.

[0021] 2. The halophilic Bacillus GY6 also exhibits multiple plant-promoting properties, including phosphorus solubilization, potassium release, and the secretion of plant growth hormones. Regarding nutrient activation, the strain can effectively acidify the rhizosphere microenvironment by secreting various organic acids (such as gluconic acid and citric acid), thereby dissolving insoluble inorganic phosphorus compounds (such as calcium phosphate) and potassium silicate minerals in the soil and converting them into available phosphorus (H2PO4) that can be directly absorbed by plants. - / HPO4 2- ) and readily available potassium (K + This process can significantly improve the phosphorus and potassium nutrition efficiency of soybeans in continuously cropped or infertile soils, alleviating physiological stress caused by nutrient fixation. Regarding hormone regulation, strain GY6 possesses a complete indole-3-pyruvate (IPyA) synthesis pathway, capable of efficiently converting tryptophan precursors into biologically active IAA. The secreted IAA can act as an exogenous signaling molecule to directly stimulate the division and elongation of soybean root cells, promoting the development of lateral roots and primary root hairs, thereby expanding the root absorption surface area and enhancing the plant's ability to acquire water and nutrients. This provides a solid material basis for pod formation and grain filling, effectively increasing the number of pods, grains, and soybean weight per plant, providing effective microbial support for green yield increase and quality improvement in soybeans.

[0022] 3. Liquid microbial agents were prepared using strain GY6. The dosage of the agent applied to the soil was 20-25 L / mu, significantly lower than the commonly used field application rate of 50-60 L / mu for conventional microbial pesticides. This reduction in agent dosage lowers production costs and labor input for field application, enhancing the technology's economic viability and potential for wider adoption. Furthermore, precise control of the agent dosage minimizes structural disturbances to the native soil microbial community, helping to maintain the stability and biodiversity of the rhizosphere microecology and avoiding potential ecological risks from excessive introduction of exogenous microorganisms.

[0023] 4. The use of microbial inoculants to replace pesticides exhibits significant environmentally friendly characteristics in farmland ecosystems. This functional strain GY6, screened from the natural environment, completes its life cycle in the soil and can be completely biodegraded by the indigenous microbial community without producing toxic metabolic residues, thus maintaining the stability and safety of the soil micro-ecosystem.

[0024] In summary, strain GY6 achieves a synergistic improvement in crop yield and quality through disease control and healthy crop growth, providing an effective approach for green soybean yield enhancement. The prepared microbial agent also plays a positive role in reducing agricultural non-point source pollution, promoting soil health, and even building a sustainable farmland ecosystem. Detailed Implementation

[0025] The present invention will be further described below through embodiments.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0027] Example 1

[0028] Strain screening

[0029] Soybean plants from fields infected with root rot were collected. The entire root system, including the soil, was dug up, and large clods of soil and other useless debris were removed to obtain the complete root system and rhizosphere soil. 5 g of rhizosphere soil from each sample was placed in an Erlenmeyer flask containing 100 mL of sterile physiological saline and placed on a shaker at 160 r·min. -1 Shake for 0.5 h to obtain the rhizosphere soil bacterial suspension. Take 1 mL of the above bacterial suspension and add it to 9 mL of sterile physiological saline containing nystatin, then serially dilute to 10⁻⁶. -7 Concentration. The colonies were diluted and spread on LB plates, and those with different growth morphologies were purified separately.

[0030] Bacteria isolated from soybean rhizosphere soil were activated, and the antagonistic ability of bacterial strains against different soybean root rot pathogens was determined using the plate confrontation culture method. The soybean root rot pathogens tested were obtained from soybean rhizosphere soil and were: Fusarium solani (…). Fusarium solani L1, Fusarium solani ( Fusarium solani L2, *Equisetum hyemale* ( Fusarium horsetail L3.

[0031] The specific steps are as follows: A 5 mm diameter mycelial block of each root rot pathogen was inoculated in the center of a PDA plate, and bacterial strains for testing were placed at equidistant points on both sides of the block. Plates inoculated only with the pathogen served as a control. All plates were incubated at 28°C for 5 days. The diameter of the pathogen colonies was measured, and the inhibition rate of each bacterial strain was calculated. Strains with high inhibition rates were selected and purified by repeated streaking on LB agar plates to obtain pure cultures of antagonistic strains.

[0032] The selected antagonistic strains were inoculated into LB liquid medium and cultured under constant temperature and shaking until the logarithmic growth phase. 25 μL of the bacterial suspension was dropped into the center of a clean glass slide, and an equal volume of soybean lectin solution was added. The mixture was gently mixed and allowed to stand at room temperature for 30 min. After air-drying and staining, the agglutination reaction was observed under an optical microscope. Strains exhibiting obvious agglutination were selected and identified as antagonistic strains with soybean affinity.

[0033] The above LB medium formula is as follows: 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar, and distilled water to a final volume of 1000 mL, adjusting the pH to 7.0-7.2.

[0034] The formula for calculating the antibacterial rate is:

[0035] ;

[0036] The GY6 strain used in this application is a soybean root rot biocontrol strain obtained using this screening method. Plate confrontation experiments showed that strain GY6 had inhibitory effects on all three obtained soybean root rot pathogens, as shown in Table 1.

[0037] Table 1. Inhibition rate of strain GY6 against three root rot pathogens.

[0038]

[0039] Example 2

[0040] Identification of strains

[0041] The strain GY6 was classified and identified by combining morphological observation, physiological and biochemical characteristic analysis and 16S rRNA gene sequence comparison.

[0042] Morphological observations included Gram staining, and physiological and biochemical assays were performed in accordance with the "Manual of Systematic Identification of Common Bacteria" and the "Bergey's Manual of Bacterial Identification". Specifically, these assays included starch hydrolysis, catalase, VP, MR, gelatin liquefaction, glucose oxidation fermentation, and H2S production. The results are shown in Table 2.

[0043] BLAST alignment analysis of the 16S rRNA gene sequence of strain GY6 was performed based on the NCBI database, combined with... gyrB / gyrA A phylogenetic tree was constructed using gene sequences, and its morphological characteristics and physiological and biochemical properties were combined to ultimately identify it as a halophilic Bacillus. Bacillus halotolerans The sequence information of related genes of strain GY6 is shown in the sequence listing (SEQ ID NO:1).

[0044] Table 2. Partial physiological and biochemical characteristics of strain GY6

[0045]

[0046] Example 3

[0047] Determination of IAA production and phosphorus and potassium solubilization capabilities of strain GY6

[0048] 1. Inoculate strain GY6 at a rate of 2% into nitrogenous liquid medium and incubate at 28°C and 160 r·min. -1 The culture was shaken and cultured for 5 days under the specified conditions. After the culture was completed, the fermentation broth was centrifuged and the supernatant was collected. An appropriate amount of the supernatant was mixed with Salkowski's colorimetric reagent at a volume ratio of 1:2 and reacted in the dark at 25°C for 30 min. An uninoculated nitrogenous liquid medium was used as a blank control, and the absorbance value was measured at a wavelength of 530 nm. The IAA production capacity of strain GY6 was calculated based on this.

[0049] Salkowski's colorimetric reagent formulation is: 1 mL 0.5 mol·L⁻¹ -1 Add FeCl3 solution to 50 mL of 35% HClO4 solution, mix well and set aside.

[0050] The nitrogenous liquid culture medium formula is as follows: 10.0 g sucrose, 2.0 g K2HPO4, 0.5 g MgSO4·7H2O, 0.1 g NaCl, 0.5 g yeast extract, 0.5 g CaCO3, and distilled water to a final volume of 400 mL, adjusting the pH to 7.0.

[0051] 2. Inoculate strain GY6 at a rate of 2% into an inorganic phosphorus liquid medium with Ca3(PO4)2 as the sole phosphorus source, and incubate at 28℃ and 160 r·min. -1 Under the specified conditions, the culture was shaken for 7 days. After the culture was completed, 1 mL of fermentation broth was taken and incubated at 8000 r·min. -1 Centrifuge for 10 min and collect the supernatant. Accurately pipette 10 μL of the supernatant, add 1 mL of molybdenum antimony colorimetric reagent, and dilute to 10 mL with distilled water. Mix well and let stand at room temperature for 15 min. Measure the absorbance at 700 nm to evaluate the phosphate-solubilizing ability of the strain.

[0052] The formula for the inorganic phosphorus liquid culture medium is as follows: 10 g glucose, 5 g Ca3(PO4)2, 0.3 g KCl, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.03 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, diluted with distilled water to 1000 mL, and the pH value adjusted to 7.2.

[0053] 3. Inoculate strain GY6 at a rate of 2% into potassium-solubilizing liquid medium and incubate at 28°C and 160 r·min. -1 Under the specified conditions, the culture was shaken for 5 days. After the culture was completed, 10 mL of the bacterial suspension was accurately pipetted, 0.4 mL of 30% hydrogen peroxide solution was added, and the mixture was digested at 121℃ for 30 min at 4000 r·min. -1 Centrifuge for 20 min, collect the supernatant, and bring the volume to 100 mL with distilled water. Measure the absorbance at 420 nm using the molybdenum-antimony colorimetric method to calculate the water-soluble potassium content and assess the strain's potassium-solubilizing ability.

[0054] The formula for potassium-solubilizing liquid culture medium is as follows: 5.0 g sucrose, 2.0 g Na2HPO4, 0.05 g FeCl3, 0.1 g CaCO3, 1.0 g potassium feldspar powder, 0.2 g MgSO4·7H2O, diluted with distilled water to 1000 mL, and the pH value adjusted to 7.0-7.5.

[0055] The measurement results are shown in Table 3.

[0056] Table 3 Growth-promoting characteristics of strain GY6

[0057]

[0058] As shown in Table 3, strain GY6 has the ability to produce IAA, solubilize phosphorus and potassium, indicating that this strain has the potential to promote plant growth.

[0059] Example 4

[0060] Pot experiment

[0061] 1. Preparation of microbial agents

[0062] The GY6 strain was inoculated into 5 mL LB liquid culture tubes and cultured at 28°C for 20 h. Then, it was inoculated into 180 mL LB liquid culture medium at a volume ratio of 1% and placed on a shaker at 28°C and 180 r·min. -1 After shaking culture for 16-20 h, GY6 liquid bacterial agent was obtained, OD 600 =1.0.

[0063] 2. Pot experiment

[0064] This experiment consisted of 7 treatment groups: control (CK) without inoculation, inoculation with pathogen L1 alone (L1), inoculation with pathogen L1 and GY6 agent together (L1+GY6), inoculation with pathogen L2 alone (L2), inoculation with pathogen L2 and GY6 agent together (L2+GY6), inoculation with pathogen L3 alone (L3), and inoculation with pathogen L3 and GY6 agent together (L3+GY6). Each group had 6 replicates.

[0065] Select plump, uniformly sized soybean seeds and sow them in flowerpots filled with nutrient soil. At sowing time and during the first true leaf unfolding stage, each co-inoculation treatment group was watered with 10 mL of GY6 bacterial suspension. The control group was watered with an equal volume of sterile culture medium at the same time. After the first pair of true leaves had fully unfolded, all treatments were uniformly inoculated with 10 mL of 1×1... CFU·mL -1 Pathogen suspension was used. Thirty days after inoculation, the disease incidence in each treatment plant was investigated, the disease rate was calculated, and the disease index and control effect were determined. The occurrence of soybean root rot was assessed according to GB / T 17980.88-2004, "Classification and Investigation Methods for Soybean Root Rot Disease."

[0066] Grading standards for soybean root rot:

[0067] Grade 0: No disease spots on the base of the stem and main root;

[0068] Grade 1: A few lesions on the base of the stem and the main root;

[0069] Grade 3: There are many lesions at the base of the stem or on the main root, and the lesion area accounts for 1 / 4 to 1 / 2 of the total area of ​​the stem and root;

[0070] Grade 5: Numerous and large lesions on the base of the stem and the main root, with the lesion area accounting for 1 / 2 to 3 / 4 of the total area of ​​the stem base and roots;

[0071] Level 7: Patches of disease on the base of the stem and main root merge together, forming a phenomenon of wrapping around the stem, but the root system is not dead;

[0072] Level 9: Root necrosis, withered or dead above-ground parts of the plant.

[0073] The calculation formula is as follows:

[0074] Disease incidence rate (%) = Number of diseased plants / Total number of plants planted × 100%;

[0075] Disease index = [Σ(number of diseased plants at each level × disease severity level) / (total number of plants × highest disease severity level)] × 100;

[0076] Prevention and control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.

[0077] The results are shown in Table 4.

[0078] Table 4. Control efficacy of GY6 inoculant against soybean root rot in pot experiments.

[0079]

[0080] As shown in Table 4, GY6 achieved control effects of 77.78%, 72.73%, and 71.42% against soybean root rot caused by three root rot pathogens, L1, L2, and L3, respectively, all of which were higher than 70%, demonstrating its broad-spectrum disease resistance characteristics and its important application value in the biological control of soybean root rot.

[0081] Example 5

[0082] The effects of GY6 on soybean growth

[0083] The method for preparing the microbial agent used in Example 5 is the same as that in Example 4.

[0084] The experiment was divided into two groups: an experimental group treated with the fungicide and a control group treated without the fungicide. Each group was replicated three times.

[0085] Plump and uniformly sized soybean seeds were sown in flowerpots filled with nutrient soil. At sowing time and during the first true leaf unfolding stage, 10 mL of GY6 bacterial suspension was applied to the treatment group, while the control group was applied with an equal volume of sterile culture medium at the same time. Thirty days after the last treatment, the root length, fresh weight of stems, leaves, and roots, and dry weight of the soybean plants in each treatment were measured. The results are shown in Table 5.

[0086] Table 5 Effects of GY6 on soybean growth

[0087]

[0088] Note: This indicates that the differences between treatments are significant ( p <0.05).

[0089] As shown in Table 5, the application of GY6 inoculant significantly increased the fresh weight of soybean stems and leaves, fresh weight of roots, dry weight of stems and leaves, and dry weight of roots, by 63.71%, 89.55%, 63.83%, and 92.31% respectively compared with the control group without inoculant application, reaching a significant level. p <0.05). These results indicate that GY6 inoculant significantly increases root biomass while also enhancing the root system's ability to absorb water and nutrients by optimizing root architecture and improving metabolic activity. This synergistic promotion of root system structure and function ultimately lays the physiological foundation for increasing soybean yield potential.

[0090] Example 6

[0091] field trials

[0092] The method for preparing the inoculant used in Example 6 is the same as in Example 4.

[0093] The experiment was divided into two groups: an experimental group treated with GY6 inoculant and a control group without inoculant treatment. In the experimental group, soybean seeds were treated with GY6 inoculant before sowing. When the first pair of true leaves of the soybean seedlings had just unfolded, 10 mL of GY6 inoculant diluted 10 times with water was applied to the base of each seedling stem. In the control group, soybean seeds were treated with LB medium before sowing. When the first pair of true leaves of the soybean seedlings had just unfolded, 10 mL of LB medium diluted 10 times with water was applied to the base of each seedling stem. After 100 days of growth, the disease severity of the plants was recorded, and the disease index and control effect were calculated. The results are shown in Table 6.

[0094] Meanwhile, the plant height, bottom pod height, number of nodes, number of effective branches, number of pods per plant, number of seeds per plant, weight of soybeans per plant, and weight of 100 seeds were measured for each soybean plant. The results are shown in Table 7. The number of 0-pod, 1-pod, 2-pod, 3-pod, and 4-pod plants, as well as their length and width, were also counted for each plant. The results are shown in Table 8.

[0095] Table 6. Control efficacy of GY6 inoculant against soybean root rot in field trials.

[0096]

[0097] As shown in Table 6, the disease index of the treatment group inoculated with GY6 inoculant was 20.10%, significantly lower than that of the control group (84.07%), with a control effect of 76.09%. The results indicate that GY6 inoculant has a significant control effect on soybean root rot, which is closely related to its stable colonization ability in the rhizosphere and its continuous antagonistic effect on the pathogen.

[0098] Table 7. Effects of GY6 inoculant on soybean agronomic traits and yield in field trials.

[0099]

[0100] Note: This indicates that the differences between treatments are highly significant. p <0.01).

[0101] As shown in Table 7, the experimental group treated with GY6 inoculant showed significantly higher plant height, number of effective branches, number of pods per plant, number of seeds per plant, and bean weight per plant compared to the control group without inoculant treatment. These increases were 14.90%, 260.00%, 81.19%, 89.35%, and 74.64% respectively. The height of the bottom pods was significantly lower than that of the control group without inoculant treatment. p<0.01). These changes indicate that the GY6 inoculant significantly enhances soybean yield-forming capacity by optimizing plant spatial configuration and reproductive allocation. The substantial increase in the number of effective branches directly improves the number of fertilization sites; the significant increases in the number of pods, seeds, and soybean weight per plant reflect expanded sink capacity and enhanced nutrient accumulation; and the moderate reduction in bottom pod height helps improve plant lodging resistance. In summary, the GY6 inoculant not only promotes the synergistic enhancement of soybean vegetative and reproductive growth but also lays an important foundation for achieving high and stable yields.

[0102] Table 8. Effects of GY6 inoculant on soybean pods in field trials.

[0103]

[0104] Note: This indicates that the differences between treatments are significant ( p <0.05), This indicates that the differences between treatments are highly significant. p <0.01).

[0105] As shown in Table 8, the experimental group treated with GY6 inoculant showed a 90.91% increase in the number of two pods and a 6.98% increase in pod width compared to the control group without inoculant treatment, reaching a significant level. p <0.05), the number of 3 pods increased by 96.43% compared with the control group without fungicide, reaching a highly significant level ( p <0.01). These results indicate that the microbial agent optimized the sink structure by promoting the formation of high-grain-number pods, and the significant increase in pod width further suggests that the agent may have improved pod morphology, which is beneficial for increasing grain filling. In summary, the GY6 microbial agent enhances the yield potential of soybeans from multiple dimensions by synergistically regulating key traits such as pod number, pod shape, and grain number.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A salt-resistant Bacillus ( Bacillus halotolerans It was named GY6 and deposited at the China Center for Type Culture Collection on November 12, 2025, with accession number CCTCC M 20252537.

2. A biocontrol agent, characterized in that, The biocontrol agent includes the salt-tolerant Bacillus GY6 as described in claim 1.

3. The biocontrol agent as described in claim 2, characterized in that, The biocontrol agent is a liquid agent, which is produced by fermentation of halophilic Bacillus GY6.

4. The biocontrol agent as described in claim 3, characterized in that, The liquid bacterial agent contains 1×10⁻⁶ live bacteria. 8 -9×10 8 CFU·mL -1 Within the range.

5. A method for preparing the biocontrol agent as described in claim 3 or 4, characterized in that, The preparation method of liquid bacterial agent is as follows: S1. Activation of bacterial strain: Inoculate GY6 strain into LB liquid medium and incubate at 28-32℃ for 14-20 h to obtain activated bacterial solution; S2. Fermentation culture: The activated bacterial solution from step S1 is inoculated into 180 mL of LB liquid medium at a volume ratio of 1-5%, and placed on a shaker at 28-32℃ and 160-200 r·min. -1 After shaking culture for 14-20 h, liquid bacterial agent was obtained.

6. A microbial compound fertilizer, characterized in that, It includes the salt-tolerant Bacillus GY6 as described in claim 1 and excipients that are well compatible with the salt-tolerant Bacillus GY6.

7. The microbial compound fertilizer as described in claim 6, characterized in that, The auxiliary materials include any one or more combinations of fermentation substrates, organic and / or inorganic fertilizers permitted by soil fertility science, and slow-release adsorption carriers.

8. The application of the biocontrol agent according to any one of claims 2-4 or the microbial compound fertilizer according to any one of claims 6-7 in the prevention and control of soybean root rot and / or the promotion of soybean growth and development.

9. A method for promoting soybean growth and / or preventing soybean root rot, characterized in that, Apply an effective amount of the biocontrol agent as described in any one of claims 3-4 to soybeans, or apply the microbial compound fertilizer as described in any one of claims 6-7 to soybeans.

10. The method as described in claim 9, characterized in that, Soybeans are irrigated with the biocontrol agent as described in any one of claims 3-4, wherein the application rate of the biocontrol agent is 20-25 L per acre.

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