Bacillus complex bacterial community and application thereof in corn disease prevention and growth promotion

Abstract

This invention discloses a Bacillus complex and its application in maize disease prevention and growth promotion. Two different species of Bacillus were isolated and screened from the rhizosphere of maize plants highly resistant to stalk rot: *Bacillus belyss* (…). Bacillus velezensis IPPMD-360 and Bacillus simplex ( Bacillus simplex IPPMD-315. Experiments show that the simple Bacillus IPPMD-315 can significantly promote the growth and biofilm formation of Bacillus belyssima IPPMD-360. The combined microbial community, after inoculation treatment of maize seeds, can promote the growth of maize seedlings and has a good inhibitory effect on the occurrence of maize stalk rot. Moreover, the growth-promoting and disease-preventing effects are significantly better than those of Bacillus belyssima IPPMD-360 alone. The biocontrol agent of this invention is simple to cultivate and can provide new materials for the development of high-efficiency biological agents and microbial fertilizers. The bacteria used are safe for humans and animals, have no environmental pollution problems, and the cultivation conditions are simple, easy to preserve, and suitable for development and application.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases, specifically relating to a Bacillus complex and its application in maize disease prevention and growth promotion. Background Technology

[0002] Corn stalk rot is a prevalent and significant soil-borne disease in major corn-producing areas of my country. In recent years, with climate change and the increasing scale of corn planting in my country, soil degradation caused by years of cultivation, variety changes, and shifts in planting systems have led to the continuous accumulation of pathogens in the field, creating conditions conducive to the occurrence and spread of stalk rot. The incidence rate is generally 5%–10%, but can reach 20%–30% in severe cases, and as high as 50%–60% in some areas, resulting in a yield reduction of 25%, and in severe cases, even total crop failure. Under natural conditions, corn stalk rot is mostly caused by the co-infection of multiple pathogens, mainly Fusarium and Pythium. It is a disease that affects the entire growth cycle, with peak development occurring during the grain-filling stage and the late milk-ripe to waxy-ripe stage. Fusarium graminearum (… Fusarium graminearum Currently, it is the dominant fungal species causing corn stem rot in my country, capable of infecting the roots and stem base of cereal crops such as corn.

[0003] Currently, the main method for controlling maize stalk rot is seed coating with chemical seed dressing agents. However, because the pathogen of maize stalk rot is abundant in the soil and its influence lasts for a long time, most chemical seed dressing agents cannot continuously control the disease throughout the entire growth period of maize. Furthermore, long-term use of chemical agents disrupts the balance of the soil microecological environment and destroys the natural "beneficial" microbial community, thus exacerbating the occurrence of the disease. Therefore, biological control is receiving increasing attention. Exploring the use of microorganisms and their secondary metabolites for soil-borne disease control can promote plant growth, increase crop yield, and achieve sustainable, green control.

[0004] Microorganisms in nature do not exist as individuals, but are dynamically changing; microbial communities involve mutual influence and communication. Synthetic communities ( SynComs Synthetic functional microbiota are increasingly being used to study related microbial activities and interactions with plant hosts. Synthetic functional microbiota refer to co-culture systems formed by two or more known microorganisms in specific, controlled environmental conditions. Compared to single-strain systems, synthetic functional microbiota possess the characteristics of communication and division of labor. Microbial community members can communicate unidirectionally, bidirectionally, or even multidirectionally through signal molecule exchange, detection, and mutual response; they can also perform different tasks through division of labor, enabling the entire microbiota to achieve complex functions that a single strain cannot accomplish. Summary of the Invention

[0005] This invention addresses the problems of poor colonization and low efficacy of existing single-strain control agents for maize stalk rot by providing a Bacillus complex with disease-preventing and growth-promoting functions for maize, and its application in maize seed treatment. This invention utilizes Bacillus belye, a bacterium isolated from the maize rhizosphere. Bacillus velezensis IPPMD-360 (CGMCC No. 36628, deposit date: November 14, 2025, depositary institution code: CGMCC-China General Microbiological Culture Collection Center) and Bacillus simplex ( Bacillus simplex IPPMD-315 (CGMCC No. 36627, deposit date: November 14, 2025, depositary code: CGMCC-China General Microbiological Culture Collection Center) is used to prepare a Bacillus compound inoculant, providing a Bacillus compound inoculant seed treatment preparation for controlling corn stalk rot.

[0006] The Bacillus belesiensis of the present invention ( Bacillus velezensis IPPMD-360 and Bacillus simplex ( Bacillus simplex IPPMD-315 was obtained from the rhizosphere of highly resistant maize varieties through isolation and purification. Experimental results of this invention show that *Bacillus simplex* IPPMD-315 significantly promotes the growth and biofilm formation of *Bacillus belyssioides* IPPMD-360. The bacterial flora synthesized by *Bacillus belyssioides* IPPMD-360 and *Bacillus simplex* IPPMD-315 has a significant growth-promoting effect on maize seedlings and inhibits the growth of *Fusarium graminearum*, the pathogen of maize stalk rot. Fusarium graminearum It also has a certain inhibitory effect, and its growth-promoting and disease-preventing effects are significantly better than those of Bacillus belysin IPPMD-360 alone.

[0007] The purpose of this invention is to provide two strains of *Bacillus belyssae*, a rhizosphere bacterium of maize. Bacillus velezensis IPPMD-360 and Bacillus simplex ( Bacillus simplex IPPMD-315; provides information on the growth-promoting effects of Bacillus complex on maize and its biocontrol efficacy against maize Fusarium stalk rot. Utilizing Bacillus belye (… Bacillus velezensis IPPMD-360 and Bacillus simplex ( Bacillus simplex The method of promoting plant growth and resisting fungal diseases by IPPMD-315 compound strain biological agent is also within the scope of protection of this invention.

[0008] The first objective of this invention is to provide Bacillus belesiensis (B. belesiensis) Bacillus velezensis IPPMD-360, with accession number CGMCC No.36628.

[0009] The second objective of this invention is to provide simple Bacillus ( Bacillus simplex IPPMD-315, with accession number CGMCC No.36627.

[0010] A third objective of this invention is to provide a complex microbial community composed of the aforementioned Bacillus belyssus IPPMD-360 and the aforementioned Bacillus simplex IPPMD-315.

[0011] A fourth object of the present invention is to provide a biological agent containing, as an active ingredient, the aforementioned Bacillus berleis IPPMD-360 and the aforementioned Bacillus simplex IPPMD-315, or a culture containing the aforementioned Bacillus berleis IPPMD-360 and the aforementioned Bacillus simplex IPPMD-315.

[0012] A fifth objective of this invention is to provide a bio-fertilizer containing the aforementioned biological agents and fertilizer.

[0013] A sixth object of the present invention is to provide a seed treatment agent comprising the aforementioned biological agent and seed coating agent excipients.

[0014] A seventh object of the present invention is to provide the use of the biological agent in at least one of the following (1) to (3):

[0015] (1) Promotes corn seed germination;

[0016] (2) Promotes corn growth;

[0017] (3) Control corn stalk base rot.

[0018] Preferably, the corn stalk base rot is caused by Fusarium graminearum (…). Fusarium graminearum Corn stalk base rot caused by )

[0019] The eighth object of the present invention is to provide a method for promoting maize growth and preventing maize stalk rot, comprising the following steps: applying the biological agent to maize seeds or maize plants.

[0020] Preferably, the biological agent is applied to corn seeds by seed dressing or coating; and applied to corn plants by root irrigation.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The Bacillus berreatus IPPMD-360 and Bacillus simplex IPPMD-315 of this invention have significant growth-promoting effects on corn seedlings and also have a certain inhibitory effect on corn stalk rot. They are highly efficient biological agents and biological fertilizers with good development and application prospects.

[0023] The preparation of the Bacillus vesiculosus IPPMD-360 and Bacillus simplex IPPMD-315 biological agents of the present invention is simple and easy, and can be widely promoted and applied.

[0024] The Bacillus berberis IPPMD-360 and Bacillus simplex IPPMD-315 provided by this invention are safe and non-toxic to humans and animals, have good stability, and are environmentally friendly.

[0025] Preservation Instructions

[0026] The simple Bacillus of the present invention ( Bacillus simplex IPPMD-315 was deposited on November 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 36627, depository code: CGMCC-China General Microbiological Culture Collection Center, address of depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0027] The Bacillus belesiensis of the present invention ( Bacillus velezensis IPPMD-360 was deposited on November 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 36628, depository code: CGMCC-China General Microbiological Culture Collection Center, address of depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0028] Figure 1This study analyzes the growth characteristics of *Bacillus belyssioides* IPPMD-360 and *Bacillus simplex* IPPMD-315. a) The plate confrontation growth phenotypes of strains IPPMD-360 and IPPMD-315 against *Fusarium graminearum* P067 are presented. b) The phenotypes of colony growth on LB plates for isolated strain IPPMD-360, co-cultured strains IPPMD-360 and IPPMD-315, and isolated strain IPPMD-315 are presented. c) Quantitative statistical results of colony growth area on LB plates for isolated strain IPPMD-360, co-cultured strains IPPMD-360 and IPPMD-315, and isolated strain IPPMD-315 are presented. d) Colony morphology of strains IPPMD-315 and IPPMD-360 is presented. e) Spore morphology of strains IPPMD-315 and IPPMD-360 is presented. f) The 16S morphology of strain IPPMD-315 is presented. Phylogenetic tree of rRNA gene sequence; g is the phylogenetic tree of 16S rRNA gene sequence of strain IPPMD-360.

[0029] Figure 2 The results show that Bacillus simplex IPPMD-315 promotes the growth of Bacillus belyss IPPMD-360; a) shows the phenotypes of CK, IPPMD-360, IPPMD-315, and IPPMD-360+IPPPMD-315 after crystal violet staining; b) quantifies the biofilm growth promotion effect of the different treatments in a by measuring OD values; c) shows the changes in OD values ​​of CK, IPPMD-360, IPPMD-360+IPPPMD-315, and IPPMD-315 after culturing for 24 h, with measurements taken every 1 h.

[0030] Figure 3 The results show the growth-promoting effects of the combined bacterial flora of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 on maize plants; a) the promoting effects of CK, IPPMD-360, IPPMD-360+IPPMD-315, and IPPMD-315 treatments on maize growth phenotypes after 21 days; b) the statistical results of plant height after 21 days of the above treatments; c) the statistical results of fresh weight after 21 days of the above treatments; and d) the statistical results of stem diameter after 21 days of the above treatments.

[0031] Figure 4The inhibitory effect of the Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 complex on maize stalk rot; a) shows the growth and disease resistance phenotypes of maize after 21 days of treatment with fg (i.e., Fusarium graminearum P067), IPPMD-360+fg, IPPMD-360+IPPMD-315+fg, and IPPMD-315+fg; b) shows the statistical data on the incidence of disease in the above treatments; c) shows the statistical data on the plant height in the above treatments; d) shows the phenotype of root disease in the above treatments.

[0032] Figure 5 The results show the promoting effect of seed treatment with a compound seed dressing agent of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 on the germination of maize seeds; a) shows the germination effect of CK (uncoated), CK (coated with fludioxonil), IPPMD-360, and IPPMD-360+IPPPMD-315 on seeds; b) shows the statistical results of the germination rate of the above treatments.

[0033] Figure 6 This study investigates the inhibitory effect of a combination seed dressing agent of Bacillus belyssima IPPMD-360 and Bacillus simplex IPPMD-315 on maize stem base rot and its influence on maize growth in pot experiments. (a) shows the growth phenotype of the control group (CK), after inoculation with Fusarium graminearum P067, after coating with IPPMD-360 and its combination seed dressing agent IPPMD-360+IPPPMD-315, followed by pot inoculation with Fusarium graminearum P067 21 days later. (b) shows the incidence rate of the above treatments, and (c) shows the effect of the above treatments on plant height.

[0034] Figure 7 The effects of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 combined seed dressing agents on maize seedling growth phenotype during the seedling stage were as follows: a) Effects of CK, fludioxonil coating, Penicillium benzoate seed dressing agent, IPPMD-360 coating, and combined seed dressing agent IPPMD-360+IPPMD-315 coating on maize seedling height during the seedling stage; b) Effects of CK, fludioxonil coating, Penicillium benzoate seed dressing agent, IPPMD-360 coating, and combined seed dressing agent IPPMD-360+IPPMD-315 coating on maize seedling fresh weight during the seedling stage; c) Effects of CK, fludioxonil coating, Penicillium benzoate seed dressing agent, IPPMD-360 coating, and combined seed dressing agent IPPMD-360+IPPMD-315 coating on maize seedling stem diameter during the seedling stage. Detailed Implementation

[0035] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0036] Example 1: Inhibitory effect of screened strains on Fusarium graminearum and colony confrontation test

[0037] 1.1 Culture medium preparation

[0038] PDA medium: Difco™ Potato Dextrose Agar 39 g, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0039] LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0040] 1.2 Test strains

[0041] The selected strains, numbered IPPMD-315 and IPPMD-360, were both isolated from maize rhizosphere soil. *Fusarium graminearum* (IPPMD-315) Fusarium graminearum Strain P067 was isolated from the stem base of maize plants infected with stem rot at the Jiangcheng Experimental Base in Yunnan Province and is preserved in the Maize Diseases Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. 1.3 Determination of the antifungal activity of the screened strains against Fusarium graminearum.

[0042] Fusarium graminearum P067, frozen in glycerol, was activated and allowed to grow for 3 to 5 days before use.

[0043] The screened and purified rhizosphere bacterial strains IPPMD-315 and IPPMD-360 were inoculated into LB liquid medium and cultured overnight at 28°C and 200 rpm to obtain activated bacterial solutions.

[0044] The antagonistic effects of rhizosphere bacterial strains IPPMD-315 and IPPMD-360 on the target strain Fusarium graminearum P067 were determined using the confrontation culture method. A 5 mm mycelial cake of the target strain was inoculated into the center of a PDA plate, with 10 μL of activated bacterial solution inoculated 25 mm to the left and right. Three replicates were performed for each rhizosphere bacterial activated bacterial solution. The blank control consisted only of the target strain.

[0045] The results showed that strain IPPMD-360 had a significant inhibitory effect on Fusarium graminearum P607, with an inhibition rate of 55.34%, while strain IPPMD-315 had no inhibitory effect on Fusarium graminearum P067. Figure 1 (a) in the middle.

[0046] 1.4 Group swimming confrontation experiment to verify the synergistic effect of strains IPPMD-315 and IPPMD-360

[0047] Strains IPPMD-315 and IPPMD-360, stored at -80℃, were streaked onto LB solid medium and incubated at 37℃ for 12 h. Single colonies were then transferred to liquid LB medium and cultured by shaking (37℃, 200 rpm, 12 h), followed by centrifugation (6000 rpm, 25℃, 10 min). The OD values ​​were adjusted using a microplate reader. 600 =0.5. Take 2 μL of bacterial suspension and drop it at both ends 2 cm away from the center of the petri dish. Add 2 μL of sterile water to the control end. After incubation for 24 h, count the size of the colonies.

[0048] The results showed that 2 μL of solution with OD600=1 The bacterial suspension was dropped 2 cm from the center of the plate. After 24 h of incubation, statistical data were collected. One-way ANOVA showed that strains IPPMD-315 and IPPMD-360 had no antagonistic effect on the plate. Figure 1 (b, c in the text).

[0049] 1.5 Bacterial Gram Staining

[0050] Strawberries IPPMD-315 and IPPMD-360, preserved at -80℃, were streaked onto LB agar plates and incubated at 37℃ for 12 h. 2 μL of sterile physiological saline was dropped onto a sterile glass slide. A loopful of a single colony of the bacteria was taken, spread evenly, and quickly passed over an alcohol lamp twice. After the colonies dried, they were stained with crystal violet for 1 min. The slide was then washed along one side with a wash bottle, dried with sterile filter paper, stained with iodine solution for 1 min, rinsed with the staining solution, dried, and destained with destaining solution for 30–60 s. After rinsing and drying, they were stained with safranin for 1 min, rinsed with the staining solution, and dried. Cedarwood oil was then added and the staining was observed under a microscope. Photographs were taken using a Leica microscope.

[0051] The results showed that after culturing at 37℃ for 12 h on LB medium, strain IPPMD-315 colonies were regularly round, with a slightly yellowish, uneven surface. The colonies were thicker in the center, gradually thinning towards the edges. Strain IPPMD-360 colonies typically had neat edges, mostly irregular shapes, with a few scattered small white colonies. These colonies were milky white, with a smooth surface, a loose, viscous texture, and noticeable wrinkles. Some colonies had raised wrinkles in the center. Figure 1 (d) Both strains IPPMD-315 and IPPMD-360 are Gram-positive bacteria under a microscope, and their spore morphologies differ significantly. Strain IPPMD-315 exhibits a longer rod-like shape, while strain IPPMD-360 displays a short and thick rod-like shape. Figure 1(e in the text).

[0052] 1.6 Molecular Identification

[0053] DNA extraction: After culturing strains IPPMD-315 and IPPMD-360 on LB agar plates at 37°C for 12 h, single colonies were picked with sterile toothpicks and placed in 5 mL of LB liquid medium, and cultured overnight at 37°C and 200 rpm for 12 h. The overnight culture suspensions of strains IPPMD-315 and IPPMD-360 were used as template DNA for bacterial PCR. PCR amplification of the bacterial 16S rRNA ribosomal subunit gene was performed using the following primers: 27F: TACGGYTACCTTGTTACGACTT; 1492R: AGAGTTTGATCMTGGCTCAG. PCR amplification was performed in a 30 μL reaction system containing: 1 μL each of forward and reverse primers, 15 μL of 2×Taq Master Mix, and 13 μL of ddH2O. Reaction conditions: pre-denaturation at 95℃ for 5 min, followed by 32 cycles including: denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 30 s; and final extension at 72℃ for 5 min.

[0054] Gene sequencing and sequence analysis: The target DNA fragment amplified by PCR was sent to Qingke Biotechnology Co., Ltd. for sequencing. After rigorous verification, the 16S ribosomal subunit rRNA sequence of strain IPPMD-315 is shown in SEQ ID NO.1, and the 16S ribosomal subunit rRNA sequence of strain IPPMD-360 is shown in SEQ ID NO.2.

[0055] On the NCBI website, the 16S ribosomal subunit rRNA sequence of strain IPPMD-315 was obtained and searched for and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. Through sequence comparison, strain IPPMD-315 was found to be similar to *Bacillus simplex* (…). Bacillus simplex The sequence closest to ( Figure 1 (f) This result is consistent with the morphological identification results. Combining the colony morphology characteristics, spore morphology characteristics, and molecular biological identification results, the strain IPPMD-315 we isolated is a simple Bacillus (f). Bacillus simplex Therefore, the newly screened strain was named *Bacillus simplex*. Bacillus simplex IPPMD-315.

[0056] The 16S ribosomal subunit rRNA gene sequence of strain IPPMD-360 was determined and searched for and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. Through sequence comparison, strain IPPMD-360 was found to be similar to *Bacillus belyssae* (…). Bacillus velezensis The sequence closest to ( Figure 1 The result (g) is consistent with the morphological identification results. Combining the colony morphology characteristics, spore morphology characteristics, and molecular biological identification results, the strain IPPMD-360 we isolated is *Bacillus belye* (…). Bacillus velezensis Therefore, the newly screened strain was named *Bacillus belyssus*. Bacillus velezensis IPPMD-360.

[0057] Example 2: Simple Bacillus IPPMD-315 promotes biofilm formation of Bacillus belyss IPPMD-360 2.1 Culture medium preparation

[0058] LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0059] 2.2 Crystal violet staining assay for biofilms verified that Bacillus simplex IPPMD-315 promotes biofilm formation of Bacillus belyssioides IPPMD-360.

[0060] Bacillus simplex IPPMD-315 and Bacillus bereaves IPPMD-36012 h were activated on LB solid and stored at -80℃. Single colonies were added to 10 mL of LB liquid and incubated at 37℃ for 12 h. 1 mL of the overnight culture was then transferred to 100 mL of LB liquid and incubated for another 12 h. The OD was adjusted to 0.5, and 600 μL was transferred to a 24-well plate and incubated at 37℃ for 24 h. Excess culture medium was slowly aspirated with 1 mL of insulin, and the plate was washed once with PBS. The plate was fixed with methanol for 15 min, dried at room temperature for 15 min, stained with 0.01% crystal violet for 20 min, washed three times with PBS, air-dried, and dissolved in 75% ethanol for 5 min to completely dissolve the biofilm. The solution was diluted 20-fold, and the absorbance was measured at 600 nm using a microplate reader.

[0061] The results showed that, compared with the control group, the combination of Bacillus belye IPPMD-360 and Bacillus simplex IPPMD-315 produced a deeper crystal violet staining than Bacillus belye IPPMD-360 alone. This was further confirmed by measuring the OD... 600 The OD values ​​of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 were found to be low after mixed culture. 600=0.73, while the OD of Bacillus belysin IPPMD-360 cultured alone was... 600 =0.537, indicating that Bacillus simplex IPPMD-315 can promote the formation of biofilm of Bacillus belyss IPPMD-360. Figure 2 (a and b in the text).

[0062] 2.3 Bacterial growth curve experiment to verify that Bacillus simplex IPPMD-315 promotes the formation of biofilm from Bacillus belyss IPPMD-360

[0063] Bacillus simplex IPPMD-315 and Bacillus bereaves IPPMD-360 were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. The strains were collected during the logarithmic growth phase, washed three times with LB liquid medium to thoroughly remove the fermentation broth, and the OD of the bacterial culture was adjusted. 600 =1, then dilute 100-fold and set aside. Growth curve determination was performed in 96-well growth plates, with 200 μL of the prepared bacterial solution added to each well. Five replicates were performed for each treatment to ensure data reliability. Bioscreen The fully automated growth curve analyzer was used to set the growth plate culture conditions to 37℃, 200 rpm, and shaking culture for 24 hours. The OD of the bacterial solution in each well was measured every 1 hour. 600 value.

[0064] The results showed that the OD value of Bacillus simplex IPPMD-315 and Bacillus belycei IPPMD-360 after co-culture was higher than that of Bacillus belycei IPPMD-360 alone. Bacillus simplex IPPMD-315 could promote the increase of the OD value of Bacillus belycei IPPMD-360. Figure 2 (c in the text)

[0065] Example 3: The combination of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 promotes the growth of maize plants.

[0066] 3.1 Culture medium preparation

[0067] LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0068] 3.2 Test strains

[0069] Bacillus belyssus IPPMD-360, Bacillus simplex IPPMD-315.

[0070] 3.3 Pot experiment to verify the growth-promoting effects of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 on maize plants.

[0071] Mix nutrient soil and vermiculite at a volume ratio of 3:1, and sterilize by dry heat at 121℃ for 3 hours. Inoculate *Bacillus belyssima* IPPMD-360 and *Bacillus simplex* IPPMD-315 separately into LB liquid medium, incubate overnight at 37℃ and 200 rpm with shaking, centrifuge at 6000 rpm for 10 min at room temperature, wash three times with sterile water, and adjust OD. 600 =1, reserved. Plant 3 uniformly sized corn seeds in each pot. 12 pots constitute one treatment group, for a total of 4 treatment groups. The treatment solutions and the amount of each corn plant treated are as follows: A – Blank control (15 mL water), B – Inoculated with 15 mL of *Bacillus belyssum* IPPMD-360 bacterial solution, C – Inoculated with 7.5 mL of *Bacillus belyssum* IPPMD-360 bacterial solution + 7.5 mL of *Bacillus simplex* IPPMD-315 bacterial solution, D – Inoculated with 15 mL of *Bacillus simplex* IPPMD-315 bacterial solution. At sowing, inoculate the corn seeds with the above treatment solutions A and D. Maintain soil moisture throughout the growing process. Observe plant growth 21 days after inoculation.

[0072] The results showed that, compared with the blank control group A, the fresh weight of maize plants in group B increased by 3.97%, the plant height increased by 9.25%, and the stem diameter was not significantly different from that in group A; compared with the blank control group A, the fresh weight of maize plants in group C increased by 56.6%, the plant height increased by 23.01%, and the stem diameter increased by 10%; compared with the blank control group A, the fresh weight of maize plants in group D increased by 6.77%, the plant height increased by 1.23%, and group C had no effect on promoting stem diameter. Figure 3 Compared to group B, group C corn plants showed a 50.59% increase in fresh weight, a 12.58% increase in plant height, and a 10.43% increase in stem diameter. Compared to group D, group C corn plants showed a 46.64% increase in fresh weight, a 21.52% increase in plant height, and an 11.80% increase in stem diameter. Figure 3 ).

[0073] Example 4: Pot experiment to verify the control efficacy of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 against maize Fusarium stalk rot.

[0074] 4.1 Culture medium preparation

[0075] LB medium: 10 g peptone, 5 g sodium chloride, 10 g yeast extract, 15 g agar powder, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0076] PDA medium: Difco™ Potato Dextrose Agar 39 g, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0077] Mung bean soup culture medium: 10 g mung beans, boiled in boiling water for 20 min, filtered through three layers of gauze, water added to make up to 1 L, and autoclaved at 121℃ for 15 min.

[0078] 4.2 Test strains

[0079] Bacillus belyssus IPPMD-360, Bacillus simplex IPPMD-315.

[0080] Fusarium graminearum ( Fusarium graminearum Strain P067 was isolated from the stem base of maize plants infected with stem rot at the Jiangcheng Experimental Base in Yunnan Province and is preserved in the Maize Diseases Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0081] 4.3 Pot experiment to verify the control efficacy of Bacillus belyssioides IPPMD-360 and Bacillus simplex IPPMD-315 against maize stalk rot.

[0082] Mix nutrient soil and vermiculite at a volume ratio of 3:1, and sterilize by dry heat at 121℃ for 3 h. Inoculate the tested strain *Fusarium graminearum* P067 into the sporulation medium (mung bean soup) and incubate at 25℃ and 150 rpm for 7 days. Centrifuge *Fusarium graminearum* P067 at 6000 rpm for 10 min at room temperature, wash three times with sterile water, and adjust the spore suspension to 1×10⁻⁶. 5 Bacillus belye IPPMD-360 and Bacillus simplex IPPMD-315 were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm (12–18 h). After culture, the culture was centrifuged at 6000 rpm for 10 min at room temperature, washed three times with sterile water, and the OD of the culture was adjusted. 600 =1.

[0083] Three uniformly sized corn seeds were planted in each pot. Twelve pots were considered one treatment group, for a total of four treatment groups. The treatment solutions and the amounts used per corn plant were as follows: A – (Control) 15 mL of *Fusarium graminearum* P067 spore suspension + 15 mL of water; B – 15 mL of *Bacillus belyssum* IPPMD-360 bacterial suspension and 15 mL of *Fusarium graminearum* P067 spore suspension; C – 7.5 mL of *Bacillus belyssum* IPPMD-360 + 7.5 mL of *Bacillus simplex* IPPMD-315 bacterial suspension and 15 mL of *Fusarium graminearum* P067 spore suspension; D – 15 mL of *Bacillus simplex* IPPMD-315 bacterial suspension and 15 mL of *Fusarium graminearum* P067 spore suspension. At sowing, the treatment solutions A and D were inoculated next to the corn seeds. Throughout the growing process, the soil was kept moist. Observe the plant growth 21 days after inoculation.

[0084] The results showed that, observing the growth of the four groups of maize plants, group A maize plants infected with the tested strain Fusarium graminearum P067 experienced wilting of the outer stem and root rot symptoms. Compared with the control group A, the incidence of stem rot in group B maize decreased by 24%; compared with the control group A, the incidence of stem rot in group C maize decreased by 38%; and compared with the control group A, the incidence of stem rot in group D maize decreased by 8%. Furthermore, we found that despite inoculation with Fusarium graminearum P607, group B and group C still promoted maize plant height compared to group A, increasing it by 23.07% and 45% respectively, while group D showed no significant difference compared to group A. Figure 4 ).

[0085] Example 5: Seed germination test to verify the promoting effect of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 on maize germination.

[0086] 5.1 Test formulation

[0087] Bacillus belyssin IPPMD-360 seed coating agent and Bacillus belyssin IPPMD-360 + Bacillus simplex IPPMD-315 seed coating agent, with a total effective viable count of ≥2.0 × 10⁻⁶. 9 CFU / mL; the recommended dosage is 5 mL of seed coating agent to 15 mL of water for 1 kg of corn seeds, and mix well.

[0088] The chemical seed dressing agent, fludioxonil, is a 25 g / L fludioxonil suspension seed dressing agent (Cuiying), with a dosage of 1.5 mL / kg of seeds. The manufacturer is Syngenta Nantong Crop Protection Co., Ltd.

[0089] 5.2 The promoting effect of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 on maize germination

[0090] Take seeds of the maize variety Zhengdan 958, disinfect the surface with 75% alcohol, rinse thoroughly with sterile water, and take the seed coating agent according to the dosage (5 mL of seed coating agent per kg of maize seeds, diluted with 15 mL of sterile water). Dilute the seed coating agent with three times the required volume of sterile water (to ensure the seed coating agent evenly coats the maize seeds), then mix it thoroughly with the maize seeds, and air dry for later use. For chemical seed coating with fludioxonil: take the fludioxonil suspension seed coating agent according to the dosage (1.5 mL of fludioxonil suspension seed coating agent per kg of maize seeds, diluted with 15 mL of sterile water), and dilute it with the corresponding proportion of sterile water. Mix it thoroughly with the maize seeds, and air dry for later use. Add an appropriate amount of sterile water to sterilized filter paper, replenishing the sterile water as needed during seed culture. Calculate the seed germination rate after 5 days.

[0091] The results showed that the germination rate of seeds treated with a combination of *Bacillus belyssus* IPPMD-360 and *Bacillus simplex* IPPMD-315 was 86.63%, while the germination rate of seeds treated with *Bacillus belyssus* IPPMD-360 alone was 81.1%, the germination rate of the control group (CK) was 71.1%, and the germination rate of seeds treated with the chemical seed dressing agent fludioxonil was 76.67%. This indicates that *Bacillus simplex* IPPMD-315 promoted the germination rate of maize seeds treated with *Bacillus belyssus* IPPMD-360. Figure 5 ).

[0092] Example 6: Seed dressing pot experiment to verify the control efficacy of Bacillus belyssima IPPMD-360 and Bacillus simplex IPPMD-315 against maize stalk rot.

[0093] 6.1 Culture medium preparation

[0094] PDA medium: Difco™ Potato Dextrose Agar 39 g, add water to a final volume of 1 L; autoclave at 121°C for 15 min.

[0095] Mung bean soup culture medium: 10 g mung beans, boiled in boiling water for 20 min, filtered through three layers of gauze, water added to make up to 1 L, and autoclaved at 121℃ for 15 min.

[0096] 6.2 Seed dressing pot experiment to verify the control efficacy of Bacillus vesicularis IPPMD-360 and Bacillus simplex IPPMD-315 against maize stalk rot.

[0097] The tested strain *Fusarium graminearum* P067 was inoculated into mung bean soup medium for sporulation and cultured at 25°C and 150 rpm for 7 days for later use. *Fusarium graminearum* P067 was centrifuged at 6000 rpm for 10 min at room temperature, washed three times with sterile water, and the spore suspension was adjusted to 1×10⁻⁶. 5 per mL.

[0098] Mix nutrient soil and vermiculite in a volume ratio of 3:1, and sterilize by dry heat at 121℃ for 3 hours for later use.

[0099] Take corn variety B73 seeds, disinfect the surface with 75% alcohol, rinse thoroughly with sterile water, take 5 mL of seed coating agent per kg of seeds according to the seed coating agent concentration, dilute the seed coating agent with 3 times the volume of sterile water (to ensure that the seed coating agent can evenly coat the corn seeds), then mix it with the corn seeds, and let it dry for later use.

[0100] Three uniformly sized corn seeds were planted in each pot. Twelve pots were considered one treatment group, resulting in four treatment groups: A – (blank control) Corn seeds treated with 15 mL of water + 15 mL of Fusarium graminearum P067 spore suspension; B – Corn seeds treated with Bacillus belysium IPPMD-360 + 15 mL of Fusarium graminearum P067 spore suspension; C – Corn seeds treated with Bacillus belysium IPPMD-360 + Bacillus simplex IPPMD-315 + 15 mL of Fusarium graminearum P067 spore suspension. The specific treatment involved treating the seeds first, then applying the Fusarium graminearum P067 spore suspension to each corn seed at sowing time via root irrigation. Throughout the growing process, the soil was kept moist. Plant growth was observed 21 days after inoculation.

[0101] The results showed that, observing the growth of the four groups of maize plants, group A maize plants infected with the tested strain Fusarium graminearum P067 experienced wilting of the outer stems, root rot, and significantly inhibited plant height. Compared with group A, the incidence of stalk rot in group B maize decreased by 10.33%; compared with group A, the incidence of stalk rot in group C maize decreased by 21.67%. Figure 6 Furthermore, one-way ANOVA revealed that inoculation with Fusarium graminearum P067 spore suspension significantly inhibited plant height. The plant height in group C increased by 30.05% compared to group A (inoculated only with Fusarium graminearum P067 spore suspension), the plant height in group B increased by 16.38% compared to group A, and the plant height in group C increased by 16.67% compared to group B. Figure 6 The results indicate that the combination of Bacillus belye IPPMD-360 and Bacillus simplex IPPMD-315 has a good disease resistance effect against Fusarium graminearum P067 and also promotes plant height.

[0102] Example 7: Field trial of seed dressing agents to verify the promoting effect of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 on maize growth phenotype.

[0103] 7.1 Field experiments to verify the promoting effects of Bacillus belyssus IPPMD-360 and Bacillus simplex IPPMD-315 on maize plant growth phenotypes.

[0104] Bacillus bereaves IPPMD-360 seed coating agent and Bacillus bereaves IPPMD-360 + Bacillus simplex IPPMD-315 seed coating agent (these two seed coating agents differ from Penicillium baicalensis seed coating agents in the strains used, while the other excipients are the same), with a total effective viable count ≥ 2.0 × 10⁻⁶. 9 CFU / mL; the recommended dosage is 5 mL of seed coating agent to 15 mL of water for 1 kg of corn seeds, and mix well.

[0105] Penicillium baicale seed coating agent is a suspension seed coating agent processed by Muen (Beijing) Biotechnology Co., Ltd. The recommended dosage is 5 mL of seed coating agent to 15 mL of water for 1 kg of corn seeds, mixed well, and used as a biological seed coating agent control.

[0106] The chemical seed dressing agent, fludioxonil, is a 25 g / L fludioxonil suspension seed dressing agent (Cuiying). The dosage is 1.5 mL / kg of corn seeds, diluted with 15 mL of water and mixed well. The manufacturer is Syngenta Nantong Crop Protection Co., Ltd.

[0107] Seeds of maize variety B73 were surface-sterilized with 75% alcohol and then thoroughly rinsed with sterile water. Following the dosage standards for each seed dressing agent, the following seed dressing agents were diluted accordingly: Bacillus bereaves IPPMD-360, Bacillus bereaves IPPMD-360 + Bacillus simplex IPPMD-315, Penicillium baicaleum seed dressing, and fludioxonil seed dressing. Each agent was then mixed with maize seeds, dried, and stored separately. Five treatments were included: uncoated (CK), seed dressed with fludioxonil, seed dressed with Bacillus bereaves IPPMD-360, seed dressed with Bacillus bereaves IPPMD-360 and Bacillus simplex IPPMD-315, and seed dressed with Penicillium baicaleum. Each treatment was planted in a single plot, 15 m long and 12 m wide. Furrows were machine-made, with a plant spacing of 25 cm, a row spacing of 60 cm, and a sowing depth of 5 cm. Seeds were sown manually, one seed at a time. Based on the corn growth cycle, after sowing, a five-point sampling method was used to investigate the growth of corn under different seed dressing agents. Five plants were sampled at each point, and plant height, fresh weight, and stem diameter were measured.

[0108] The results showed that, compared with Bacillus belycei IPPMD-315 alone, coating Bacillus belycei IPPMD-360 with Bacillus simplex significantly promoted maize plant height, fresh weight, and stem diameter during the seedling stage. Figure 7 ).

[0109] In summary, pot experiments showed that the combined application of *Bacillus berreatus* IPPMD-360 and *Bacillus simplex* IPPMD-315 via root irrigation promoted maize plant growth; the combined *Bacillus berreatus* IPPMD-360 + *Bacillus simplex* IPPMD-315 bacterial suspension had a certain control effect on maize stalk rot; and the combined *Bacillus berreatus* IPPMD-360 + *Bacillus simplex* IPPMD-315 seed dressing agent promoted maize seed germination and plant growth. The application of the combined *Bacillus berreatus* IPPMD-360 + *Bacillus simplex* IPPMD-315 not only improved the diversity of rhizosphere microorganisms in maize but also promoted the growth of maize seedlings (plant height, fresh weight, and stem diameter). It also provided some control over soil-borne diseases like stalk rot through biological control, avoiding the environmental pollution caused by chemical control methods, making it more environmentally friendly and efficient.

Claims

1. Simple Bacillus ( Bacillus simplex IPPMD-315, characterized in that, The accession number is CGMCCNo.36627.

2. A complex microbial community, characterized in that, Bacillus belesiensis ( Bacillus velezensis The composition consists of IPMMD-360 and the simple Bacillus IPMMD-315 as described in claim 1, wherein the preservation number of the Bacillus belyssus IPMMD-360 is CGMCC No. 36628.

3. A biological agent, characterized in that, The active ingredient is a culture containing Bacillus belyssima IPPMD-360 and the simple Bacillus IPPMD-315 as described in claim 1, or a culture containing Bacillus belyssima IPPMD-360 and the simple Bacillus IPPMD-315 as described in claim 1; the preservation number of Bacillus belyssima IPPMD-360 is CGMCC No. 36628.

4. A bio-fertilizer, characterized in that, It contains the biological agent and fertilizer described in claim 3.

5. A seed treatment agent, characterized in that, It contains the biological agent and seed coating agent excipients as described in claim 3.

6. The use of the biological agent according to claim 3 in at least one of the following (1) to (3): (1) Promotes corn seed germination; (2) Promotes corn growth; (3) Control corn stalk base rot.

7. The application according to claim 6, characterized in that, The aforementioned corn stalk base rot is caused by Fusarium graminearum (… Fusarium graminearum Corn stalk base rot caused by ) 8. A method for promoting maize growth and preventing maize stalk rot, characterized in that, Includes the following steps: Apply the biological agent of claim 3 to corn seeds or corn plants.

9. The method according to claim 8, characterized in that, The biological agent is applied to corn seeds by seed dressing or coating; the biological agent is applied to corn plants by root irrigation.

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