Bacillus safensis AHAU-WU410 and application thereof in prevention and treatment of bacterial wilt of corn and in promotion of corn plant growth

Abstract

The present application relates to a kind of bacillus safensis AHAU-WU410 and its application in the prevention and treatment of corn bacterial wilt, promote the growth of corn plant, belong to biological control technical field, the live pure culture of bacillus safensis (Bacillus safensis) AHAU-WU410 is preserved in China typical culture collection of Wuhan University, and the preservation date is August 4, 2025, and the preservation number is CCTCC NO: M 20251769, bacillus safensis AHAU-WU410 has significant antagonism to the pathogenic bacteria of corn bacterial wilt Pantoea stewartii, also can promote the growth of corn plant by secreting IAA and exerting phosphorus solubilization effect.The present application has screened and identified bacillus safensis AHAU-WU410, and the prevention and treatment effect, biological control mechanism and growth-promoting function of the biocontrol bacteria have been verified and analyzed, which provides a new direction and theoretical basis for the development of corn bacterial disease control related green biocontrol agent.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to a Bacillus sabensis AHAU-WU410 and its application in controlling bacterial wilt of maize and promoting maize plant growth. Background Technology

[0002] Maize (Zea mays L.) belongs to the genus Zea in the family Poaceae and is one of the world's highest-yielding food crops. Stewart's bacterial wilt of corn is a common disease caused by Pantoeastewartii ssp. Stewartii (hereinafter referred to as PSS). The pathogen can infect maize at all stages of growth, and symptoms include severe spotting and wilting on leaves and stems. The vascular bundles are filled with bright yellow bacterial slime, leading to the death of leaves, flowers, fruits, stems, and even the entire plant, resulting in significant yield reduction. Therefore, research and control of the pathogen of maize bacterial wilt are of great significance for maintaining global food security.

[0003] Biological control is a method of pest control that utilizes interspecific relationships between organisms, such as introducing natural enemies or developing biological agents, to achieve control effects. Compared to chemical control methods, it has advantages such as being environmentally friendly, highly safe, low-cost, and preventing pathogens from developing resistance, making it more suitable for the needs of sustainable agricultural development. Developing biological control bacteria is one of the core pathways of biological control. Bacillus sp., with its broad-spectrum antibacterial properties and strong environmental adaptability, has shown great application potential in the field of crop disease control.

[0004] Bacillus safortus is a species of Bacillus. Currently, there are studies on the use of Bacillus safortus to suppress rice blast and eucalyptus bacterial wilt in the field. However, there are few reports on the use of Bacillus safortus to control bacterial wilt of maize caused by Pantotheca stevia. In this regard, this invention proposes a Bacillus safortus AHAU-WU410 and its application in the control of bacterial wilt of maize and the promotion of maize plant growth. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus sabensis AHAU-WU410 and its application in controlling bacterial wilt of maize and promoting maize plant growth in order to solve the above problems.

[0006] The present invention achieves the above objectives through the following technical solutions: This invention provides a live pure culture of Bacillus safensis AHAU-WU410, which is deposited at the China Center for Type Culture Collection, Wuhan University, on August 4, 2025, with accession number CCTCC NO: M 20251769.

[0007] This invention also provides the application of Bacillus sabensis AHAU-WU410 in the control of bacterial wilt of maize.

[0008] As a further optimization of the present invention, Bacillus saffron AHAU-WU410 has a significant antagonistic effect on Pantoea stewartii ssp. stewartii, the pathogen of bacterial wilt of maize.

[0009] As a further optimization of the present invention, the antagonistic effect of Bacillus sabovellae AHAU-WU410 against Pantothenia gravis includes: (1) Inhibit the activity of Pantotheca stearothermia through short-distance physical contact or delivery of highly effective bactericidal substances; (2) Inhibit the expression of pan-Stokes virulence genes, including fliA, crp, pgm, cheY, fadD and hrpA; (3) Enhance the expression of disease resistance genes WRKY and PR-1 in maize to improve maize’s disease resistance.

[0010] This invention also provides the application of Bacillus salsa AHAU-WU410 in promoting maize plant growth. Bacillus salsa AHAU-WU410 promotes maize plant growth by secreting IAA and exerting phosphorus solubilization.

[0011] As a further optimization of the present invention, promoting corn growth refers to promoting the increase of corn plant height.

[0012] This invention also provides the application of Bacillus sabensis AHAU-WU410 in the prevention and control of bacterial wilt disease in maize and the promotion of maize plant growth.

[0013] The present invention also provides a biocontrol agent containing Bacillus sabriophyte AHAU-WU410.

[0014] The beneficial effects of this invention are as follows: This invention employs the inhibition zone method to screen bacterial strains with pathogen-inhibiting functions obtained from diseased tissues of maize bacterial diseases. The resulting strain, T056-2-4, exhibits strong inhibitory activity against *Pantotheca stearothermiae*, the causative agent of maize bacterial wilt, and was identified as *Bacillus safoetida*. Analysis of the biocontrol mechanism of T056-2-4 revealed that it not only inhibits the activity of *Pantotheca stearothermiae* through short-distance physical contact with cells or by delivering highly effective bactericidal substances, but also inhibits the expression of the core virulence gene of *Pantotheca stearothermiae* and enhances the expression of the resistance genes WRKY and PR-1, thereby achieving a significant control effect against maize bacterial wilt. Furthermore, this invention verifies that the T056-2-4 biocontrol strain can secrete IAA and possess phosphate-solubilizing capabilities, thus promoting maize plant growth. Multiple studies have comprehensively verified the application value of T056-2-4 biocontrol bacteria in controlling bacterial wilt of maize and promoting maize plant growth, providing a new direction and theoretical basis for the development of green biocontrol agents related to the control of maize bacterial diseases. Finally, the T056-2-4 biocontrol bacteria were microbially preserved and renamed Bacillus safensis AHAU-WU410. Attached Figure Description

[0015] Figure 1 Analysis of the antagonistic ability of biocontrol bacteria T056-2-4 against Pantotheca stearothera Pss; Figure 2 Colony morphology analysis, Gram staining analysis, and phylogenetic tree analysis based on 16S rRNA and gyrB sequences of biocontrol bacteria T056-2-4 were performed. Figure 3 Comparative analysis of the antagonistic ability of biocontrol bacterium T056-2-4 and other common types of Bacillus against Pantotheca stevia Pss (Bacillus safensis T056-2-4: Bacillus saffron T056-2-4; Bacillus velezensis T054-1-4: Bacillus belezensis T054-1-4; Bacillus stratosphericus T094-8: Bacillus stratosphericus T094-8; Bacillus subtilis T073-2-2: Bacillus subtilis T073-2-2). Figure 4 Analysis of the IAA production capacity, phosphorus solubilization capacity and growth-promoting capacity of biocontrol bacteria T056-2-4; Figure 5 Analysis of extracellular degradative enzyme activity in biocontrol bacteria T056-2-4; Figure 6Analysis of the inhibitory effect of biocontrol bacteria T056-2-4 on the toxicity of pathogenic bacteria Pss; Figure 7 Analysis of the biocontrol effect of biocontrol bacteria T056-2-4 on maize. Detailed Implementation

[0016] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] 1. Experimental materials (1) The Bacillus safensis AHAU-WU410 used in the experiment is also known as T056-2-4 biocontrol bacteria. Its live pure culture is deposited at the China Center for Type Culture Collection of Wuhan University on August 4, 2025, with accession number CCTCC NO: M 20251769.

[0018] (2) The pathogen used in the experiment was Pantoea stewartii sp. Stewartii, the pathogen of bacterial wilt of maize; (3) The screening materials for biocontrol bacteria were collected from diseased maize tissue samples from maize-growing areas such as Anhui and Hainan. (4) The tested plant type was maize, and the maize variety was B73; (5) LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH=7.0); protease detection medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, skim milk powder 10 g / L); cellulase detection medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sodium carboxymethyl cellulose 10 g / L); inorganic phosphorus solubilizing detection medium (inorganic phosphorus bacteria medium 31.4 g / L); organic phosphorus solubilizing detection medium (organic phosphorus bacteria medium 31.6 g / L). (6) Solid culture medium can be prepared by adding 1.5% (w / v) agar powder to the liquid culture medium. All culture media should be sterilized at 121℃ for 20 min. Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products.

[0019] 2. Experimental Methods 2.1 Isolation, purification, screening, analysis, and identification of biocontrol bacteria for bacterial wilt of maize 2.1.1 Isolation and purification of candidate biocontrol bacteria Nearly one hundred bacterial disease tissue samples from maize-growing areas in Anhui and Hainan provinces were aseptically minced and homogenized with sterile water to obtain a stock solution. Pss was activated to the logarithmic growth phase using LB medium, and after centrifugation and washing, the OD600 of the bacterial culture was adjusted to 0.01. The disease tissue homogenate and Pss bacterial culture were mixed at a 1:2 ratio and co-cultured for 24-48 h. After sterilization at 85℃ for 15 min, 100 μL was plated on LB agar plates and incubated at 30℃ for 48-72 h. Single colonies were picked, streaked for purification, and then stored in glycerol tubes at -80℃ using the shake method.

[0020] 2.1.2 Screening of candidate biocontrol bacteria The candidate representative strains identified in section 2.1.1 were screened using the inhibition zone method to identify candidate biocontrol bacteria with antagonistic activity against Pantotheca stearothera Pss, as follows: Preparation of PSS indicator bacteria screening plates: Take 3 μL of freshly cultured representative candidate biocontrol bacteria suspension and spot-inoculate the suspension onto a PSS indicator bacteria screening plate. Repeat the process three times for each tested strain. After spot-inoculating the candidate biocontrol bacteria, invert the plates and incubate them in a 30℃ constant temperature incubator for 2 days. Observe the formation of antagonistic zones on each PSS indicator bacteria screening plate.

[0021] The results are as follows Figure 1 As shown, strain T056-2-4 has a strong antagonistic effect against Pantoea stewartii ssp. stewartii, thus identifying strain T056-2-4 as a biocontrol strain for bacterial wilt of maize (Pantoeastewartii ssp. stewartii), and selecting this strain for subsequent experiments.

[0022] 2.1.3 Morphological observation and Gram staining analysis of biocontrol strains T056-2-4 bacterial strain was activated by shaking, and 30 μL of fresh bacterial solution was streaked onto LB agar plates in three zones. The plates were then incubated at 30℃, and bacterial morphology was observed. Results showed that on LB agar plates, the T056-2-4 biocontrol bacteria exhibited colony characteristics similar to typical Bacillus sabinatus: raised colonies with relatively regular edges and a smooth surface. After a period of time, the colony surface became rough (e.g., ...). Figure 2 (As shown in A).

[0023] Gram staining was performed on biocontrol bacteria T056-2-4. The bacteria observed under a microscope were dark purple, indicating they were Gram-positive bacteria. The bacterial cells were rod-shaped (e.g., ...). Figure 2As shown in B).

[0024] 2.1.4 Identification of biocontrol bacteria Purified streaked single colonies of strain T056-2-4 were taken, and conserved gene sequences were amplified using universal 16S rRNA primers and gyrB primers (as shown in Table 1). The obtained PCR products were sent to General Biotech (Anhui) Co., Ltd. for sequencing after agarose gel electrophoresis. The sequencing results were submitted to the NCBI website for BLAST homology sequence comparison analysis to construct a phylogenetic tree.

[0025] Table 1: Universal primer sequences for 16S rRNA and gyrB ; A phylogenetic tree constructed based on the 16S rRNA and gyrB gene sequences of the biocontrol bacterium T056-2-4 (as shown in SEQ ID No. 1 and SEQ ID No. 2) is as follows: Figure 2 As shown in C and D, according to the phylogenetic tree, the biocontrol bacterium T056-2-4 is *Bacillus safensis*. A live pure culture of biocontrol bacterium T056-2-4 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, and renamed *Bacillus safensis* AHAU-WU410, with a deposit date of August 4, 2025, and accession number CCTCC NO: M 20251769. For ease of labeling, it will continue to be referred to as T056-2-4 below.

[0026] 2.2 Research on the growth-promoting ability, antibacterial ability, and biocontrol mechanism of biocontrol bacteria 2.2.1 Analysis of the antibacterial ability of biocontrol bacteria The biocontrol bacterium T056-2-4 was compared with several other types of Bacillus species in terms of its PSS antagonistic ability against Pantotheca stevia. The comparison objects included: T054-1-4 (Bacillus velezensis T054-1-4), T094-8 (Bacillus stratosphericus T094-8), and T073-2-2 (Bacillus subtilis T073-2-2).

[0027] The results are as follows Figure 3 As shown in A and B, the T056-2-4 biocontrol bacteria produced the largest antagonistic zone against Pantotheca stearothera Pss, indicating the strongest antagonistic effect.

[0028] 2.2.2 Detection of auxin yield, phosphorus solubilization capacity, and growth-promoting effect of biocontrol bacteria on potted maize The biocontrol bacterium T056-2-4 was activated using LB liquid medium. The bacterial culture was transferred at a 1% inoculum to LB liquid medium containing 100 μL of tryptophan stock solution and cultured at 37℃ with a shaker at 120 rpm / min for 48 h. After centrifugation, the supernatant was collected, and 50 μL of 83% orthophosphate and 4 mL of Salkowski's reagent were added for color development. The absorbance at 530 nm was measured, and the concentration and color change were calculated based on the standard curve to determine the biocontrol bacterium's IAA production capacity. The results showed that the T056-2-4 biocontrol bacterium can secrete IAA (3-indoleacetic acid, a plant growth regulator) into the environment, which can cause the colorimetric reagent to develop color (…). Figure 4 A).

[0029] Three µL of seed culture of biocontrol bacteria T056-2-4 was spotted in the center of a phosphorus-containing medium plate, with three replicates. The presence or absence of a clear zone was used to determine the phosphorus-solubilizing ability of the biocontrol bacteria. The results showed that biocontrol bacteria T056-2-4 could produce a degradation halo on phosphorus-containing plates, exhibiting a strong phosphorus-solubilizing effect. Figure 4 B).

[0030] An appropriate amount of bacterial solution was serially diluted with LB medium to adjust the OD600 value to 1.0. Uniformly growing maize seedlings were selected and transplanted into pre-treated pots. A quantitative inoculation method was used, with 20 mL of bacterial solution slowly poured along the pot wall. The control group was watered with sterile water. The growth and height differences of the maize plants were observed and recorded. Results showed that the maize plant height after 8 days of treatment with the biocontrol bacteria T056-2-4 was significantly higher than that of the water-treated control group. Figure 4 C, D).

[0031] The above experimental results indicate that T056-2-4 biocontrol bacteria has a certain ability to promote plant growth.

[0032] 2.2.3 Detection of extracellular degradative enzyme activity in biocontrol bacteria Three µL of seed culture of biocontrol bacterium T056-2-4 was spotted in the center of cellulase and protease activity assay plates, with three replicates per group. The presence or absence of a clear zone was used to determine the extracellular degradative enzyme activity of the biocontrol bacterium. The results showed that biocontrol bacterium T056-2-4 had strong extracellular cellulase and extracellular protease activities. Figure 5 ).

[0033] 2.2.4. Confrontation experiment and quantitative fluorescence analysis of the expression of the pathogen's Pss virulence gene. Confrontation experiment: Biocontrol bacteria T056-2-4 with OD600=1.0 and pathogenic bacteria Pss were prepared. 3 μL of Pss bacterial solution was inoculated into a square culture medium. After 1 day of culture, 3 μL of T056-2-4 bacterial solution was inoculated at 1 cm, 2 cm and 3 cm away from the pathogen, respectively. The inhibition zone, colony morphology and cell morphology were observed, and the relationship between antagonistic ability and cell contact distance was analyzed.

[0034] The results showed that the colony morphology of Pss exhibited progressive damage as the contact distance between colony cells shortened. When the confrontation distance was close, abnormal phenomena such as degradation and lightening of color appeared at the edge of the Pss colony. Under the microscope, the pathogen cells were found to be damaged, shrunken into irregular granules, and lost their cell integrity. Figure 6 (A, B) indicates that the biocontrol bacteria can inhibit the activity of pathogens through short-distance physical contact or the delivery of highly effective bactericidal substances.

[0035] Analysis of Pss virulence gene expression inhibition: Pss culture broths from the T056-2-4 biocontrol bacteria fermentation supernatant treatment group and the normal control group were set up. Bacterial cells were collected from both groups, and total RNA was extracted and reverse-transcribed into cDNA as a template. Literature review was conducted, and reported pathogen virulence genes fliA (encoding Flagellar Sigma Factor), crp (encoding Global regulator), pgm (encoding Phosphoglucose mutase), cheY (encoding Chemotaxis protein), fadD (encoding Acyl-CoA synthetase), and hrpA (encoding Hrppilus structural protein), as well as the internal reference gene 16S, were selected. Specific primer sequences were designed (as shown in Table 2), and quantitative real-time PCR was performed using SYBR Green dye. The expression of Pss virulence genes inhibited by the biocontrol bacteria was quantitatively analyzed.

[0036] Table 2: Primer sequences for quantitative real-time analysis of Pss internal reference gene 16S and reported toxic genes. ; The results of the quantitative fluorescence experiment showed that, compared with the untreated control group, the expression levels of core virulence genes of Pss, including fliA, crp, pgm, cheY, fadD, and hrpA, were significantly reduced in the T056-2-4 biocontrol bacteria treatment group, indicating a significant inhibition state. Figure 6 C).

[0037] 2.2.5 Pot test for disease resistance efficacy and quantitative fluorescence analysis of maize disease resistance gene expression Potted plant efficacy experiment: A mixed bacterial solution (OD600=1.0) was prepared by mixing PSS bacterial solution and T056-2-4 biocontrol bacterial solution in equal volumes at a 1:1 ratio. Three-week-old B73 maize plants with uniform growth were inoculated on their leaf sheaths. The treatment group was injected with 0.5-1 mL of the mixed bacterial solution, while the control group was injected with an equal volume of PSS bacterial solution. The disease incidence was observed and recorded after 5-10 days, and the control effect of T056-2-4 biocontrol bacteria was analyzed. The results showed that compared with single inoculation with the pathogen PSS, the experimental group inoculated with the mixed PSS and T056-2-4 biocontrol bacteria showed significantly reduced lesion expansion and leaf wilting. Figure 7 A) indicates that the T056-2-4 biocontrol bacteria has a strong inhibitory effect on Pantotheca stearothermia.

[0038] Analysis of induced disease resistance gene expression: RNA was extracted from maize leaves treated with T056-2-4 biocontrol bacteria at different time points and from untreated maize leaves for qRT-PCR amplification. The amplification results were analyzed to detect the expression levels of disease resistance genes WRKY and PR-1, clarifying the biocontrol mechanism of T056-2-4 biocontrol bacteria on maize. Results showed that compared with the untreated control group, the expression levels of disease resistance genes WRKY and PR-1 were significantly increased in maize leaves treated with T056-2-4 biocontrol bacteria. Figure 7 (B) This result indicates that the T056-2-4 biocontrol bacteria can effectively improve the disease resistance of maize.

[0039] 3. Conclusion In summary, T056-2-4 biocontrol agent (i.e., Bacillus safranin AHAU-WU410) has good biocontrol and disease resistance effects on crops and also has a certain growth-promoting ability. This discovery lays the foundation for the subsequent development of related green biocontrol agents for the prevention and control of bacterial diseases in maize.

[0040] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of Bacillus safensis AHAU-WU410 in the control of bacterial wilt in maize, characterized in that: The live pure culture of Bacillus sarfusae AHAU-WU410 is deposited at the China Center for Type Culture Collection, Wuhan University, on August 4, 2025, with accession number CCTCC NO: M 20251769. The described Bacillus sarcodactylis AHAU-WU410 has a significant antagonistic effect against Pantoeastewartii ssp. stewartii, the pathogen of bacterial wilt in maize; The antagonistic effect of *Bacillus sarcodactylis* AHAU-WU410 against *Panthera steudensis* includes: (1) Inhibit the activity of Pantotheca stearothermia through short-distance physical contact or delivery of highly effective bactericidal substances; (2) Inhibit the expression of pan-Stokes virulence genes, including fliA, crp, pgm, cheY, fadD and hrpA; (3) Enhance the expression of disease resistance genes WRKY and PR-1 in maize to improve maize’s disease resistance.

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