A type of Bacillus atrophus, a biocontrol agent and its application

Abstract

This invention isolated and identified a strain of *Bacillus atrophicus* SW from wheat root tissue. Through colony morphology, genomic phylogenetic tree analysis, average nucleotide similarity analysis, and digital DNA-DNA hybridization analysis, it was identified as *Bacillus atrophicus* SW. Plate confrontation experiments demonstrated that the *Bacillus atrophicus* SW strain has a strong inhibitory effect on the growth of *Fusarium graminearum*, and the minimum concentration at which *Bacillus atrophicus* SW inhibits *Fusarium graminearum* growth was determined. Furthermore, the SW strain can produce siderophores, which may help enhance plant nutrient absorption and disease resistance. In in vitro control experiments and greenhouse pot experiments on wheat, the *Bacillus atrophicus* SW strain demonstrated good application effects in the biological control of wheat stem rot, showing its potential and application value in the biological control of wheat diseases. The *Bacillus atrophicus* SW strain also showed good antagonistic effects against *Sclerotinia sclerotiorum*, further demonstrating its broad application potential in agricultural disease control.

Description

Technical Field

[0001] This invention relates to the field of biocontrol strains, specifically to a Bacillus atrophus, a biocontrol agent, and their applications. Background Technology

[0002] Plant fungal diseases pose a serious threat to global food security and agricultural sustainability. In particular, wheat stem rot caused by *Fusarium pseudograminearum* is one of the most destructive diseases affecting wheat globally. Climate change is expected to exacerbate the frequency and severity of wheat stem rot. This disease primarily affects the stem base and roots of wheat plants, leading to wilting, lodging, and reduced yields. More seriously, the pathogen can also produce mycotoxins such as deoxynivalenol and zearalenone, contaminating wheat grains and posing significant risks to human and animal health.

[0003] Plant endophytic microorganisms can colonize internal plant tissues without causing significant harm to the host. They offer various benefits to plant growth and health, including nitrogen fixation, phosphorus solubilization, synthesis of plant hormones, production of siderophores, induction of systemic resistance, and antagonism against plant pathogens. Although the effectiveness of endophytic bacteria as plant probiotics has been extensively studied and confirmed, research literature on specific endophytic bacteria targeting wheat stem base rot is relatively limited. Therefore, screening and expanding an endophytic strain resource bank targeting wheat stem base rot is particularly important. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus atrophus species that has antagonistic effects against Fusarium graminearum and Sclerotinia sclerotiorum and its application in the prevention and control of wheat stem rot and tomato sclerotinia disease.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A type of Bacillus atrophaeus (Bacillus atrophaeus SW strain), classified and named Bacillus atrophaeus, was deposited at the China General Microbiological Culture Collection Center on April 15, 2024, with accession number CGMCC 30329.

[0007] This invention isolated and identified a strain of *Bacillus atrophicus* SW from wheat root tissue. Plate confrontation experiments demonstrated that the *Bacillus atrophicus* SW strain possesses a strong inhibitory effect on the growth of *Fusarium graminearum*, and the minimum concentration at which the *Bacillus atrophicus* SW strain inhibits *Fusarium graminearum* growth was determined. Combined with in vitro control efficacy tests on wheat and greenhouse pot experiments, the good application effect of the *Bacillus atrophicus* SW strain in the biocontrol of wheat stem base rot was demonstrated. It can be used for the biocontrol of fungal diseases in wheat, providing a new strain resource for the biocontrol fungal bacteria resource bank for wheat fungal diseases. The *Bacillus atrophicus* SW strain of this invention has an antagonistic effect against *Botrytis californica*, and its application to diseases caused by *Botrytis californica* can help control fungal diseases in various crops.

[0008] The Bacillus atrophus SW strain of the present invention can secrete siderophores, which have broad application prospects in plant nutrition and inducing plant resistance.

[0009] Furthermore, the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, China General Microbiological Culture Collection Center.

[0010] Furthermore, the nucleotide sequence of Bacillus atrophus SW is shown in SEQ ID NO:1.

[0011] Furthermore, another objective of this application is to provide a biocontrol agent containing the aforementioned Bacillus atrophus SW.

[0012] Furthermore, another object of the present invention is to provide a method for preparing the above-mentioned biocontrol agent, comprising the following steps:

[0013] The above-mentioned Bacillus atrophus SW was inoculated into LB liquid medium, and centrifuged when the culture reached the end of the logarithmic phase. Excess LB medium was washed away with sterile PBS buffer, and the bacterial cells were resuspended in sterile PBS buffer until the OD 600 reached 1.7-1.8, which is the biocontrol agent.

[0014] Another object of the present invention is to provide a biological pesticide or biological fertilizer prepared from the above-mentioned Bacillus atrophus SW or from the above-mentioned biocontrol agent.

[0015] Another object of the present invention is to provide a biocontrol agent or antibacterial agent prepared from the above-mentioned Bacillus atrophus SW or from the above-mentioned biocontrol agent.

[0016] Another object of the present invention is to provide the above-mentioned Bacillus atrophus SW or the above-mentioned biocontrol agent, or the above-mentioned biopesticide or biofertilizer, or the above-mentioned biocontrol agent or antibacterial agent for any of the following applications:

[0017] It is used to antagonize plant pathogens; to control wheat stem base rot; and to control sclerotinia stem rot in tomato caused by Sclerotinia sclerotiorum.

[0018] Furthermore, the plant pathogens include Fusarium pseudograss or Sclerotinia sclerotiorum.

[0019] Another object of the present invention is to provide the application of the above-mentioned Bacillus atrophus SW in the preparation of biocontrol agents, biopesticides, biofertilizers, biocontrol agents, antibacterial agents or plant immune inducers for the prevention and control of plant diseases.

[0020] Furthermore, the plant disease is wheat stem base rot or sclerotinia stem rot of tomato caused by Sclerotinia sclerotiorum.

[0021] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0022] 1. This invention isolated and identified a strain of *Bacillus atrophicus* SW from wheat root tissue. Plate confrontation experiments demonstrated that the *Bacillus atrophicus* SW strain possesses a strong inhibitory effect on the growth of *Fusarium graminearum*, and the minimum concentration at which *Bacillus atrophicus* SW strain inhibits *Fusarium graminearum* growth was determined. Combined with in vitro control efficacy tests on wheat and greenhouse pot experiments, the invention demonstrated the good application effect of *Bacillus atrophicus* SW strain in the biocontrol of wheat stem base rot, indicating its potential for use in the biocontrol of wheat fungal diseases and providing a new strain resource for the biocontrol fungal resource bank of wheat fungal diseases.

[0023] 2. The Bacillus atrophicus SW strain of the present invention has an antagonistic effect against Botrytis californica and can be applied to diseases caused by Botrytis californica, which helps to control fungal diseases of various crops.

[0024] 3. The Bacillus atrophus SW strain of the present invention can secrete siderophores, which have broad application prospects in plant nutrition and inducing plant resistance. Attached Figure Description

[0025] Figure 1 This is a colony diagram of Bacillus atrophus SW.

[0026] Figure 2 This is a phylogenetic tree of the genome of Bacillus atrophus SW.

[0027] Figure 3 The average nucleotide similarity analysis of Bacillus atrophus SW.

[0028] Figure 4 This is a morphological image of Bacillus atrophus SW under a scanning electron microscope.

[0029] Figure 5This is a plate antagonistic graph showing the interaction between Bacillus atrophus SW and Fusarium pseudograss.

[0030] Figure 6 The minimum concentration of Bacillus atrophus SW to inhibit Fusarium pseudograss.

[0031] Figure 7 The effect of Bacillus atrophus SW on Fusarium wilt infection of wheat grains (Figure a shows Fusarium wilt infection of wheat grains; Figure b shows Fusarium wilt infection of wheat grains treated with SW strain; Figure c shows Fusarium wilt infection of wheat grains treated with the chemical pesticide fludioxonil).

[0032] Figure 8 To demonstrate the in vitro control efficacy of Bacillus atrophus SW against Fusarium graminearum-infected wheat seedlings (Figure a shows the negative control, i.e., wheat seedlings infected with Fusarium graminearum treated with sterile phosphate buffer; Figure b shows wheat seedlings infected with Fusarium graminearum treated with SW strain).

[0033] Figure 9 The disease index of Bacillus atrophus SW in the greenhouse for wheat stem base rot.

[0034] Figure 10 A plate confrontation diagram of Bacillus atrophus SW antagonizing Sclerotinia sclerotiorum.

[0035] Figure 11 The clear zone is a siderophore produced by Bacillus atrophus SW. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Example 1

[0039] Isolation of Bacillus atrophus SW strain

[0040] Bacillus atrophicus SW strain is a strain of Bacillus atrophicus isolated from the root tissue of healthy wheat plants collected in the field in Fan County, Puyang City, Henan Province.

[0041] The method for isolating the Bacillus atrophus SW strain includes the following steps:

[0042] (1) Wash the dust off the surface of the root tissue of the wheat sample with pure water, disinfect the sample surface, and grind it into a homogenate in a sterile mortar in a clean bench.

[0043] (2) Mix the sample with sterile phosphate buffer (PBS) and perform serial dilutions. Take the different dilutions and spread them on tryptone soybean agar (TSA) plates. Incubate them upside down in a 30°C incubator.

[0044] (3) When colonies grow on the plate, isolate, purify and preserve the strain based on the size and morphology of the colonies growing on the culture medium.

[0045] The colony diagram of Bacillus atrophus SW is attached. Figure 1 .

[0046] Strain identification of Bacillus atrophus SW

[0047] Bacillus atrophus SW was sent to Guangdong Megabio Technology Co., Ltd. for whole-genome second-generation + third-generation sequencing. A phylogenetic tree was constructed from the genome (see appendix). Figure 2 ) and average nucleotide similarity analysis (see appendix) Figure 3 We presume that the antagonistic bacterium of this invention is Bacillus atrophus, and name it Bacillus atrophus SW.

[0048] The 16S rRNA sequence SEQ ID NO:1 is published as follows:

[0049] >Bacillus atrophus SW 16S ribosomal RNA

[0050]

[0051] Morphological observation of Bacillus atrophus under SW scanning electron microscopy

[0052] Samples for scanning electron microscopy (SEM) of *Bacillus atrophicus* SW were prepared by fixing the bacterial suspension with glutaraldehyde and dehydrating it using different ethanol gradients. The suspension was then washed with anhydrous ethanol and resuspended for later use. After CO2 drying, the *Bacillus atrophicus* SW samples were sputter-coated with gold and then observed under a scanning electron microscope. The morphology of *Bacillus atrophicus* SW under a scanning electron microscope is shown in the appendix. Figure 4 It has a short rod-like shape.

[0053] Antagonistic test of Bacillus atrophus SW against Fusarium pseudobulb.

[0054] 4.1 Plate antagonism test of Bacillus atrophus SW against Fusarium graminearum

[0055] This invention uses the plate confrontation method to determine the antagonistic ability of Bacillus atrophus SW against Fusarium pseudograss.

[0056] The culture medium used was potato dextrose agar (PDA) medium, which consisted of: 200 g / L peeled potatoes, 20 g / L glucose, 20 g / L agar, and autoclaved at 115°C for 20 min.

[0057] Preparation of *Bacillus atrophus* SW bacterial culture: A biocontrol strain stored at -80℃ was inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 48 h with shaking to obtain a seed culture. A sterile inoculation loop was used to streak the seed culture onto LB agar medium and cultured at 37℃ for 48 h to obtain fresh single colonies. Single colonies were picked and inoculated into LB liquid medium and cultured at 37℃ and 220 rpm for 48 h. Excess LB medium was washed away with sterile PBS buffer, and the bacterial cells were resuspended in sterile PBS buffer to induce OD. 600 When the concentration reaches 1.6 to 1.7, it is considered a biocontrol agent.

[0058] Pick a 5mm diameter Fusarium graminearum mycelium cake from the center of a PDA medium plate, and inoculate the above-mentioned biocontrol agent 2.5cm from the center. Use sterile phosphate buffer as a negative control. Incubate upright in a 26℃ incubator for 5-7 days. When the control group of Fusarium graminearum has completely covered the plate, calculate the inhibition rate of each tested species.

[0059] The formula for calculating the inhibition rate is: Inhibition rate = (Coronary diameter of control group / Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0060] Table 1. Inhibition rate of strain SW against Fusarium oxysporum

[0061]

[0062] As shown in Table 1 and Figure 5 As shown, Bacillus atrophicus SW has a strong antagonistic effect on Fusarium pseudograss, further confirming that Bacillus atrophicus SW is an antagonistic species to Fusarium pseudograss.

[0063] 4.2 Minimum concentration of *Bacillus atrophus* SW to inhibit *Fusarium graminearum*

[0064] Fusarium graminearum mycelium was inoculated into sodium carboxymethyl cellulose liquid medium and cultured at 26°C and 180 rpm for 7 days. The culture solution was filtered through three layers of sterile gauze, centrifuged at 5000 rpm for 10 minutes to collect spores, and resuspended in sterile PBS to obtain Fusarium graminearum spore solution.

[0065] Bacillus atrophus SW was administered at a rate of 5 × 10 1 -5×10 8 The final concentration gradient of CFU / mL is 10 times that of the spore concentration. 5 Mix / mL of *Fusarium graminearum* spore suspension, spot the mixture onto agar plates, and record the minimum SW bacterial concentration corresponding to no growth of *Fusarium graminearum* as the minimum inhibitory concentration (MIC). See the attached table for the experimental results. Figure 6 As shown in the figure, the minimum concentration of *Bacillus atrophus* SW to inhibit the growth of *Fusarium graminearum* is 5 × 10⁻⁶. 6 CFU / mL.

[0066] 4.3 Effects of Bacillus atrophus SW on Fusarium graminearum infection of wheat grains

[0067] Fusarium graminearum was mixed with Bacillus atrophus SW or the chemical pesticide fludioxonil, and sprayed evenly onto the surface of wheat grains. The mixture was then incubated under moist conditions for 7 days. The experimental results are attached. Figure 7 As shown in the figure, Bacillus atrophus SW can effectively inhibit the growth of Fusarium graminearum on wheat grains, but its inhibitory effect is slightly lower than that of the chemical pesticide fludioxonil.

[0068] 4.4 In vitro protective efficacy of Bacillus atrophus SW against Fusarium graminearum infection in wheat seedlings

[0069] After surface disinfection of wheat seeds, germinate for 2-3 days. Select wheat seedlings of similar growth for coleoptile infection. Remove the tips of the wheat coleoptiles with scissors to create a wound. First, spray with 10µL of *Bacillus atrophicus* SW bacterial solution. 12 hours later, inoculate the wound with 5µL of *Fusarium graminearum* spore suspension. Use sterile phosphate buffer as a negative control. Repeat the experiment three times. Observe seedling growth daily and water regularly. After 5 days, observe the control effect of *Bacillus atrophicus* SW on *Fusarium graminearum* infection in wheat seedlings. Results are attached. Figure 8As shown in the figure, Bacillus atrophus SW can help wheat seedlings resist infection by Fusarium graminearum.

[0070] 4.5 Control effect of Bacillus atrophus SW on Fusarium oxysporum infection in potted wheat.

[0071] After boiling millet in water, rinse it with cold water to remove surface sugars, drain the water, and spread the millet evenly on newspaper to air dry. Place the dried millet into Erlenmeyer flasks for sterilization, and let them sit for 12 hours to further dry the surface. Inoculate the millet-filled Erlenmeyer flasks with 5mm diameter Fusarium oxysporum mycelium cakes (cultivated for 7 days) and incubate at 26℃. After 7 days of incubation, the millet grains can be used as pathogenic millet for Fusarium oxysporum to infect potted plants.

[0072] The conventional wheat variety Jimai 22 was selected for sowing, with 15 seeds per pot. Watering was done every two days after sowing. Once the wheat seedlings emerged, a fungicide treatment was immediately applied by pouring Bacillus atrophicus SW seed solution into the pots. A negative control was used without fungicide treatment. On the 7th day after sowing, millet-infected grains were evenly spread in the pots, ensuring at least two infected grains on each wheat seedling. A layer of wheat bran was then spread on top of the infected grains, and finally, a layer of substrate soil was placed on top of the wheat bran.

[0073] Two weeks after infection, the wheat was removed from the substrate, the roots were rinsed with clean water, and after washing and drying, the plant height and fresh weight were recorded to investigate the disease results.

[0074] The grading criteria are as follows: 0: No disease in the plant; 1: The roots are obviously brown or the area below the first leaf sheath is yellow; 3: The first leaf sheath is obviously brown but not black; 7: The third leaf sheath is brown; 9: The plant's roots are rotten and the plant is dead or the leaves are withered and dead.

[0075] Based on the obtained disease severity, the disease index and control effect of each treatment are calculated using the following formula:

[0076] Disease index = 100 × ∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)

[0077] Prevention and control effect (%) = (CK disease index - treatment disease index) / (CK disease index) × 100%.

[0078] The potted disease index of Bacillus atrophus SW against wheat stem base rot is shown in [the figure]. Figure 9 The efficacy index was 80.41% ± 0.16%.

[0079] Antagonistic test of Bacillus atrophus SW against Sclerotinia sclerotiorum.

[0080] Two symmetrical points on the diameter of a PDA plate, equidistant from both sides and the center, were inoculated with *Sclerotinia sclerotiorum* mycelial cakes (5 mm in diameter) and *Bacillus atrophicus* SW seed culture at the two points, respectively. *Sclerotinia sclerotiorum* inoculation and sterile phosphate buffer were used as negative controls. The experiment was repeated in three groups. The plates were incubated upright at 26°C for 5–7 days. When the *Sclerotinia sclerotiorum* mycelia in the control group had completely covered the plate, the inhibition rate of *Bacillus atrophicus* SW against *Sclerotinia sclerotiorum* was calculated.

[0081] The formula for calculating the inhibition rate is: Inhibition rate = (colon radius of the control group / colony radius of the treatment group) / colony radius of the control group × 100%.

[0082] Table 2. Inhibition rate of strain SW against Sclerotinia sclerotiorum.

[0083]

[0084] As shown in Table 2 and Figure 10 As shown, Bacillus atrophus SW has a strong antagonistic effect on Sclerotinia sclerotiorum.

[0085] Bacillus atrophus SW secretes siderophores

[0086] The presence of siderophores in *Bacillus atrophicus* SW was evaluated using CAS agar plates. Seed culture of *Bacillus atrophicus* SW was spotted in the center of a CAS agar plate, with three replicates. The plates were incubated upright at 37°C for 3 days, and the formation of an inhibition zone around the *Bacillus atrophicus* SW was observed. The results are attached. Figure 11 As shown in the figure, Bacillus atrophus SW can secrete siderophores.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus atrophus ( Bacillus atrophaeus ), characterized in that, It is deposited at the China General Microbiological Culture Collection Center on April 15, 2024, with accession number CGMCC 30329.

2. A biocontrol agent containing Bacillus atrophus as described in claim 1.

3. A method for preparing the biocontrol agent as described in claim 2, characterized in that, The method includes the following steps: inoculating the Bacillus atrophus described in claim 1 into LB liquid medium, shaking the bacteria until the logarithmic phase is reached, centrifuging the culture, washing away excess LB medium with sterile PBS buffer, and resuspending the bacteria in sterile PBS buffer until the OD 600 reaches 1.7-1.8, which is the biocontrol agent.

4. The use of Bacillus atrophus as described in claim 1 in the preparation of a biocontrol agent for preventing and controlling wheat stem rot caused by Fusarium graminearum.

5. The use of the Bacillus atrophus of claim 1 in the preparation of a biocontrol agent for preventing and controlling sclerotinia disease of tomato caused by Sclerotinia sclerotiorum.

6. A biological pesticide or biological fertilizer, characterized in that, It is prepared from the Bacillus atrophus as described in claim 1.

7. A biocontrol agent, characterized in that, It is prepared from the Bacillus atrophus as described in claim 1.

8. An antibacterial agent, characterized in that, It is prepared from the Bacillus atrophus as described in claim 1.

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