Preparation method and application method of bacillus albolactis yc12 and nano-selenium complex microbial agent

Abstract

This invention discloses a method for preparing and applying *Bacillus albus* YC12 and a nano-selenium composite bacterial agent, specifically relating to the fields of agricultural biotechnology and microbial preparation technology. The invention provides a method for isolating and identifying this strain, a process for preparing the nano-selenium composite bacterial agent, and a method for applying the composite bacterial agent. The preparation process uses LB medium as a substrate, adds sodium selenite as a selenium source, and inoculates YC12, then cultivates at 30℃ and 150 r / min to obtain an active bacterial agent containing red nano-selenium. This composite bacterial agent possesses multiple functions including broad-spectrum antibacterial activity, nitrogen fixation, phosphorus solubilization, cellulose degradation, and selenium nutrient fortification. It exhibits high inhibition rates against *Gyromitra esculenta* and *Target Spot Bacterium tumefaciens*, and can tolerate a high-salt environment of 10% NaCl. This invention solves the problems of low strain safety, single function, unclear process, and poor environmental adaptability in existing nano-selenium synthesis technologies.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology and microbial preparation technology, and specifically discloses the preparation and application methods of Bacillus albus YC12 and nano-selenium composite bacterial agents. Background Technology

[0002] Nano-selenium, as a plant growth regulator, has significant potential in improving crop stress resistance and quality. Its preparation methods mainly include chemical reduction and microbial synthesis. Although the chemical method is relatively mature, it generally uses strong reducing agents, resulting in problems such as reagent residues, environmental pollution, and poor biocompatibility of the products, making it difficult to meet the requirements of green agriculture. While the microbial method is environmentally friendly, it is limited by bottlenecks such as the safety of available strains, limited functionality, and unclear process conditions.

[0003] Specifically, existing microorganisms used for synthesizing nano-selenium have significant drawbacks: First, the safety of the strains is insufficient, with some strains potentially producing toxins and posing biosafety risks. Second, their functions are relatively singular, often focusing only on selenium conversion or a specific growth-promoting function, lacking comprehensive performance that integrates disease control, growth promotion, and selenium nutrient fortification. Third, the synthesis process parameters are vague, lacking systematic optimization of key conditions such as selenium source concentration, temperature, and time, resulting in low and unstable conversion efficiency. Fourth, their environmental adaptability is weak, with most strains exhibiting limited resistance to salinity, drought, and other adverse conditions, restricting their widespread application in non-ideal soils.

[0004] In summary, existing technologies, such as chemical methods, lack greenness, while microbial methods have limitations in terms of strain safety, functional versatility, process clarity, and environmental adaptability. Therefore, there is an urgent need to develop a safe, multifunctional, highly adaptable microbial resource with a well-defined process, and to establish a stable and efficient method for synthesizing nano-selenium, in order to achieve multifunctional integration in agricultural applications. This is precisely the starting point and core problem that this invention aims to solve. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a method for preparing and applying Bacillus albus YC12 and nano-selenium composite bacterial agent, which solves the problems of low strain safety, single function, unclear process, poor environmental adaptability, and insufficient greenness of chemical synthesis methods in the existing microbial synthesis technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent specifically includes the following steps: S1: Take a soil sample, add sterile physiological saline to it, mix thoroughly and dilute to obtain a mixture, then spread the mixture on LB solid plate medium, invert the medium for incubation, then pick a single colony from the medium for purification, and finally identify the colony as Bacillus albus by morphological observation, Gram staining and 16S rRNA sequencing, and name it strain YC12. S2: Take tryptone, yeast extract, sodium chloride, and deionized water to make up to volume to obtain LB liquid culture medium. Then, sterilize the LB liquid culture medium at high temperature. After the culture medium cools to room temperature, add sodium selenite under aseptic conditions. S3: Pick a single colony from the YC12 plate, inoculate it into 2 mL of LB liquid medium, and shake the medium to obtain a seed culture. Then, inoculate the seed culture into LB medium containing sodium selenite and shake the medium. When the color of the culture medium changes from pale yellow to red, the culture is stopped. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined, and the residual amount of sodium selenite was measured by ascorbic acid reduction method. The selenium conversion rate was calculated, and a portion of the fermentation broth was used directly as a compound microbial agent for subsequent biological control and growth promotion experiments.

[0007] In the preferred S1 solution, weigh 1–1.5 parts by weight of soil sample, add 9 times the volume of sterile physiological saline, and mix thoroughly by shaking to prepare a 10⁻¹ dilution; then use a 10-fold serial dilution method to gradually dilute to 10⁻³–10⁻¹. 4 Prepare the dilution ratio for later use.

[0008] Preferably, in S1, 10⁻³~10⁻ 4 100 μL of the diluted mixture was spread onto LB solid plates and incubated upside down at 30°C for 24 h.

[0009] Preferably, 5-10 parts by weight of tryptone, 3-6 parts by weight of yeast extract, and 8-12 parts by weight of sodium chloride are taken from S2 and diluted to 1000 parts by weight with deionized water to obtain LB liquid culture medium.

[0010] Preferably, in step S2, the LB liquid culture medium obtained by adjusting the volume is sterilized at 121°C for 20 minutes. After the liquid culture medium cools to room temperature, 10 parts by weight of sodium selenite with a concentration of 5 mmol / L are added to it.

[0011] Preferably, in S3, a single colony is picked from a YC12 plate and inoculated into 5 parts by weight of LB liquid medium, and the medium is cultured at 20-30°C and 150 r / min for 12 h to obtain a seed culture.

[0012] Preferably, in step S3, the seed culture is inoculated into the culture medium after shaking culture at an inoculation rate of 2-5% and then placed in a shaker at 20-30℃ and 150r / min for shaking culture for 24-60h.

[0013] Preferably, when calculating the selenium conversion rate in S4, a standard curve of sodium selenite is plotted at a wavelength of 550 nm to obtain the linear equation y = 0.5787x + 0.0682. Then, the reduction rate of sodium selenite at different culture time points is calculated using the standard curve.

[0014] Preferably, testing in S4 The steps for calculating the absorbance are as follows: turn on the UV-Vis spectrophotometer and preheat it, set the wavelength to 600 nm, then take 2 mL of blank culture medium and inject it into the cuvette, wipe the outer wall to remove impurities, place it in the sample cell and zero it to 0.000 absorbance, then take 2 mL of culture medium and inject it into the cuvette, wipe it, place it in the instrument and read the stable absorbance value, and perform the same sample in parallel 3 times and take the average value.

[0015] Application methods of Bacillus albus YC12 and nano-selenium compound bacterial agent The antibacterial activity of the compound microbial agent was determined by inoculating it onto culture media containing wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The colony morphology of strain YC12 was observed and recorded on nitrogen-free Assumption medium, inorganic phosphorus medium, Congo red cellulose medium, and LB high-salt medium with 10% NaCl.

[0016] The technical effects and advantages of the preparation and application methods of Bacillus albus YC12 and nano-selenium composite bacterial agent of the present invention are as follows: 1. The invention uses YC12 strain, which is a known safe biocontrol bacterium that has been monitored to produce no toxins. The compound bacterial agent prepared by it poses no safety risk to food when applied to crops. Compared with traditional strains such as Bacillus cereus, its safety is significantly improved.

[0017] 2. This invention shows that the YC12 strain has an inhibitory effect on eight common pathogenic fungi, and has a high inhibition rate against gray mold of leeks and target spot of tobacco. The nano selenium in the compound microbial agent can further enhance crop stress resistance and improve the disease control effect.

[0018] 3. In this invention, the nitrogen-fixing, phosphorus-solubilizing, and cellulose-degrading functions of YC12 can promote the absorption of nutrients by crops, and the nano-selenium can regulate the activity of antioxidant enzymes. The synergistic effect of the two can increase the plant height and fresh weight of crops.

[0019] 4. This invention allows YC12 to still grow in high-salt environments, and the compound microbial agent can adapt to various adverse soil conditions such as slightly saline-alkali land and drought, which can expand the range by 30% compared with existing preparations.

[0020] 5. This invention employs a simple process, in which the optimal 5 mmol / L selenium source and 60 h of culture can achieve a reduction rate of over 50%. Its production cost is reduced and the formulation can be applied through conventional methods such as root irrigation and foliar spraying. Attached Figure Description

[0021] Figure 1 The results of plate confrontation between Bacillus albus YC12 and nano-selenium compound bacterial agent proposed in this invention and wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus and pepper anthracnose fungus are presented.

[0022] Figure 2 The results of plate confrontation between Bacillus albus YC12 and nano-selenium compound bacterial agent proposed in this invention and Colletotrichum gloeosporioides, Tobacco Target Spot Fungus, Equisetum equisetifolium, and Rice Blast Fungus are shown.

[0023] Figure 3 The colony morphology of Bacillus albus YC12 and nano-selenium composite bacterial agent proposed in this invention on different culture media.

[0024] Figure 4 This is a schematic diagram showing the colony morphology (A) and quantity (B) of Bacillus leucosus YC12 on LB plates of different concentrations of sodium selenite proposed in this invention.

[0025] Figure 5 This is a growth curve of Bacillus albus YC12 proposed in this invention in LB medium containing different concentrations of sodium selenite.

[0026] Figure 6 This is a graph showing the growth and transformation efficiency of Bacillus subtilis YC12 proposed in this invention in LB medium containing different concentrations of sodium selenite. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0029] Example 1 This embodiment provides a method for preparing and applying Bacillus albus YC12 and a nano-selenium composite bacterial agent. The specific implementation steps include: Experimental materials: 9 mL sterile saline, 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 5 mmol / L sodium selenite.

[0030] Experimental objective: Preparation of Bacillus albus YC12 and nano-selenium composite bacterial agent and their testing.

[0031] Experimental steps: S1: Take 1 g of soil sample, add 9 mL of sterile physiological saline and mix thoroughly. Dilute stepwise to 10⁻³~10⁻³ using a 10-fold serial dilution method. 4 Gradient; then 100 μL was spread on LB solid plate medium, and the medium was incubated upside down at 30℃ for 24 h. Single colonies with typical morphological characteristics were picked for isolation and purification. After verification of antibacterial activity, the strain was identified by colony morphology observation, Gram staining and 16S rRNA gene sequencing. It was identified as Bacillus albus and named YC12.

[0032] S2: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and deionized water to make up to 1 L to obtain LB liquid culture medium. Then take 200 mL of LB liquid culture medium and sterilize it at 121℃ for 20 min. After cooling to room temperature, add 2 mL of 5 mmol / L sodium selenite under sterile conditions. S3: Pick a loop of bacterial growth from the YC12 slant and inoculate the bacterial growth into 5 mL of LB liquid medium. Incubate the medium at 30℃ and 150 r / min for 12 h to obtain seed culture. Then, inoculate the seed culture into LB medium containing 5 mmol / L sodium selenite at a 2% inoculation rate and incubate at 30℃ and 150 r / min for 60 h. Stop the culture when the color of the culture medium changes from pale yellow to red. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined by using the ascorbic acid reduction method to determine the residual sodium selenite, and the selenium conversion rate was calculated. A portion of the fermentation broth was also used directly as a compound microbial agent for subsequent biocontrol and growth promotion experiments. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 h. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 min and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 h. Colony growth was then observed. S7: YC12 seed culture was spread onto LB solid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and then incubated at 30℃ for 48 h, observing the number of single colonies on the plates. Then, YC12 seed culture was inoculated at a 2% inoculum into LB liquid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and incubated at 30℃ with shaking at 150 rpm. Samples were taken every 12 h for analysis. Values ​​and plot growth curves; S8: YC12 was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured with shaking at 30℃ and 150 r / min for 60 h. A sodium selenite standard curve was then constructed at 550 nm, yielding the linear equation y = 0.5787x + 0.0682. =0.9976) and the reduction rate of sodium selenite at different culture time points was calculated using a standard curve.

[0033] Experimental results: See Table 1 for details.

[0034] Table 1: Test Results of Example 1 Example 1 Inhibition rate of wheat stem rot pathogen 51.32% inhibition rate of gray mold in chives 78.65% Inhibition rate of cotton wilt pathogen 28.23% inhibition rate of anthrax bacteria in chili peppers 22.09% Antibacterial rate of Colloidal anthrax 21.43% Tobacco target spot pathogen inhibition rate 76.98% Inhibition rate of *Equisetum hyemale* pathogen 30.17% Inhibition rate of rice blast fungus 62.45% refer to Figure 1 , 2 Diagram of the standoff outcome Figure 3 Colony morphology on different culture media: In Example 1, Bacillus albus YC12 was successfully isolated and identified through a series of experiments, and a composite bacterial agent containing nano-selenium was prepared. LB medium was used as the fermentation substrate, and the culture was carried out with shaking for 60 days at 30°C and 150 r / min with an addition of 5 mmol / L sodium selenite. After h, the bacterial solution changed from pale yellow to red, and the sodium selenite reduction rate reached 52.00%, confirming that YC12 possesses efficient nano-selenium synthesis capabilities. Antibacterial tests showed that this strain inhibited eight common pathogenic fungi, with inhibition rates of 78.65% and 76.98% against *Gyromitra esculenta* and *Target Spot Fungus* of tobacco, respectively. It also exhibited varying degrees of inhibitory activity against other pathogenic fungi such as *Fusarium wilt* of cotton, demonstrating significant broad-spectrum biocontrol effects. Physiological function tests showed that YC12 can form colonies on nitrogen-free media, produce clear zones on inorganic phosphorus and Congo red cellulose media, and tolerate high-salt environments of 10% NaCl. It possesses nitrogen-fixing, phosphorus-solubilizing, and cellulose-degrading functions, along with strong environmental adaptability, providing solid support for its integrated application of disease control, growth promotion, and selenium nutrient fortification. (Reference) Figure 4 , 5 6. This further reveals the tolerance and transformation characteristics of YC12 to sodium selenite: Figure 4 This indicates that it can grow normally at concentrations below 20 mmol / L; Figure 5 The growth rate was shown to slow down with increasing selenium concentration; Figure 6 This confirms that YC12 has the highest efficiency in the synthesis of nano-selenium under the condition of 5 mmol / L sodium selenite, providing experimental basis for optimizing the concentration of selenium source in the preparation of bacterial agents.

[0035] Example 2 This embodiment provides a method for preparing and applying Bacillus albus YC12 and a nano-selenium composite bacterial agent. The specific implementation steps include: Experimental materials: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 10 mmol / L sodium selenite Experimental objective: To investigate the effects of higher concentrations of selenium source on the growth of strain YC12 and its ability to synthesize nano-selenium.

[0036] Experimental steps: S1: The YC12 strain stored at -80℃ was inoculated onto LB solid medium and incubated upside down at 30℃ for 24 h to obtain single colonies.

[0037] S2: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and deionized water to make up to 1 L to obtain LB liquid culture medium. Then take 200 mL of LB liquid culture medium and sterilize it at 121℃ for 20 min. After cooling to room temperature, add 2 mL of 10 mmol / L sodium selenite under sterile conditions. S3: Pick a single colony from a YC12 plate and inoculate it into 2 mL of LB liquid medium. Incubate the medium at 30°C and 150 r / min for 12 h to obtain a seed culture. Then, inoculate the seed culture into LB medium containing 10 mmol / L sodium selenite at a 2% inoculation rate and incubate it in a shaker at 30°C and 150 r / min for 60 h. Stop the culture when the color of the culture medium changes from pale yellow to red. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined by using the ascorbic acid reduction method to determine the residual sodium selenite, and the selenium conversion rate was calculated. A portion of the fermentation broth was also used directly as a compound microbial agent for subsequent biocontrol and growth promotion experiments. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 hours. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 minutes and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 hours. Colony growth was then observed. S7: The YC12 seed culture was spread onto LB solid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and then incubated at 30°C for 48 h, observing the number of single colonies on the plates. Then, 2% of the YC12 seed culture was inoculated into LB liquid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and incubated at 30°C with shaking at 150 rpm. Samples were taken every 12 h for analysis. Values ​​and plot growth curves; S8: YC12 was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured with shaking at 30℃ and 150 r / min for 60 h. A sodium selenite standard curve was then constructed at 550 nm wavelength, yielding the linear equation y = 0.5787x + 0.0682. =0.9976) and the reduction rate of sodium selenite at different culture time points was calculated using a standard curve.

[0038] Experimental results: See Table 2 for details.

[0039] Table 2: Test Results of Example 2 Example 2 Inhibition rate of wheat stem rot pathogen 41.08% inhibition rate of gray mold in chives 63.27% Inhibition rate of cotton wilt pathogen 22.75% inhibition rate of anthrax bacteria in chili peppers 17.97% Antibacterial rate of Colloidal anthrax 17.64% Tobacco target spot pathogen inhibition rate 65.67% Inhibition rate of *Equisetum hyemale* pathogen 25.73% Inhibition rate of rice blast fungus 53.37% In Example 2, the concentration of sodium selenite was adjusted to 10 mmol / L. Sodium selenite has a certain degree of toxicity to microorganisms, and doubling the concentration will exacerbate the oxidative stress and growth inhibition of YC12 cells. The higher the concentration, the fewer the colony count and the longer the lag phase. Therefore, at high concentrations, bacterial growth is inhibited, metabolic activity decreases, and both the nano-selenium conversion efficiency and antibacterial function are significantly reduced.

[0040] Example 3 This embodiment provides a method for preparing and applying Bacillus albus YC12 and a nano-selenium composite bacterial agent. The specific implementation steps include: Experimental materials: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 5 mmol / L sodium selenite.

[0041] Experimental objective: To investigate the effect of low-temperature culture on the efficiency of nano-selenium synthesis by strain YC12.

[0042] Experimental steps: S1: The YC12 strain stored at -80℃ was inoculated onto LB solid medium and incubated upside down at 30℃ for 24 h to obtain single colonies.

[0043] S2: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and deionized water to make up to 1 L to obtain LB liquid culture medium. Then take 200 mL of LB liquid culture medium and sterilize it at 121℃ for 20 min. After cooling to room temperature, add 2 mL of 5 mmol / L sodium selenite under sterile conditions. S3: Pick a single colony from a YC12 plate and inoculate it into 2 mL of LB liquid medium. Incubate the medium at 20°C and 150 r / min for 12 h to obtain a seed culture. Then, inoculate the seed culture into LB medium containing 5 mmol / L sodium selenite at a 2% inoculation rate and incubate it in a shaker at 20°C and 150 r / min for 60 h. Stop the culture when the color of the culture medium changes from pale yellow to red. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined by using the ascorbic acid reduction method to determine the residual sodium selenite, and the selenium conversion rate was calculated. A portion of the fermentation broth was also used directly as a compound microbial agent for subsequent biocontrol and growth promotion experiments. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 hours. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 minutes and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 hours. Colony growth was then observed. S7: The YC12 seed culture was spread onto LB solid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and then incubated at 30°C for 48 h, observing the number of single colonies on the plates. Then, 2% of the YC12 seed culture was inoculated into LB liquid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and incubated at 30°C with shaking at 150 rpm. Samples were taken every 12 h for analysis. Values ​​and plot growth curves; S8: YC12 was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured with shaking at 30℃ and 150 r / min for 60 h. A sodium selenite standard curve was then constructed at 550 nm wavelength, yielding the linear equation y = 0.5787x + 0.0682. =0.9976) and the reduction rate of sodium selenite at different culture time points was calculated using a standard curve.

[0044] Experimental results: See Table 3 for details.

[0045] Table 3: Test Results of Example 3 Example 3 Inhibition rate of wheat stem rot pathogen 37.54% inhibition rate of gray mold in chives 59.36% Inhibition rate of cotton wilt pathogen 22.75% inhibition rate of anthrax bacteria in chili peppers 16.73% Antibacterial rate of Colloidal anthrax 16.37% Tobacco target spot pathogen inhibition rate 57.53% Inhibition rate of *Equisetum hyemale* pathogen 22.73% Inhibition rate of rice blast fungus 46.76% The optimal temperature for YC12 is 30℃. Low temperatures inhibit the activity of aerobic respiratory enzymes and synthetic enzymes in the bacteria, thus prolonging the lag phase and delaying the stationary phase. As the concentration of sodium selenite decreases, the synthesis of antibacterial products depends on enzymatic reactions, and low temperatures directly reduce enzyme efficiency, thus decreasing the antibacterial rate. Sodium selenite reductase activity decreases significantly with decreasing temperature.

[0046] Example 4 This embodiment provides a method for preparing and applying Bacillus albus YC12 and a nano-selenium composite bacterial agent. The specific implementation steps include: Experimental materials: 8 g tryptone, 3 g yeast extract, 10 g sodium chloride, 5 mmol / L sodium selenite.

[0047] Experimental objective: To investigate the effects of low-nutrient culture medium and low rotation speed on the preparation and function of compound microbial agents.

[0048] Experimental steps: S1: The YC12 strain stored at -80℃ was inoculated onto LB solid medium and cultured upside down at 30℃ for 24 h to obtain single colonies; S2: Take 8 g of tryptone, 3 g of yeast extract, 10 g of sodium chloride, and deionized water to make up to 1 L to obtain LB liquid culture medium. Then take 200 mL of LB liquid culture medium and sterilize it at 121℃ for 20 min. After cooling to room temperature, add 2 mL of 5 mmol / L sodium selenite under sterile conditions. S3: Pick a single colony from a YC12 plate and inoculate it into 2 mL of LB liquid medium. Incubate the medium at 30℃ and 120 r / min for 12 h to obtain a seed culture. Then, inoculate the seed culture into LB medium containing 5 mmol / L sodium selenite at a 2% inoculation rate and incubate it in a shaker at 30℃ and 120 r / min for 60 h. Stop the culture when the color of the culture medium changes from pale yellow to red. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined by using the ascorbic acid reduction method to determine the residual sodium selenite, and the selenium conversion rate was calculated. A portion of the fermentation broth was also used directly as a compound microbial agent for subsequent biocontrol and growth promotion experiments. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 hours. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 minutes and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 hours. Colony growth was then observed. S7: The YC12 seed culture was spread onto LB solid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and then incubated at 30°C for 48 h, observing the number of single colonies on the plates. Then, 2% of the YC12 seed culture was inoculated into LB liquid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and incubated at 30°C with shaking at 150 r / min. Samples were taken every 12 h for analysis. Values ​​and plot growth curves; S8: YC12 was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured with shaking at 30℃ and 150 r / min for 60 h. A sodium selenite standard curve was then constructed at 550 nm wavelength, yielding the linear equation y = 0.5787x + 0.0682. =0.9976) and the reduction rate of sodium selenite at different culture time points was calculated using a standard curve.

[0049] Experimental results: See Table 4 for details.

[0050] Table 4: Test Results of Example 4 Example 4 Inhibition rate of wheat stem rot pathogen 37.67% inhibition rate of gray mold in chives 57.46% Inhibition rate of cotton wilt pathogen 19.46% inhibition rate of anthrax bacteria in chili peppers 15.73% Antibacterial rate of Colloidal anthrax 15.73% Tobacco target spot pathogen inhibition rate 55.75% Inhibition rate of *Equisetum hyemale* pathogen 22.47% Inhibition rate of rice blast fungus 43.75% In Example 4, both insufficient nutrients and insufficient dissolved oxygen jointly inhibited YC12 metabolism. Low nutrients resulted in a lack of carbon and nitrogen sources and growth factors, leading to slow cell proliferation and insufficient raw materials for the synthesis of metabolic products. Low rotation speed reduced the dissolved oxygen content in the culture medium. Since YC12 is an aerobic bacterium, insufficient dissolved oxygen inhibits aerobic respiration, reducing ATP production and further limiting growth and metabolism.

[0051] Example 5 This embodiment provides a method for preparing and applying Bacillus albus YC12 and a nano-selenium composite bacterial agent. The specific implementation steps include:

[0052] Experimental materials: 10g tryptone, 5g yeast extract, 10g sodium chloride, 5 mmol / L sodium selenite.

[0053] Experimental objective: To investigate the effects of inoculum size on excessive bacterial cell growth and nano-selenium synthesis.

[0054] Experimental steps: S1: The YC12 strain stored at -80℃ was inoculated onto LB solid medium and cultured upside down at 30℃ for 24 h to obtain single colonies; S2: Take 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and deionized water to make up to 1 L to obtain LB liquid culture medium. Then take 200 mL of LB liquid culture medium and sterilize it at 121℃ for 20 min. After cooling to room temperature, add 2 mL of 5 mmol / L sodium selenite under sterile conditions. S3: Pick a single colony from a YC12 plate and inoculate it into 2 mL of LB liquid medium. Incubate the medium at 30°C and 150 r / min for 12 h to obtain a seed culture. Then, inoculate the seed culture into LB medium containing 5 mmol / L sodium selenite at a 5% inoculation rate and incubate it in a shaker at 30°C and 150 r / min for 60 h. Stop the culture when the color of the culture medium changes from pale yellow to red.

[0055] S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined by using the ascorbic acid reduction method to determine the residual sodium selenite, and the selenium conversion rate was calculated. A portion of the fermentation broth was also used directly as a compound microbial agent for subsequent biocontrol and growth promotion experiments. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 h. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 min and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 h. Colony growth was then observed. S5: Photographs were taken of the following pathogenic fungi: wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The growth inhibition of the prepared compound fungal agent on the above pathogenic fungi was then determined using the plate confrontation method. S6: YC12 seed culture was streaked onto nitrogen-free Assumption solid medium and placed in a constant temperature incubator at 30℃ for 72 h. Colony formation was then observed. YC12 was then inoculated onto the center of inorganic phosphorus solid medium using the spot inoculation method and placed in a 30℃ environment for 5 days. The presence of transparent hydrolysis zones around the colonies was observed. YC12 was then inoculated onto Congo red cellulose medium using the spot inoculation method and placed in a 30℃ environment for 7 days. Finally, the medium was stained with 1% sodium chloride solution for 10 min and the presence of transparent degradation zones around the colonies was observed. Finally, YC12 seed culture was spread onto LB solid medium containing 10% (w / v) sodium chloride and placed in a 30℃ environment for 48 h. Colony growth was then observed. S7: YC12 seed culture was spread onto LB solid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and then incubated at 30℃ for 48 h, observing the number of single colonies on the plates. Then, YC12 seed culture was inoculated at a 2% inoculum into LB liquid medium containing 0, 5, 10, 15, 20, and 25 mmol / L sodium selenite, and incubated at 30℃ with shaking at 150 rpm. Samples were taken every 12 h for analysis. Values ​​and plot growth curves; S8: YC12 was inoculated into LB liquid medium containing 5 mmol / L sodium selenite and cultured with shaking at 30℃ and 150 r / min for 60 h. A sodium selenite standard curve was then constructed at 550 nm, yielding the linear equation y = 0.5787x + 0.0682. =0.9976) and the reduction rate of sodium selenite at different culture time points was calculated using a standard curve.

[0056] Experimental results: See Table 5 for details.

[0057] Table 5: Test Results of Example 5 Example 5 Inhibition rate of wheat stem rot pathogen 47.43% inhibition rate of gray mold in chives 70.45% Inhibition rate of cotton wilt pathogen 27.43% inhibition rate of anthrax bacteria in chili peppers 20.72% Antibacterial rate of Colloidal anthrax 19.67% Tobacco target spot pathogen inhibition rate 27.57% Inhibition rate of *Equisetum hyemale* pathogen 18.44% Inhibition rate of rice blast fungus 59.24% In Example 5, an excessively high inoculum size led to an imbalance in nutrient competition, resulting in an initially high bacterial density and subsequent nutrient deficiency. Increasing the inoculum size from 2% to 5% resulted in a large initial bacterial count, but the LB medium had limited nutrients. Later, intense competition for nutrients caused metabolic disorders, preventing the continuous synthesis of antibacterial products. The sodium selenite reduction reaction depends on bacterial activity; later, nutrient depletion led to decreased bacterial activity and reduced reductase secretion, resulting in a slight decrease in the reduction rate.

[0058] Comparative Example 1 This embodiment provides a traditional method for preparing a selenium-based compound microbial agent, the specific implementation steps of which include: Experimental materials: Sodium selenite, ascorbic acid, 10 g peptone, 5 g yeast extract, 10 g sodium chloride, deionized water to a final volume of 1 L, and sterile physiological saline.

[0059] Experimental objective: Selenium nanoparticles were prepared by chemical reduction method, their antibacterial effect was evaluated, and compared with the nano-selenium composite bacterial agent synthesized by biological method of the present invention.

[0060] Experimental steps: S1: Weigh 100 mL of 5 mmol / L sodium selenite solution and place it in a 250 mL Erlenmeyer flask; S2: Add an equal volume of ascorbic acid solution (concentration of 10 mmol / L) to the conical flask, stir magnetically for 2 h at room temperature, and observe the color change of the solution; S3: After the reaction is complete, centrifuge the mixture at 8000 r / min for 10 min and collect the red precipitate; S4: Wash the precipitate three times with deionized water and resuspend it in 100 mL of sterile physiological saline to obtain a selenium nanoparticle suspension prepared by chemical method. S5: Wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus were inoculated into their respective culture media. Using the plate confrontation method, the above selenium nanoparticle suspension was evenly spread around the colony and placed in a 30℃ incubator for 72 h. S6: Measure the diameter of the inhibition zone of each pathogen and calculate the inhibition rate.

[0061] Experimental results: See Table 6 for details.

[0062] Table 6: Test Results of Comparative Example 1 Comparative Example 1 Inhibition rate of wheat stem rot pathogen 15.34% inhibition rate of gray mold in chives 22.45% Inhibition rate of cotton wilt pathogen 10.42% inhibition rate of anthrax bacteria in chili peppers 8.52% Antibacterial rate of Colloidal anthrax 9.54% Tobacco target spot pathogen inhibition rate 20.43% Inhibition rate of *Equisetum hyemale* pathogen 12.57% Inhibition rate of rice blast fungus 18.45% Comparative Example 1, prepared using a traditional chemical reduction method, showed significantly lower antibacterial effects than the nano-selenium composite bacterial agents biosynthesized using Bacillus albus YC12 in Examples 1-5 of this invention. Chemically synthesized selenium particles exhibit uneven particle size, poor surface modification, and lack the synergistic effect of bacterial metabolites, resulting in weaker antibacterial activity. Furthermore, the chemical method also suffers from reagent residues and environmental pollution, lacking the advantages of biological methods such as being green, efficient, and functionally diverse.

[0063] Example 1 uses standard optimized conditions. Under suitable selenium concentration and temperature, YC12 cells grow well, sodium selenite reductase activity is high, nano-selenium synthesis efficiency reaches 52.00%, and cell metabolism is active, secreting abundant antibacterial substances, achieving significant inhibition of a variety of pathogens, especially against gray mold of leeks and target spot of tobacco, with an inhibition rate of nearly 80%, demonstrating the dual functions of efficient biocontrol and selenium conversion.

[0064] Example 2 used a high selenium stress condition of 10 mmol / L sodium selenite. The high selenium concentration caused oxidative stress on YC12, which inhibited bacterial growth and metabolic enzyme activity, resulting in a decrease in nano-selenium conversion efficiency and a reduction in the synthesis of antibacterial substances. The antibacterial rate of each pathogen was lower than that in Example 1, indicating that excessive selenium source is not conducive to the function of bacteria.

[0065] Example 3 uses low-temperature culture conditions. Low temperature inhibits the activity of YC12 respiratory enzymes and synthases, prolongs the lag phase, and reduces cell density and metabolic intensity. This results in insufficient synthesis of nano-selenium and accumulation of antibacterial products, and further decreases the various antibacterial rates, indicating that temperature is a key factor affecting cell activity and function.

[0066] Example 4 employs low-nutrient and low-oxygen conditions. Insufficient carbon and nitrogen sources limit bacterial proliferation, and reduced dissolved oxygen inhibits aerobic metabolism. This dual stress leads to slow growth of YC12, insufficient ATP synthesis, limited nano-selenium conversion and antibacterial substance synthesis, and a general weakening of the antibacterial effect.

[0067] Example 5 used a high inoculum of 5%. The high initial bacterial density led to rapid nutrient consumption, and the intense competition for nutrients in the later stage caused metabolic disorders, decreased bacterial activity, discontinuous synthesis of antibacterial products, and a slight decrease in the reduction efficiency of nano-selenium. This indicates that the inoculum should be moderate to maintain metabolic balance.

[0068] Comparative Example 1 uses a chemical reduction method to prepare selenium nanoparticles. The selenium particles synthesized by this method have unmodified surfaces, uneven particle sizes, and lack the synergistic effect of YC12 cells and their metabolites, resulting in significantly lower antibacterial activity than the biological method. The chemical method also suffers from problems such as reagent residues and environmental unfriendliness, and does not possess the green, efficient, and functionally diverse characteristics of the biological method.

[0069] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0070] In conclusion, 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, 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 method for preparing Bacillus albus YC12 and nano-selenium composite bacterial agent, characterized in that, Specifically, the following steps are included: S1: Take a soil sample, add sterile physiological saline to it, mix thoroughly and dilute to obtain a mixture, then spread the mixture on LB solid plate medium, invert the medium for incubation, then pick a single colony from the medium for purification, and finally identify the colony as Bacillus albus by morphological observation, Gram staining and 16S rRNA sequencing. S2: Take tryptone, yeast extract, sodium chloride, and deionized water to make up to volume to obtain LB liquid culture medium. Then, sterilize the LB liquid culture medium at high temperature. After the culture medium cools to room temperature, add sodium selenite under aseptic conditions. S3: Pick a loop of bacterial growth from the YC12 slant, inoculate it into LB liquid medium, and shake the medium to obtain a seed culture. Then, inoculate the seed culture into LB medium containing sodium selenite and shake it. When the color of the culture medium changes from pale yellow to red, the culture is stopped. S4: After the culture is completed, take a sample of the culture medium for testing. The value was determined, and the residual amount of sodium selenite was measured by ascorbic acid reduction method. The selenium conversion rate was calculated, and a portion of the fermentation broth was used directly as a compound microbial agent for subsequent biological control and growth promotion experiments.

2. The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, Weigh 1 to 1.5 parts by weight of the sample in S1 and dilute it thoroughly with 0.5 to 1.0 parts by weight of sterile physiological saline.

3. The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, In S1, 100 μL of the mixture was spread onto LB solid plate medium and incubated upside down at 30°C for 24 h.

4. The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, Take 5-10 parts by weight of tryptone, 3-6 parts by weight of yeast extract, and 8-12 parts by weight of sodium chloride from S2 and bring the volume to 1000 parts by weight with deionized water to obtain LB liquid culture medium.

5. The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, In S2, the LB liquid culture medium obtained by adjusting the volume was sterilized at 121°C for 20 min. After the liquid culture medium cooled to room temperature, 10 parts by weight of sodium selenite with a concentration of 5 mmol / L were added to it.

6. The preparation method of Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, A single colony was picked from the YC12 plate in S3 and inoculated into 2 mL of LB liquid medium. The medium was then cultured at 20-30℃ and 150 r / min for 12 h to obtain the seed culture.

7. The method for preparing Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, In S3, the seed culture was inoculated into the culture medium after shaking culture at an inoculation rate of 2-5% and then placed in a shaker at 20-30℃ and 150r / min for shaking culture for 24-60 h.

8. The method for preparing Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, When calculating the selenium conversion rate as described in S4, a standard curve of sodium selenite was plotted at a wavelength of 550 nm to obtain the linear equation y = 0.5787x + 0.0682. Subsequently, the reduction rate of sodium selenite at different culture time points was calculated using the standard curve.

9. The method for preparing Bacillus albus YC12 and nano-selenium composite bacterial agent as described in claim 1, characterized in that, Testing in S4 The steps for calculating the absorbance are as follows: turn on the UV-Vis spectrophotometer and preheat it, set the wavelength to 600 nm, then take 2 mL of blank culture medium and inject it into the cuvette, wipe the outer wall to remove impurities, place it in the sample cell and zero it to 0.000 absorbance, then take 2 mL of culture medium and inject it into the cuvette, wipe it, place it in the instrument and read the stable absorbance value, and perform the same sample in parallel 3 times and take the average value.

10. The method of applying Bacillus albus YC12 and nano-selenium composite bacterial agent as described in any one of claims 1-9, characterized in that, The antibacterial activity of the compound microbial agent was determined by inoculating it onto culture media containing wheat stem rot fungus, leek gray mold fungus, cotton wilt fungus, pepper anthracnose fungus, collodion fungus, tobacco target spot fungus, horsetail falcatum fungus, and rice blast fungus. The colony morphology of strain YC12 was observed and recorded on nitrogen-free Assumption medium, inorganic phosphorus medium, Congo red cellulose medium, and LB high-salt medium with 10% NaCl.

Images