Optimized preparation process of biochar fungicide for preventing and treating delonix regia root rot

Abstract

The invention discloses an optimized preparation process of a charcoal fungicide for preventing and treating delonix regia root rot, and belongs to the field of microbial fermentation optimization. The process comprises the following steps: firstly, carrying out bacterial liquid culture (culture at 32 DEG C and 200rpm) on Pseudomonas aeruginosa and Bacillus licheniformis through an LB liquid culture medium, then carrying out liquid culture medium optimized fermentation on 3% of bacterial liquid (culture at 32 DEG C and 220rpm for 24 hours under the conditions of 3% of sucrose, 0.5% of ammonium sulfate, 1% of beef extract and 2% of potassium chloride) so as to obtain optimized fermentation liquor, and carrying out fermentation on the optimized fermentation liquor so as to obtain the Pseudomonas aeruginosa and Bacillus licheniformis. And mixing the optimized fermentation liquor and charcoal according to the mass ratio of 5: 1, and standing at normal temperature for 24 hours to obtain the fungicide. In a greenhouse experiment, the control effect on the delonix regia root rot reaches 83.53%. The biochar fungicide has the characteristics of high efficiency, environment friendliness, no pollution and the like, is suitable for preventing and treating root diseases of delonix regia and other garden plants, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant disease control, specifically to a method for controlling diseases in Delonix regia (…). Delonix regia An optimized preparation process for biochar inoculants for root rot was developed. This biochar inoculant effectively inhibits the growth of pathogens by using antagonistic microbial strains, falling under the category of biological control technology. It is suitable for green control of diseases in garden plants, especially those caused by Ganoderma lucidum (Ganoderma). Ganoderma multipileum Root rot caused by ) Background Technology

[0002] Phoenix Tree ( Delonix regia Delonix regia is a tropical and subtropical ornamental plant widely used in landscaping and urban beautification, and is popular for its beautiful tree shape and vibrant flowers. However, with the expansion of the planting area of ​​Delonix regia, its disease problems have become increasingly prominent, especially those caused by Ganoderma lucidum fungi (Ganoderma lucidum). Ganoderma multipileum Root rot, caused by soil-borne pathogens such as *Pterocarpus indicus*, severely impacts the healthy growth and ornamental value of the plant. Root rot infects the plant's root system, damaging its nutrient transport system and leading to root decay, potentially causing the death of the entire plant. This problem not only poses a challenge to landscape management but also threatens the sustainable development of urban greening.

[0003] Currently, the main methods for controlling root rot in flame trees include chemical control and soil disinfection. However, long-term use of chemical agents can easily lead to drug resistance in pathogens and cause significant environmental pollution, disrupting the soil's ecological balance. With increasing environmental awareness, green and safe control methods are receiving more and more attention, leading to the emergence of biological control technologies, which are gradually becoming an important alternative to chemical control.

[0004] Biological control utilizes antagonistic microorganisms in nature to inhibit the growth of pathogens or to reduce disease by inducing systemic resistance in plants. Bacillus ( Bacillus spp.) and Pseudomonas ( Pseudomonas Bacillus spp. are common biological control strains, widely used in the control of agricultural and horticultural plant diseases due to their ability to produce various antimicrobial substances and their strong environmental adaptability. Bacillus spp. have the ability to produce antimicrobial peptides, antibiotics, and lysozyme, which can directly inhibit the growth of pathogens and enhance plant defense by inducing systemic resistance. Pseudomonas spp., on the other hand, inhibit the spread of pathogens by competing for nutrients and space, and by secreting volatile organic compounds.

[0005] In recent years, biochar inoculants, as a novel biocontrol agent that loads functional microorganisms onto a biochar carrier, have demonstrated significant advantages in agriculture and the environment. Biochar offers significant advantages in agricultural applications due to its cost-effectiveness, biomass availability, and physical and chemical properties. With its large specific surface area and high porosity, biochar is considered an excellent cell immobilization carrier material, providing a beneficial microenvironment for microorganisms and effectively protecting them from environmental stresses such as ultraviolet radiation, drought, and extreme pH, thus significantly improving the survival rate and stability of the inoculant. Since biochar and biocontrol bacteria can regulate soil metabolites and microbial communities, respectively, the synergistic effect of biochar and biocontrol bacteria may be better than using either alone. Furthermore, biochar inoculants use biochar as a carrier, making the raw materials widely available. The preparation process is green and environmentally friendly, aligning with sustainable development principles. The long-term stability of biochar in soil can reduce the frequency of inoculant application, lowering agricultural production costs. Therefore, biochar inoculants, with their excellent carrier properties, enhanced microbial function, improved soil environment, synergistic effects, and environmental friendliness, have broad application prospects in agriculture and the environment.

[0006] Although biological control technologies have been widely applied in various crops, research on biological control of root rot in flame trees remains relatively limited. Root rot caused by Ganoderma lucidum fungi, in particular, is difficult to control due to the complexity and diversity of its pathogens. Therefore, there is an urgent need to develop targeted biochar agents with significant antibacterial effects and optimize their preparation processes to improve control efficacy in practical applications. Summary of the Invention

[0007] The main objective of this invention is to provide an optimized process for biochar inoculants used to prevent and control root rot of the Delonix regia, thereby achieving effective prevention and control of root rot in Delonix regia.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] An optimized preparation process for a biochar agent for preventing root rot in phoenix trees, characterized by the following steps:

[0010] Step 1: Prepare Pseudomonas aeruginosa (… Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis Bacillus licheniformis and Pseudomonas aeruginosa fermentation broth were prepared by bacterial culture and liquid fermentation.

[0011] Step 2: Optimize the fermentation broth culture medium of Bacillus licheniformis and Pseudomonas aeruginosa to obtain the optimized fermentation broth;

[0012] Step 3, the Pseudomonas aeruginosa obtained in Step 2 ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The fermentation broth and biochar were mixed and the loading process was carried out for 24 hours to ensure that the bacterial solution was fully adsorbed on the surface of the biochar and to obtain the biochar inoculant.

[0013] Further, the method for culturing the bacterial culture in step one is as follows: *Pseudomonas aeruginosa* (… Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The cells were streaked onto LB solid medium using the streak plating method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water. The concentration was adjusted to 10⁻¹⁰ at a 1:1 ratio. 8 CFU / ml bacterial suspension;

[0014] Further, the liquid fermentation culture method in step one is as follows: *Pseudomonas aeruginosa* (… Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The bacterial culture was inoculated into LB liquid medium and cultured at 32℃ and 220rpm for 24h.

[0015] Furthermore, in step one, the carbon source of the liquid fermentation medium is sucrose, the composite nitrogen source of the medium is beef extract and ammonium sulfate, and the inorganic salt of the medium is potassium chloride;

[0016] Furthermore, in step one, the concentration of sucrose as the carbon source in the liquid fermentation medium is 3%, the concentration of ammonium sulfate as the composite nitrogen source is 0.5%, the concentration of beef extract is 1%, and the concentration of potassium chloride as the inorganic salt is 2%.

[0017] Furthermore, the biochar produced in step three is straw charcoal;

[0018] Furthermore, the *Pseudomonas aeruginosa* in step three ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The optimal mass ratio of fermentation broth to biochar was 5:1.

[0019] Application of biochar inoculants prepared by any of the above methods in the prevention and control of root rot disease of *Delonix regia* caused by *Ganoderma lucidum*.

[0020] Furthermore, the pathogen causing the root rot of the phoenix tree is Ganoderma lucidum.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. Highly effective antibacterial effect: The two microbial strains significantly inhibited the growth of Ganoderma lucidum through synergistic effect, with an antibacterial rate of 72.8%.

[0023] 2. Green and environmentally friendly: This control agent is prepared from natural microorganisms, avoiding the use of chemical agents, reducing environmental pollution, suitable for long-term use, and has good environmental performance.

[0024] 3. Promote plant growth: This invention can not only prevent and control root rot, but also promote plant growth and enhance its resistance by improving the health of the root system.

[0025] 4. Wide applicability: The biochar agent of the present invention is not only suitable for root rot of phoenix trees, but can also be widely used for the prevention and control of root diseases of other garden plants, and has broad application prospects. Attached Figure Description

[0026] Figure 1 The study investigated the antibacterial effects of Pseudomonas aeruginosa, Bacillus licheniformis, and their mixed plate interactions. CK was the control for Ganoderma lucidum, FH-3 represented the plate interaction effect of Bacillus licheniformis, FH-7 represented the plate interaction effect of Pseudomonas aeruginosa, and FH3 and FH-7 represented the plate interaction effect of the mixed plate interaction of Bacillus licheniformis and Pseudomonas aeruginosa.

[0027] Figure 2 The following is a preferred embodiment of the present invention: the effect of carbon source, nitrogen source and inorganic salt on the antibacterial rate of fermentation broth, where a is the effect of different carbon sources on the antibacterial rate of fermentation broth, b is the effect of different carbon sources on the antibacterial rate of fermentation broth, and c is the effect of different inorganic salts on the antibacterial rate of fermentation broth.

[0028] Figure 3 These are the results of an orthogonal experiment on the nutrient components of the fermentation broth culture medium;

[0029] Figure 4 To assess the biocontrol effect of biochar inoculants on root rot of Delonix regia, a represents the control group, and b represents the group treated with biochar inoculants. Detailed Implementation

[0030] The invention will now be described in further detail with reference to examples.

[0031] Unless otherwise specified, the percentage sign "%" in this invention refers to the mass percentage; however, the percentage of antibacterial rate, unless otherwise specified, refers to (the diameter of the control Ganoderma lucidum colony - the diameter of the treated Ganoderma lucidum colony) / (the diameter of the control Ganoderma lucidum colony - 9) × 100%.

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] The embodiments of the present invention include the following steps: the biochar agent is made from Bacillus licheniformis, Pseudomonas aeruginosa, and straw biochar. The preparation method includes seed liquid culture of Bacillus licheniformis and Pseudomonas aeruginosa, liquid fermentation, optimization of fermentation medium, and mixing of the optimized fermentation liquid with biochar to prepare the biochar agent, thereby improving the control effect.

[0034] Example 1

[0035] Antibacterial activity verification test of Pseudomonas aeruginosa and Bacillus licheniformis.

[0036] The pathogen used in this experiment was Ganoderma lucidum, the pathogen causing root rot of the phoenix tree. Ganoderma multipileum).

[0037] The plate confrontation method was used to detect Pseudomonas aeruginosa. Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The effects of this on the growth of Ganoderma lucidum are explained in the following steps:

[0038] 1. Preparation of PDA solid culture medium: Weigh 200g of peeled potatoes, add 1000mL of distilled water and mix well. Boil over low heat for 30min and filter with double-layer gauze. Add 20g of glucose and 20g of agar, and bring the volume to 1000mL. Autoclave at 121℃ for 20min. Set aside for later use. 2. Preparation of LB liquid culture medium (1L): Weigh 5g of yeast extract, 10g of tryptone, and 10g of sodium chloride. Adjust the pH to 7.0 with NaOH. Autoclave at 121℃ for 20min. Set aside for later use.

[0039] 2. Preparation of LB liquid culture medium (1L): Weigh 5g yeast extract, 10g tryptone, 10g sodium chloride, and 20g agar. Adjust the pH to 7.0 with NaOH. Autoclave at 121℃ for 20 minutes.

[0040] 3. Bacterial culture: Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The cells were streaked onto LB solid medium using the streak plating method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water to adjust the concentration to 10%. 8 CFU / ml bacterial suspension;

[0041] 4. Inoculate a 6mm diameter Ganoderma lucidum mycelium cake in the center of a PDA plate. Using a pipette, aspirate 8µL of the culture medium of the test strain and inoculate it 2cm away from the Ganoderma lucidum mycelium cake. Then incubate upside down in a 25℃ incubator for 7-10 days.

[0042] 5. After culturing for 7-10 days, observe the inhibitory effect of the antagonistic strains on Ganoderma lucidum on the plates. The inhibitory ability of the strains on Ganoderma lucidum is determined by measuring the diameter of the inhibition zone. The results show that Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The antibacterial rates were 71.6% and 65.4% respectively, and the combined antibacterial rate was 72.8%. Figure 1 ).

[0043] Example 2

[0044] The screening and construction of biochar carriers includes the following steps:

[0045] 1. Preparation of PDA solid culture medium: Weigh 200g of peeled potatoes, add 1000mL of distilled water and mix well. Boil over low heat for 30min and filter with double-layer gauze. Add 20g of glucose and 20g of agar, and bring the volume to 1000mL. Autoclave at 121℃ for 20min. Set aside. 2. Preparation of LB liquid culture medium (1L): Weigh 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, and 20g of agar. Adjust the pH to 7.0 with NaOH. Autoclave at 121℃ for 20min. Set aside.

[0046] 2. *Pseudomonas aeruginosa* ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The cells were streaked onto LB solid medium using the streak plating method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water, with the concentration adjusted to 10-. 8 CFU / ml bacterial culture.

[0047] 3. Weigh 5 g of each of the finely powdered straw charcoal (SB), rice husk charcoal (RHB), bamboo charcoal (BB), and wood charcoal (WB) prepared by anaerobic pyrolysis of raw materials at 500 ℃, add them to a 250 mL Erlenmeyer flask, autoclave at 121 ℃ for 30 min, add 25 mL of mixed bacterial solution, and place at 30 ℃, shake at 170 rpm for 2 h. Centrifuge at 1000 rpm for 10 min, and determine the number of bacteria fixed on the carrier using the plate counting method.

[0048] 4. As shown in Table 1, straw charcoal (SB) is the best carrier. The number of loaded bacteria from high to low is SB, RHB, WB and BB. Among them, the number of loaded bacteria in SB is 36.8% higher than that in RHB and 72.7% higher than that in BB.

[0049] Table 1. Number of bacteria loaded on different biochars.

[0050] Biochar SB RHB BB WB <![CDATA[Number of bacteria loaded (10 8 CFU / mL)]]> 15.13±0.43 11.37±0.82 8.76±0.27 10.67±0.53

[0051] 5. Four types of biochar materials—stalk charcoal (SB), rice husk charcoal (RHB), bamboo charcoal (BB), and wood charcoal (WB)—were selected and added to 250 mL Erlenmeyer flasks containing 50 mL of deionized water at material-to-liquid ratios of 1:25, 1:10, 1:5, and 1:2, respectively. The flasks were placed in a constant-temperature shaker at 200 rpm for 1 hour at room temperature to ensure thorough extraction. After extraction, the mixture was transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 min to remove suspended particles. The supernatant was filtered through a 0.22 μm sterile filter to obtain a sterile extract, which was then aliquoted and stored at 4 °C for later use. A 9 mm layer of Ganoderma lucidum mycelium was placed in the center of a PDA plate, and Oxford cups were placed around it 3 cm from the center. 100 μL of the sterile extract was added to each cup. The plates were incubated at 28 °C for 5–7 days, and mycelial growth was observed. Sterile water treatment served as a control. Each treatment was repeated in triplicate.

[0052] 6. As shown in Table 2, low-concentration extracts did not significantly inhibit the mycelial growth of *Ganoderma lucidum* covered with biochar. Under high-concentration conditions, SB extract had an inhibitory effect on the growth of *Ganoderma lucidum* covered with biochar, with the inhibition zone diameter of the 1:5 concentration group being 1.1 cm. Meanwhile, the extracts of the other biochars (RHB, BB, WB) did not show significant inhibitory effects on *Ganoderma lucidum* covered with biochar at the same concentration.

[0053] Table 2 Number of bacteria loaded with different biochar

[0054] Diameter of the inhibition zone (cm) 1:25 1:10 1:5 1:2 SB 0.4 0.5 1.1 0.5 RHB 0.1 0.2 0.4 0.2 BB 0.1 0.3 0.6 0.4 WB 0.2 0.1 0.5 0.3

[0055] Example 3

[0056] Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis Optimization of liquid culture medium fermentation includes the following steps:

[0057] 1. Preparation of PDA solid medium: Weigh 200g of peeled potatoes, add 1000mL of distilled water and mix well. Boil over low heat for 30 minutes and filter through double-layer gauze. Add 20g of glucose and 20g of agar, and bring the volume to 1000mL. Autoclave at 121℃ for 20 minutes. Set aside. 2. Preparation of LB liquid medium (1L): Weigh 5g of yeast extract, 10g of tryptone, and 10g of sodium chloride. Adjust the pH to 7.0 with NaOH. Autoclave at 121℃ for 20 minutes. Set aside. 3. Preparation of LB solid medium (1L): Weigh 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, and 20g of agar. Adjust the pH to 7.0 with NaOH. Autoclave at 121℃ for 20 minutes. Set aside.

[0058] 2. Preparation of bacterial culture: *Pseudomonas aeruginosa* (… Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis FH-7 cells were streaked onto LB solid medium using the streak plate method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water, with the concentration adjusted to 10-1. 8 CFU / ml bacterial culture.

[0059] 3. Determination of Antibacterial Activity of Fermentation Broth: Using Ganoderma lucidum as an indicator bacterium and the inhibition rate of the fermentation broth as an indicator of antibacterial activity, a single-factor experimental design was employed to optimize the optimal fermentation medium composition for the production of antibacterial active substances. Mycelial cakes were collected from the edge of Ganoderma lucidum colonies using a 9mm diameter punch and inoculated onto the center of a PDA medium plate. Simultaneously, four Oxford cups were placed at four equidistant locations (2cm from the mycelial cake), and 150μL of fermentation broth was added. After 5 days of incubation in a dark incubator at 28℃, the antagonistic effect of the fermentation broth on Ganoderma lucidum was observed. The diameter of the inhibition zone was measured using a vernier caliper with a cross-sectional method, and the inhibition rate was used as an indicator of antibacterial activity. Inhibition rate (%) = (Diameter of control Ganoderma lucidum colony - Diameter of treated Ganoderma lucidum colony) / (Diameter of control Ganoderma lucidum colony - 9) × 100%.

[0060] 4. Screening of components in the liquid fermentation medium: The carbon source of the LB liquid medium was replaced with corn flour, glucose, sucrose, rice flour, and wheat flour, respectively; the nitrogen source of the LB medium was replaced with beef extract, peptone, soybean flour, ammonium sulfate, and peanut flour, respectively; and the inorganic salts of the LB medium were replaced with potassium dihydrogen phosphate, potassium chloride, calcium carbonate, sodium chloride, and sodium dihydrogen phosphate, respectively. 3% of the bacterial suspension prepared in step one was inoculated into the above medium at a 1:1 ratio. After culturing at 220 r / min for 24 h in a dark, constant-temperature shaker at 32℃, the antibacterial activity of the fermentation broth was measured.

[0061] From the results Figure 2 It can be seen that the optimal carbon source for liquid fermentation medium is sucrose, the optimal compound nitrogen source for the medium is beef extract and ammonium sulfate, and the optimal inorganic salt for the medium is potassium chloride.

[0062] 5. Screening of liquid fermentation medium component concentrations: The optimal carbon source, nitrogen source, and inorganic salts screened in step 3 were used to construct fermentation media. Three gradients of carbon source concentrations (1%, 2%, and 3%), three concentrations of nitrogen source concentrations (0.5%, 1%, and 1.5%), and three concentrations of inorganic salts (1%, 1.5%, and 2%) were established. A 3% inoculum of the bacterial suspension prepared in step 1 was inoculated into these media. After culturing at 32℃ in the dark on a shaker at 220 rpm for 24 h, the antibacterial activity of the fermentation broth was measured.

[0063] As shown in Table 3, the factor with the greatest impact on the antibacterial rate of the fermentation broth was the organic nitrogen source, beef extract (C), while the factor with the least impact was the inorganic salt, potassium chloride (D). The combination with the best antibacterial rate among the nine combinations was A3B2C1D3. Figure 3 Therefore, the optimal culture medium formula for liquid fermentation was determined to be 3% sucrose, 0.5% ammonium sulfate, 1% beef extract, and 2% potassium chloride.

[0064] Example 4

[0065] A method for preparing a biochar agent includes the following steps:

[0066] 1. *Pseudomonas aeruginosa* ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The cells were streaked onto LB solid medium using the streak plating method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water to adjust the concentration to 10%. 8 CFU / ml bacterial suspension;

[0067] 2. Preparation of liquid fermentation medium: 3% sucrose, 0.5% ammonium sulfate, 1% beef extract, and 2% potassium chloride. Sterilize at 121 degrees Celsius before use.

[0068] 3. Take 3% of the bacterial culture prepared above and inoculate it into the above liquid fermentation medium. Incubate at 32℃ and 220rpm for 24h to prepare the optimized fermentation broth.

[0069] 4. Mix the above-mentioned optimized fermentation broth with straw charcoal at a mass ratio of 5:1, and place it at room temperature for 24 hours to ensure that the bacterial solution is fully adsorbed on the surface of the biochar, thus obtaining the biochar inoculant.

[0070] Example 5

[0071] The pot experiment using biochar inoculant included the following steps:

[0072] One-year-old healthy, soil-grown Delonix regia plants were selected. After rinsing them thoroughly with tap water, the roots were soaked in 75% alcohol for 2 minutes, followed by rinsing with sterile water 3-5 times. The plants were then transplanted into autoclaved soil. Fourteen days after transplanting, 10g of the prepared biochar agent was added to the roots as a treatment group via root drenching. A blank control group received 10mL of sterile distilled water. Each treatment was replicated in triplicate, with three plants per replicate. Seven days later, 50mL of the prepared pathogen spore suspension was injected into the roots using a needle puncture method. After 21 days of greenhouse cultivation, disease incidence was observed, and the disease index and control effect were calculated.

[0073] Table 3 shows the control effect of biochar inoculant on root rot of Delonix regia in a potted experiment. In the control group treated with pathogens, the roots of Delonix regia turned black and rotted, the root system was underdeveloped, and the disease was severe. Figure 4 After treatment with biochar agent, the Delonix regia plants inoculated with pathogens and covered with Ganoderma lucidum grew well, with fewer and less severe disease outbreaks compared to the control group, and a relative control efficacy of 83.53%.

[0074] Table 3 shows the control effect of biochar inoculants on root rot of Delonix regia in pot experiments.

[0075] deal with Disease index Prevention and control efficacy (%) Comparison with CK 72.58±2.47a — Biochar inoculant 12.15±1.91b 83.53±3.58

[0076] The method of this invention can effectively prevent and control root rot in flame trees, reduce the use of chemical agents, protect the ecological environment, and improve plant health and growth quality. The biochar agent provided by this invention is not only suitable for the prevention and control of diseases in flame trees, but can also be widely applied to the prevention and control of root diseases in other garden plants, demonstrating broad application prospects and economic value.

Claims

1. An optimized preparation process for a biochar agent used to control root rot in phoenix trees, characterized in that, The process includes the following steps: (1) Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis Fermentation broths of Bacillus licheniformis and Pseudomonas aeruginosa were prepared by culturing bacterial culture and liquid fermentation. (2) The fermentation broth culture medium of Bacillus licheniformis and Pseudomonas aeruginosa was optimized to prepare an optimized fermentation broth, which consists of the following components: 1) Carbon sources: corn flour, glucose, sucrose, rice flour, and wheat flour; 2) Nitrogen sources: beef extract, peptone, soybean flour, ammonium sulfate, and peanut flour; 3) Inorganic salts: potassium dihydrogen phosphate, potassium chloride, calcium carbonate, sodium chloride, and sodium dihydrogen phosphate; 4) The carbon source concentration is 1-3%, the composite nitrogen source concentration is 0.5-1.5%, and the inorganic salt concentration is 1-2%.

2. (3) The Pseudomonas aeruginosa obtained in step (2) ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The fermentation broth was optimized and mixed with biochar. The mixture was then placed at room temperature for 24 hours to ensure that the bacterial solution was fully adsorbed onto the surface of the biochar, thus obtaining the biochar inoculant.

3. The preparation process according to step (1) of claim 1, characterized in that, The method for culturing the bacterial culture is as follows: *Pseudomonas aeruginosa* (Pseudomonas aeruginosa) and Bacillus licheniformis (Bacillus licheniformis) The cells were streaked onto LB solid medium using the streak plating method and incubated at 30°C for 24 hours. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 32°C in the dark on a shaker at 200 rpm for 24 hours. The supernatant was then separated, cells were collected, washed, and resuspended in sterile distilled water. The concentration was adjusted to 10⁻¹⁰ at a 1:1 ratio. 8 CFU / ml bacterial culture.

4. The preparation process according to step (1) of claim 1, characterized in that, The liquid fermentation culture method is as follows: *Pseudomonas aeruginosa* (Pseudomonas aeruginosa) and Bacillus licheniformis (Bacillus) licheniformis) The bacterial culture was inoculated at 3% in liquid fermentation medium and cultured at 32℃ and 220rpm for 24h.

5. The preparation process in step (2) according to claim 1, characterized in that, The preferred carbon source for the liquid fermentation medium is sucrose, the preferred composite nitrogen source is beef extract and ammonium sulfate, and the preferred inorganic salt is potassium chloride. The preferred carbon source sucrose concentration is 3%, the preferred composite nitrogen source ammonium sulfate concentration is 0.5%, the preferred beef extract concentration is 1%, and the preferred inorganic salt potassium chloride concentration is 2%.

6. The preparation process of step (3) according to claim 1, characterized in that, The Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Bacillus licheniformis ( Bacillus licheniformis The optimal mass ratio of fermentation broth to biochar was 5:

1.

7. The preparation process according to step (3) of claim 1, characterized in that, The biochar is straw charcoal.

8. A biochar agent prepared by the process according to any one of claims 1-6, characterized in that, Used to prevent and treat root rot of the phoenix tree caused by Ganoderma lucidum.

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