Combined preparation for preventing and treating morchella diseases and application

Abstract

The invention discloses a combined preparation for preventing and treating morchella diseases and application of the combined preparation, and belongs to the technical field of edible mushroom culture. The combined preparation comprises armillaria luteo-virens polysaccharide F3 and antibacterial peptide Thanatin, and sterile water is used as a carrier. Experimental results show that when the armillaria luteo-virens polysaccharide F3 and the antibacterial peptide Thanatin are combined for use, a remarkable synergistic inhibition effect is achieved on paecilomyces varioti causing white mold of morchella esculenta. In addition, the combined preparation has no obvious negative influence on the growth of mycelia of the morchella esculenta, and shows good safety. Therefore, the invention provides an efficient, safe and environment-friendly prevention and treatment strategy for the white mold of the morchella esculenta, which is beneficial to reducing the use of chemical pesticides, delaying the generation of drug resistance of pathogenic bacteria and promoting the healthy and sustainable development of the morchella esculenta industry.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi cultivation technology, and in particular relates to a combination preparation and its application for the prevention and control of morel diseases. Background Technology

[0002] Morel mushrooms (Morchella spp.) are a prized edible fungus, highly sought after in the market for their unique flavor and rich nutritional value. In recent years, with advancements in cultivation techniques and increasing market demand, the scale of artificial cultivation of morels has been continuously expanding. However, during cultivation, disease problems have become one of the important factors restricting high and stable yields. In particular, fungal diseases severely affect the mycelial growth, fruiting body development, and ultimately, the yield and quality of morels.

[0003] Among the many diseases, white mold is a common and serious disease in morel cultivation, mainly caused by Paecilomyces penicillatus. This disease usually occurs in cultivation environments with high humidity and poor ventilation, manifesting as white, fluffy mycelium on the surface of the stipe or cap, which leads to the rotting of the fruiting body and loss of commercial value, thus causing significant economic losses to the morel industry.

[0004] Currently, the main control methods for white mold disease include environmental control, chemical control, and biological control. However, the use of traditional chemical agents easily leads to residue problems and environmental pollution, and the pathogens may also develop drug resistance. While biological control has potential, its actual application effectiveness is still unstable. Therefore, there is an urgent need to develop an efficient, safe, and sustainable control method or combination formulation to effectively control white mold disease caused by *Paecilomyces cerevisiae*, ensuring the healthy cultivation and industrial development of morel mushrooms. Summary of the Invention

[0005] The purpose of this invention is to provide a combination formulation and application for the prevention and control of morel diseases, thereby achieving green and efficient prevention and control of morel white mold disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution: First, the present invention provides a combination preparation for the prevention and control of morel white mold disease, the combination preparation being composed of Armillaria mellea polysaccharide F3, antimicrobial peptide Thanatin, and sterile water; The CAS number for the antimicrobial peptide Thanatin is 214542-43-5; The Armillaria mellea polysaccharide F3 is an Armillaria mellea polysaccharide with a molecular weight of 30-50 kDa obtained by ultrafiltration membrane separation.

[0007] Preferably, the concentration of Armillaria mellea polysaccharide F3 is 0.5-2.0 mg / mL, and the concentration of the antimicrobial peptide Thanatin is 20-40 µM.

[0008] Preferably, the Armillaria mellea polysaccharide F3 is prepared by the following method: (1) The fruiting body powder of Armillaria mellea was defatted to obtain defatted fungal powder; (2) Mix defatted bacteria powder with distilled water at a mass-to-volume ratio of 1:25, extract at 40 kHz ultrasonic frequency and 60℃ for 30 minutes, and then continue to extract in a 90℃ water bath for 2 hours to obtain the extract; (3) After the extract is concentrated under reduced pressure, ethanol is slowly added for alcohol precipitation, and the precipitate is collected by centrifugation; (4) Dissolve the precipitate in distilled water and remove the protein using the Sevage method to obtain a crude polysaccharide solution; (5) After the crude polysaccharide solution is filtered through a microporous membrane, it is passed through a 100kDa and a 50kDa molecular weight cutoff filter membrane in sequence, and then the 50kDa filtrate is passed through a 30kDa molecular weight cutoff filter membrane and the retentate is collected. (6) The retentate was freeze-dried to obtain Armillaria mellea polysaccharide F3.

[0009] Preferably, the white mold disease is caused by Paecilomyces longifolia.

[0010] Secondly, the present invention provides the application of the aforementioned combination formulation in the preparation of a drug for the prevention and treatment of morel white mold disease, characterized in that the combination formulation is used to prevent and treat morel white mold disease caused by Paecilomyces pubescens.

[0011] Furthermore, the present invention provides an antibacterial agent for inhibiting the growth of Paecilomyces longifolia, characterized in that the antibacterial agent is composed of 0.5-2.0 mg / mL Armillaria mellea polysaccharide F3, 20-40 µM antimicrobial peptide Thanatin, and sterile water; The Armillaria mellea polysaccharide F3 was prepared according to the above preparation method; The CAS number for the antimicrobial peptide Thanatin is 214542-43-5.

[0012] Next, the present invention provides an application of a composition in the preparation of an antimicrobial agent that inhibits the growth of Paecilomyces cerevisiae, characterized in that the composition consists of 0.5-2.0 mg / mL Armillaria mellea polysaccharide F3 and 20-40 µM antimicrobial peptide Thanatin.

[0013] Finally, the present invention provides the application of Armillaria mellea polysaccharide in the preparation of Paecilomyces longifolia antibacterial agent, characterized in that the Armillaria mellea polysaccharide is one of Armillaria mellea polysaccharide F2, Armillaria mellea polysaccharide F3 or Armillaria mellea polysaccharide F4; The *Armillaria mellea* polysaccharide F2, F3, and F4 were prepared by the following method: (1) The fruiting body powder of Armillaria mellea was defatted to obtain defatted fungal powder; (2) Mix defatted bacteria powder with distilled water at a mass-to-volume ratio of 1:25, extract at 40 kHz ultrasonic frequency and 60℃ for 30 minutes, and then continue to extract in a 90℃ water bath for 2 hours to obtain the extract; (3) After the extract is concentrated under reduced pressure, ethanol is slowly added for alcohol precipitation, and the precipitate is collected by centrifugation; (4) Dissolve the precipitate in distilled water and remove the protein using the Sevage method to obtain a crude polysaccharide solution; (5) After filtering the crude polysaccharide through a microporous membrane, it was passed through a 100kDa molecular weight cutoff filter membrane and a 50kDa molecular weight cutoff filter membrane in sequence. The 50kDa retentate was collected to obtain the Armillaria mellea polysaccharide F2 solution. (6) Pass the 50kDa filtrate through a 30kDa molecular weight cutoff filter membrane, collect the 30kDa cutoff solution, and obtain the Armillaria mellea polysaccharide F3 solution; (7) Pass the 30kDa filtrate through a 10kDa molecular weight cutoff filter membrane, collect the 10kDa cutoff solution, and obtain Armillaria mellea polysaccharide F4 solution; (8) Freeze-dry the Armillaria mellea polysaccharide F2 solution, Armillaria mellea polysaccharide F3 solution and Armillaria mellea polysaccharide F4 solution to obtain Armillaria mellea polysaccharide F2, Armillaria mellea polysaccharide F3 and Armillaria mellea polysaccharide F4.

[0014] The beneficial effects of this invention are as follows: The combination formulation of this invention, composed of Armillaria mellea polysaccharide F3 (molecular weight 30-50 kDa) and the antimicrobial peptide Thanatin, exhibits a significant synergistic antibacterial effect against Paecilomyces cirrhosa, which causes morel white mold disease. Experiments have shown that the inhibition rate after combination (up to 86.26%) far exceeds that of the single components, and the synergistic index (q value) is significantly higher than 1.15, confirming a non-linearly enhanced antibacterial effect.

[0015] At the same time, this combination formulation effectively inhibits pathogens without significantly negatively impacting the mycelial growth of morel mushrooms, ensuring their healthy growth and meeting the needs of green agricultural production.

[0016] Furthermore, the synergistic effect of the two components allows for a reduction in the dosage of a single active ingredient, thereby effectively lowering control costs. Simultaneously, the combined action of multiple targets and mechanisms helps delay the development of resistance in Paecilomyces pubescens, improving the sustainability of control efforts.

[0017] In summary, this invention utilizes naturally sourced Armillaria mellea polysaccharides and antimicrobial peptides to provide a non-chemical pesticide biological control solution, avoiding the environmental pollution and agricultural product residue problems of traditional chemical agents, and providing a safe and efficient solution for the healthy development of the morel mushroom industry. Attached Figure Description

[0018] Figure 1 The results show the effects of Armillaria mellea polysaccharides F1, F2, F3, and F4 on the mycelial growth of Paecilomyces longifolia. Figure 1 Image A shows the results of Armillaria mellea polysaccharide F1, image B shows the results of Armillaria mellea polysaccharide F2, image C shows the results of Armillaria mellea polysaccharide F3, and image D shows the results of Armillaria mellea polysaccharide F4. Figure 2 The figure shows the effect of the antimicrobial peptide Thanatin on the growth of Paecilomyces longifolia hyphae. Figure 3 The figure shows the results of the inhibitory effect of Armillaria mellea polysaccharide F3 combined with the antimicrobial peptide Thanatin on Paecilomyces cirrhosa. Figure 4 To evaluate the safety of combined antibacterial agents on the growth of morel mycelium. Detailed Implementation

[0019] Example 1 Raw material: Armillaria luteovirens (Aalb. et Schw:Fr.) Sacc. fruit powder (roots removed, dried, and passed through a 40-mesh sieve).

[0020] Reagents: petroleum ether, 95% ethanol, Sevage reagent (chloroform: n-butanol = 4:1), 0.45 μm microporous membrane (Millipore), 100 kDa, 50 kDa, 30 kDa, and 10 kDa molecular weight cutoff filter membranes (Millipore).

[0021] Preparation steps: (1) Add the dried yellow-green Armillaria fruiting body powder to petroleum ether at a solid-liquid ratio of 1:10 (w / v), and use an ultrasonic extractor to treat it for 30 minutes at 40°C. Then centrifuge (3000 rpm for 10 minutes), discard the supernatant, and obtain defatted bacterial powder. (2) Mix the above defatted bacteria powder with distilled water at a mass-to-volume ratio of 1:25 (w / v) and extract for 30 minutes using an ultrasonic extractor at a ultrasonic frequency of 40 kHz and a temperature of 60°C. (3) The mixture was then transferred to a 90°C water bath and heated for 2 hours to obtain the extract; (4) The extract was concentrated under reduced pressure at 60°C to 1 / 4 of its original volume. Four times the volume of 95% ethanol was slowly added, and the mixture was thoroughly mixed. The mixture was then allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation. (5) Dissolve the obtained precipitate in an appropriate amount of distilled water to prepare a 20 mg / mL solution, and remove the protein using the Sevage method: add an equal volume of Sevage reagent (chloroform: n-butanol = 4:1), shake vigorously to mix, let stand to separate the layers, take off the upper aqueous phase, repeat the operation until there is no white protein layer at the interface, and obtain the crude polysaccharide solution. (6) After filtering the crude polysaccharide solution through a 0.45 μm microporous membrane, filter it through a 100 kDa filter membrane (pressure 0.25 MPa, temperature 25℃, the same below), collect the retentate a, and record it as polysaccharide component F1; (7) Pass the filtrate a through a 50kDa filter membrane, collect the retentate b, and record it as polysaccharide component F2; (8) Pass the filtrate b through a 30kDa filter membrane, collect the retentate c, and record it as polysaccharide component F3; (9) Pass the filtrate c through a 10 kDa filter membrane, collect the retentate d, and record it as polysaccharide component F4; (10) The obtained polysaccharide components F1, F2, F3 and F4 were placed in a freeze-drying bottle and freeze-dried to obtain Armillaria mellea polysaccharide F1, Armillaria mellea polysaccharide F2, Armillaria mellea polysaccharide F3 and Armillaria mellea polysaccharide F4.

[0022] Example 2 Experimental materials: The *Paecilomyces longifolia* strain (CCMJ2836) was obtained from Jilin Agricultural University, preserved in potato dextrose agar (PDA) slant medium, and activated for use in experiments. Armillaria mellea polysaccharides F1, F2, F3, and F4 (lyophilized powder) were prepared into sterile aqueous solutions of different concentrations (e.g., 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL) and sterilized by passing them through a 0.22 μm filter membrane.

[0023] Potato glucose agar (PDA) medium: used for fungal culture and antibacterial tests.

[0024] Experimental steps (1) Streak the Paecilomyces longifolia preserved on the PDA slant onto the PDA plate and incubate at 25°C for 7 days until the colonies are fully developed. (2) Rinse the colonies on the PDA plate with sterile physiological saline, collect the spore suspension, and adjust the spore concentration to approximately 1 × 10⁻⁶ using a hemocytometer. 6 CFU / mL, for later use; (3) Cool the sterilized PDA medium to about 50°C, and add appropriate amounts of polysaccharide solution to make the final concentrations 0.5 mg / mL, 1.0 mg / mL and 2.0 mg / mL respectively; (4) After mixing thoroughly, pour into a sterile petri dish, let it solidify and set aside for later use. At the same time, set up a blank control group without polysaccharides. (5) Use a sterile punch (5 mm in diameter) to take the bacterial cake from the edge of the activated colony, inoculate it into the center of the drug-containing PDA plate, cover the petri dish, and set up 3 biological replicates for each treatment; (6) Place all plates in a constant temperature incubator at 25℃ for 7 days. After the culture is completed, measure the colony diameter (unit: mm) using the cross-cross method, calculate the average colony diameter, and calculate the inhibition rate (inhibition rate (%) = (colony diameter of control group - colony diameter of experimental group) / colony diameter of control group × 100%).

[0025] The results are as follows ( Figure 1 ): The average colony diameter of the control group was 84.73±3.02, indicating that *Paecilomyces longifolia* had good growth under the culture conditions of this invention. For Armillaria mellea polysaccharide F1, the average colony diameter was 82.47±3.37 at a concentration of 0.5 mg / mL, 82.63±2.86 at a concentration of 1.0 mg / mL, and 81.86±3.30 at a concentration of 2.0 mg / mL. The calculated inhibition rates were 2.67%, 2.48%, and 3.39%, respectively. The t-test results showed that the differences were all greater than 0.05.

[0026] For Armillaria mellea polysaccharide F2, the average colony diameter was 76.06±2.84 at a concentration of 0.5 mg / mL, 69.72±3.35 at a concentration of 1.0 mg / mL, and 65.56±3.55 at a concentration of 2.0 mg / mL. The calculated inhibition rates were 10.23%, 17.72%, and 22.63%, respectively. The t-test results showed that the differences were all less than 0.05.

[0027] For Armillaria mellea polysaccharide F3, the average colony diameter was 71.50±3.08 μm at a concentration of 0.5 mg / mL, 60.18±2.72 μm at a concentration of 1.0 mg / mL, and 54.31±3.02 μm at a concentration of 2.0 mg / mL. The calculated inhibition rates were 15.61%, 29.09%, and 35.90%, respectively. The t-test results showed that the differences were all less than 0.05.

[0028] For Armillaria mellea polysaccharide F4, the average colony diameter was 79.87±3.70 at a concentration of 0.5 mg / mL, 74.71±2.89 at a concentration of 1.0 mg / mL, and 66.18±3.66 at a concentration of 2.0 mg / mL. The calculated inhibition rates were 5.74%, 11.82%, and 21.89%, respectively. The t-test results showed that the differences were all less than 0.05.

[0029] The results above show that Armillaria mellea polysaccharide F2, F3, and F4 all have a certain inhibitory effect on Paecilomyces cerevisiae, effectively inhibiting its growth, with F3 showing the most significant antibacterial effect. However, Armillaria mellea polysaccharide F1 does not inhibit the growth of Paecilomyces cerevisiae, possibly due to its large molecular weight or structural characteristics that are not suitable for penetrating the fungal cell wall.

[0030] In conclusion, some components of Armillaria mellea polysaccharides (especially F2, F3, and F4) may serve as potential natural antifungal substances.

[0031] Example 3 Although Example 2 demonstrated that Armillaria mellea polysaccharides F2, F3, and F4 could inhibit the growth of Paecilomyces cerevisiae to some extent, the effect was not particularly ideal. Therefore, this invention attempts to combine antimicrobial peptides with them in order to achieve a better inhibitory effect on Paecilomyces cerevisiae.

[0032] Thanatin is an inducible cationic antimicrobial peptide (CAS No. 214542-43-5, purchased from MCE catalog No.: HY-P5601). Thanatin exhibits broad-spectrum activity against Gram-negative bacteria, Gram-positive bacteria, and various fungi. However, there are currently no reports on the ability of Thanatin to inhibit Paecilomyces pilosa. Therefore, this invention first investigates its inhibitory effect on Paecilomyces pilosa.

[0033] (1) Streak the Paecilomyces longifolia preserved on the PDA slant onto the PDA plate and incubate at 25°C for 7 days until the colonies are fully developed. (2) Rinse the colonies on the PDA plate with sterile physiological saline, collect the spore suspension, and adjust the spore concentration to approximately 1 × 10⁻⁶ using a hemocytometer. 6 CFU / mL, for later use; (3) Cool the sterilized PDA medium to about 50°C and add appropriate amounts of Thanatin to make their final concentrations 5µM, 10µM, 20µM and 40µM respectively. (4) After mixing thoroughly, pour into a sterile petri dish, let it solidify and set aside for later use. At the same time, set up a blank control group without polysaccharides. (5) Use a sterile punch (5 mm in diameter) to take the bacterial cake from the edge of the activated colony, inoculate it into the center of the drug-containing PDA plate, cover the petri dish, and set up 3 biological replicates for each treatment; (6) Place all plates in a 25℃ constant temperature incubator for 7 days. After the incubation, measure the colony diameter (unit: mm) using the cross-cross method, calculate the average colony diameter, and calculate the inhibition rate at the same time.

[0034] from Figure 2 The results showed that, compared to the control group's average colony diameter of 81.76±3.73, the average colony diameters of *Paecilomyces cerevisiae* at concentrations of 20µM and 40µM were 68.44±3.51 (inhibition rate 16.29%) and 58.53±3.54 (inhibition rate 28.41%), respectively, which were significantly lower than the control group, and the t-test results were less than 0.05. This indicates that the antimicrobial peptide thanatin can inhibit the growth of *Paecilomyces cerevisiae* at concentrations of 20µM and 40µM.

[0035] Example 4 This invention selects the most effective Armillaria mellea polysaccharide F3 and combines it with the antimicrobial peptide Thanatin to enhance the inhibitory effect of Armillaria mellea polysaccharide F3 on Paecilomyces cerevisiae.

[0036] (1) Streak the Paecilomyces longifolia preserved on the PDA slant onto the PDA plate and incubate at 25°C for 7 days until the colonies are fully developed. (2) Rinse the colonies on the PDA plate with sterile physiological saline, collect the spore suspension, and adjust the spore concentration to approximately 1 × 10⁻⁶ using a hemocytometer. 6 CFU / mL, for later use; (3) Cool the sterilized PDA medium to approximately 50°C and process it according to the following groups: Polysaccharide F3 alone group 1: final concentration 0.5 mg / mL Armillaria mellea polysaccharide F3; Group 2: Polysaccharide F3 alone: ​​final concentration 1.0 mg / mL Armillaria mellea polysaccharide F3; Group 3, using polysaccharide F3 alone: ​​final concentration 2.0 mg / mL Armillaria mellea polysaccharide F3; Thanatin monotherapy: final concentration 40µM thanatin. Polysaccharide-antimicrobial peptide combination group 1: final concentration 0.5 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; Polysaccharide-antimicrobial peptide combination group 2: final concentration 1.0 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; Polysaccharide-antimicrobial peptide combination group 3: final concentration 2.0 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; (4) After mixing thoroughly, pour into a sterile Petri dish, let it solidify and set aside. At the same time, set up a blank control group with PDA culture medium that does not contain polysaccharides and antimicrobial peptides. (5) Use a sterile punch (5 mm in diameter) to take the bacterial cake from the edge of the activated colony, inoculate it into the center of the drug-containing PDA plate, cover the petri dish, and set up 3 biological replicates for each treatment; (6) Place all plates in a 25℃ constant temperature incubator for 7 days. After the incubation, measure the colony diameter (unit: mm) using the cross-cross method, calculate the average colony diameter, and calculate the inhibition rate at the same time.

[0037] Table 1. Inhibitory effect of Armillaria mellea polysaccharide F3 combined with antimicrobial peptide Thanatin on Paecilomyces cerevisiae.

[0038] To determine whether the combination of Armillaria mellea polysaccharide F3 prepared in this invention and antimicrobial peptides exhibits a synergistic effect, the Bliss independence model formula was used. I expected(%) =( I A + I B - I A × I B The expected inhibition rate of the combination group was calculated by multiplying the result by 100%, and the results are shown in Table 2. Table 2. Expected inhibition rate and the difference between actual results

[0039] The actual inhibition rates of all combination groups were significantly higher than the values ​​predicted by the Bliss model, with differences of 10.04% (combination group 1), 19.37% (combination group 2), and 33.79% (combination group 3), respectively. This indicates that the combined use of polysaccharide F3 and antimicrobial peptide Thanatin is not a simple additive effect, but rather produces a synergistic antimicrobial effect.

[0040] Meanwhile, the q values ​​calculated using King's formula q = Ea+b / (Ea + Eb - Ea*Eb) are as follows: 1.26 for polysaccharide-antimicrobial peptide combination group 1, 1.40 for polysaccharide-antimicrobial peptide combination group 2, and 1.64 for polysaccharide-antimicrobial peptide combination group 3, all of which are significantly higher than 1.15.

[0041] Meanwhile, the results show that the higher the concentration of polysaccharide F3, the more significant the synergistic effect when used in combination with the antimicrobial peptide. This may be because the polysaccharide enhances the penetration ability of the antimicrobial peptide by disrupting the integrity of the bacterial cell membrane. Due to the different target sites of the two, the antibacterial effect is enhanced non-linearly. This synergistic effect can reduce the dosage of single drugs, thereby alleviating the cost pressure of using the antimicrobial peptide Thanatin and delaying the development of bacterial resistance, while achieving a significant inhibitory effect on Paecilomyces pilosa.

[0042] Example 5 A combination formulation A for the prevention and control of morel white mold disease The formulation of this combination preparation A consists of 0.5 mg / mL Armillaria mellea polysaccharide F3, 40 µM antimicrobial peptide Thanatin, and sterile water.

[0043] Example 6 A combination formulation B for the prevention and control of morel white mold disease The formulation of this combination preparation B consists of 1 mg / mL Armillaria mellea polysaccharide F3, 40 µM antimicrobial peptide Thanatin, and sterile water.

[0044] Example 7 A combination formulation C for the prevention and control of morel white mold disease The formulation of this combination preparation C consists of 2 mg / mL Armillaria mellea polysaccharide F3, 40 µM antimicrobial peptide Thanatin, and sterile water.

[0045] Example 8 To test the safety of the above-mentioned combined antimicrobial agent for morel mushrooms, this embodiment tested the inhibitory effect of the above-mentioned combined antimicrobial agent on the mycelial growth of morel mushrooms.

[0046] (1) Place the six-sister morel mushrooms ( Morchella sextelata Wipe the surface of the fruiting body with 75% alcohol for 10 seconds, then rinse three times with sterile water to remove residual disinfectant. (2) Use a sterile scalpel to cut a small piece of the stipe and inoculate it onto a PDA plate. Incubate at 22°C in the dark. Once the hyphae have grown, pick and purify the piece. (3) Inoculate the purified mycelium into PDA slant medium, and after it matures, store it in a 4℃ refrigerator for a short period of time for later use. (4) Cool the sterilized PDA medium to approximately 50°C and process it according to the following groups: Polysaccharide-antimicrobial peptide combination group 1: final concentration 0.5 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; Polysaccharide-antimicrobial peptide combination group 2: final concentration 1.0 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; Polysaccharide-antimicrobial peptide combination group 3: final concentration 2.0 mg / mL Armillaria mellea polysaccharide F3 + final concentration 40 µM antimicrobial peptide Thanatin; (5) After mixing thoroughly, pour into a sterile petri dish, let it solidify and set aside. At the same time, set up a control group that does not contain polysaccharides and antimicrobial peptides. (6) Use a sterile punch (5 mm in diameter) to take the bacterial cake from the edge of the activated colony, inoculate it into the center of the drug-containing PDA plate, cover the petri dish, and set up 3 biological replicates for each treatment; (7) Place all plates in a 25℃ constant temperature incubator for 7 days. After the culture is completed, observe the growth, take pictures, and calculate the relative mycelial diameter based on the control group.

[0047] from Figure 4 The results showed that, compared with the control group, the relative hyphal diameters of the polysaccharide-antimicrobial peptide combination groups 1, 2, and 3 all exhibited significant changes. This result indicates that the combined antimicrobial agent of polysaccharides and antimicrobial peptides used in this invention effectively inhibits pathogens (such as *Paecilomyces pubescens*) without negatively impacting the normal growth of *Morchella esculenta*, and can be used for the prevention and control of white mold disease in *Morchella esculenta*.

Claims

1. A combination preparation for the prevention and control of morel white mold disease, said combination preparation being composed of Armillaria mellea polysaccharide F3, antimicrobial peptide Thanatin, and sterile water; The CAS number for the antimicrobial peptide Thanatin is 214542-43-5; The Armillaria mellea polysaccharide F3 is an Armillaria mellea polysaccharide with a molecular weight of 30-50 kDa obtained by ultrafiltration membrane separation.

2. The combination formulation according to claim 1, characterized in that, The concentration of Armillaria mellea polysaccharide F3 is 0.5-2.0 mg / mL, and the concentration of the antimicrobial peptide Thanatin is 20-40 µM.

3. The combination formulation according to claim 2, characterized in that, The Armillaria mellea polysaccharide F3 was prepared by the following method: (1) The fruiting body powder of Armillaria mellea was defatted to obtain defatted fungal powder; (2) Mix defatted bacteria powder with distilled water at a mass-volume ratio of 1:25, extract at 40 kHz ultrasonic frequency and 60℃ for 30 minutes, and then continue to extract in a 90℃ water bath for 2 hours to obtain the extract; (3) After the extract is concentrated under reduced pressure, ethanol is slowly added for alcohol precipitation, and the precipitate is collected by centrifugation; (4) Dissolve the precipitate in distilled water and remove the protein using the Sevage method to obtain a crude polysaccharide solution; (5) After the crude polysaccharide solution is filtered through a microporous membrane, it is passed through a 100kDa and a 50kDa molecular weight cutoff filter membrane in sequence, and then the 50kDa filtrate is passed through a 30kDa molecular weight cutoff filter membrane and the retentate is collected. (6) The retentate was freeze-dried to obtain Armillaria mellea polysaccharide F3.

4. The combination formulation according to claim 3, characterized in that, The white mold disease mentioned is caused by Paecilomyces longifolia.

5. The use of the combination formulation according to any one of claims 1-4 in the preparation of a medicament for the prevention and treatment of morel white mold disease, characterized in that, The combined formulation is used to prevent and treat morel white mold disease caused by Paecilomyces longifolia.

6. An antibacterial agent for inhibiting the growth of *Paecilomyces pubescens*, characterized in that, The antibacterial agent consists of 0.5-2.0 mg / mL Armillaria mellea polysaccharide F3, 20-40 µM antimicrobial peptide Thanatin, and sterile water; The Armillaria mellea polysaccharide F3 was prepared according to the preparation method described in claim 3; The CAS number for the antimicrobial peptide Thanatin is 214542-43-5.

7. The use of a composition in the preparation of an antibacterial agent that inhibits the growth of Paecilomyces pubescens, characterized in that, The composition consists of 0.5-2.0 mg / mL Armillaria mellea polysaccharide F3 and 20-40 µM antimicrobial peptide Thanatin.

8. The application of Armillaria mellea polysaccharide in the preparation of Paecilomyces longifolia antibacterial agent, characterized in that, The Armillaria mellea polysaccharide is one of Armillaria mellea polysaccharide F2, Armillaria mellea polysaccharide F3 or Armillaria mellea polysaccharide F4; The *Armillaria mellea* polysaccharide F2, F3, and F4 were prepared by the following method: (1) The fruiting body powder of Armillaria mellea was defatted to obtain defatted fungal powder; (2) Mix defatted bacteria powder with distilled water at a mass-volume ratio of 1:25, extract at 40 kHz ultrasonic frequency and 60℃ for 30 minutes, and then continue to extract in a 90℃ water bath for 2 hours to obtain the extract; (3) After the extract is concentrated under reduced pressure, ethanol is slowly added for alcohol precipitation, and the precipitate is collected by centrifugation; (4) Dissolve the precipitate in distilled water and remove the protein using the Sevage method to obtain a crude polysaccharide solution; (5) After filtering the crude polysaccharide through a microporous membrane, it was passed through a 100kDa molecular weight cutoff filter membrane and a 50kDa molecular weight cutoff filter membrane in sequence. The 50kDa retentate was collected to obtain the Armillaria mellea polysaccharide F2 solution. (6) Pass the 50kDa filtrate through a 30kDa molecular weight cutoff filter membrane, collect the 30kDa cutoff solution, and obtain the Armillaria mellea polysaccharide F3 solution; (7) Pass the 30kDa filtrate through a 10kDa molecular weight cutoff filter membrane, collect the 10kDa cutoff solution, and obtain Armillaria mellea polysaccharide F4 solution; (8) Freeze-dry the Armillaria mellea polysaccharide F2 solution, Armillaria mellea polysaccharide F3 solution and Armillaria mellea polysaccharide F4 solution to obtain Armillaria mellea polysaccharide F2, Armillaria mellea polysaccharide F3 and Armillaria mellea polysaccharide F4.

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