Composition for preventing and treating bacterial diseases of edible mushrooms and preparation method thereof

Abstract

The invention relates to the technical field of prevention and treatment of bacterial diseases of crops, in particular to a composition for preventing and treating bacterial diseases of edible mushrooms, which is prepared from the following raw material components in percentage by mass: 25 to 35 percent of bacteriophage preparation, 20 to 25 percent of bacillus subtilis liquid, 10 to 15 percent of allicin, 10 to 15 percent of carvacrol extract, 8 to 10 percent of berberine and the balance of auxiliaries, the bacteriophage preparation and the bacillus subtilis liquid are used as main raw materials, and the bacteriophage preparation is prepared and amplified according to the types of bacterial diseases existing in an edible mushroom planting area, so that target pathogenic bacteria can be accurately and specifically split, and the prevention and treatment effect on the bacterial diseases of the edible mushrooms is ensured; the bacteriophage preparation and the bacillus subtilis liquid can secrete antibacterial peptides and competitively inhibit colonization of pathogenic bacteria, so that the prevention and treatment effect on bacterial diseases of the edible mushrooms is further improved, the bacteriophage preparation and the bacillus subtilis liquid can be degraded in the environment, drug residues are avoided, and the edible mushroom eating safety after prevention and treatment is guaranteed.

Description

Technical Field

[0001] This invention relates to the field of bacterial disease control technology for crops, specifically to a composition and preparation method for controlling bacterial diseases of edible fungi. Background Technology

[0002] Edible fungi are large fungi that can be consumed by humans. Specifically, edible fungi are mushrooms that can be eaten; mushrooms refer to a class of large fungi that can form large, fleshy fruiting bodies or sclerotia-like tissues that can be eaten or used medicinally. In biological classification, edible fungi belong to the kingdom Fungi, with the vast majority belonging to the subphylum Basidiomycota and a few to the subphylum Ascomycota. Their characteristic feature is the formation of fleshy or gelatinous fruiting bodies. They can be divided into wild and cultivated types. Bacterial diseases often occur during the cultivation of edible fungi.

[0003] The occurrence of diseases in edible fungi is a significant factor restricting the large-scale development of edible fungi cultivation. Bacterial diseases, in particular, not only severely reduce yields but also affect the quality of edible fungi products. During edible fungi cultivation, bacterial diseases occur frequently and are highly explosive, capable of spreading rapidly and causing severe economic losses. Bacterial diseases are referred to by farmers as the "cancer of edible fungi" and by those in the edible fungi industry as an international challenge.

[0004] To prevent damage to edible fungi caused by bacterial diseases, bacterial disease control compositions are often used. Currently, most bacterial diseases in edible fungi are controlled using chemical agents. While chemical agents offer advantages such as rapid effectiveness, they suffer from poor targeting and drug residues. Drug residues inside the fungi can harm consumers. Therefore, we propose a composition and preparation method for controlling bacterial diseases in edible fungi. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a composition and preparation method for preventing and controlling bacterial diseases of edible fungi, which can effectively solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a composition for preventing and controlling bacterial diseases of edible fungi, comprising the following raw material components in the following mass percentages: phage preparation 25%-35%, Bacillus subtilis liquid 20%-25%, allicin 10%-15%, carvacrol extract 10%-15%, berberine 8%-10%, and the remainder being adjuvants.

[0007] Preferably, the phage preparation method is as follows: a. Obtain host bacteria from environmental samples (sewage, soil, leachate from diseased mushroom tissue) or known bacteriophage reservoirs, and place the obtained host bacteria into a culture medium; b. Incubate under shaking at a suitable temperature until the logarithmic growth phase; c. Take sewage / soil suspension or diseased mushroom tissue homogenate, centrifuge to obtain supernatant, filter with a 0.22-0.3μm filter membrane to remove bacteria, obtain filtrate that may contain bacteriophages, and directly dilute known bacteriophages for use; d. Infection and culture were carried out using the double-layer plate method. After the phage plaques were generated, the plaques were removed and placed in liquid culture medium for amplification and harvesting. f. Choose the appropriate saving method according to your usage needs.

[0008] Preferably, the preparation method of the Bacillus subtilis liquid is as follows: Strain activation: Bacillus subtilis was inoculated into LB medium and cultured at 25℃-30℃ with shaking for 20-24 hours; Fermentation propagation: Transfer the inoculum to a fermenter at a rate of 3%-5% and culture with aeration for 48-72 hours; Centrifugal concentration: Centrifuge at 8000 rpm for 10-15 minutes, then resuspend the bacterial sludge in sterile water to 10¹. 0 CFU / mL.

[0009] A method for preparing the composition described above includes the following steps: S1. Preparation of the main raw materials of the composition Based on the varieties of edible fungi cultivated and the types of common bacterial diseases in the cultivation areas, bacteriophage preparations were prepared. S2, Amount of raw materials Weigh each raw material according to its percentage content by unit mass. S3, Preliminary mixing of raw materials The phage preparation, Bacillus subtilis liquid, allicin, carvacrol extract and berberine were added to the mixing container in sequence and mixed. S4. Addition of additives Add the dispersant and pH adjuster to deionized water and mix. Add the mixed liquid to a mixing container and bring the volume to 100%. Mix the liquid with the raw materials in the mixing container. S5, The final composition is formed. Based on the usage requirements of the composition, a suitable storage method is determined to obtain a composition for preventing and controlling bacterial diseases of edible fungi.

[0010] Preferably, in step S1, the prepared phage can be a single phage preparation or a mixture of multiple phages. After obtaining the single phage preparation or the mixture of multiple phages, it is stored at 3℃-5℃.

[0011] Preferably, in step S3, the stirring speed in the mixing container is 200-250 r / min, and the temperature of the mixing container is 5℃-10℃.

[0012] Preferably, in step S4, the pH value of the mixed solution is adjusted to 6-6.5 using a pH adjuster.

[0013] Preferably, in step S5, after the composition is prepared, if it is to be stored for a short period of time, it is to be stored in a low-temperature environment; if it is to be stored for a long period of time, it is to be stored in 50% glycerol.

[0014] The technical solution provided by this invention has the following advantages compared with the known prior art: 1. This invention employs a biological method to control bacterial diseases of edible fungi. The main components are bacteriophage preparations and Bacillus subtilis liquid. The bacteriophage preparations are prepared and amplified based on the types of bacterial diseases present in the edible fungi cultivation area, enabling precise and specific lysis of target pathogens and ensuring effective control of bacterial diseases in edible fungi. The Bacillus subtilis liquid secretes antimicrobial peptides, competitively inhibiting pathogen colonization, thereby further improving the control effect on bacterial diseases in edible fungi. Furthermore, both the bacteriophage preparations and the Bacillus subtilis liquid are biodegradable in the environment, leaving no drug residues and ensuring the safety of the treated edible fungi when consumed.

[0015] 2. This invention adds allicin, carvacrol extract, and berberine to a composition mainly composed of bacteriophage preparations and Bacillus subtilis liquid. Berberine can interfere with bacterial DNA replication and has broad-spectrum antibacterial effect, while allicin and carvacrol extract can play a good role in inhibition and killing. This can be used to prevent and control edible fungi during their growth process, avoiding their invasion by pathogenic diseases. Furthermore, the preparation process of this composition is simple, the production cost is low, and it has high value for promotion and application. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0017] Figure 1This is a schematic diagram of the process flow of the present invention; Figure 2 This is a schematic diagram comparing the effects of the biological method of this invention with the traditional chemical method. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0019] The present invention will be further described below with reference to embodiments. Example

[0020] Please refer to Figure 1-2 A composition for preventing bacterial diseases of edible fungi, comprising the following raw material components in the following mass percentages: 25% bacteriophage preparation, 20% Bacillus subtilis liquid, 10% allicin, 10% carvacrol extract, 8% berberine, and the remainder of adjuvants.

[0021] The preparation method of bacteriophage preparation is as follows: a. Obtain host bacteria from environmental samples (sewage, soil, leachate from diseased mushroom tissue) or known bacteriophage reservoirs, and place the obtained host bacteria into a culture medium; b. Incubate under shaking at a suitable temperature until the logarithmic growth phase; c. Take sewage / soil suspension or diseased mushroom tissue homogenate, centrifuge to obtain supernatant, filter with a 0.22μm filter membrane to remove bacteria, obtain filtrate that may contain bacteriophages, and directly dilute known bacteriophages for use; d. Infection and culture were carried out using the double-layer plate method. After the phage plaques were generated, the plaques were removed and placed in liquid culture medium for amplification and harvesting. f. Choose the appropriate saving method according to your usage needs.

[0022] The preparation method of Bacillus subtilis liquid is as follows: Strain activation: Bacillus subtilis was inoculated into LB medium and cultured at 25°C with shaking for 20 hours; Fermentation propagation: Transfer to a fermenter at a 3% inoculum and culture with aeration for 48 hours; Centrifugal concentration: Centrifuge at 8000 rpm for 10 min, then resuspend the bacterial sludge in sterile water to 10¹. 0 CFU / mL.

[0023] The main additives are dispersants, pH adjusters, deionized water, and glycerin.

[0024] A method for preparing the above-described composition includes the following steps: S1. Preparation of the main raw materials of the composition Based on the varieties of edible fungi cultivated and the types of common bacterial diseases in the cultivation areas, bacteriophage preparations were prepared. In step S1, the prepared phage can be a single phage preparation or a mixture of multiple phages. After obtaining the single phage preparation or the mixture of multiple phages, it is stored at 3°C.

[0025] S2, Amount of raw materials Weigh each raw material according to its percentage content by unit mass. S3, Preliminary mixing of raw materials The phage preparation, Bacillus subtilis liquid, allicin, carvacrol extract and berberine were added to the mixing container in sequence and mixed. In step S3, the stirring speed in the mixing container is 200 r / min, and the temperature of the mixing container is 5℃.

[0026] S4. Addition of additives Add the dispersant and pH adjuster to deionized water and mix. Add the mixed liquid to a mixing container and bring the volume to 100%. Mix the liquid with the raw materials in the mixing container. In step S4, the pH of the mixed solution is adjusted to 6 using a pH adjuster.

[0027] S5, The final composition is formed. Based on the usage requirements of the composition, a suitable storage method is determined to obtain a composition for preventing and controlling bacterial diseases of edible fungi. Example

[0028] Please refer to Figure 1-2 A composition for preventing bacterial diseases of edible fungi, comprising the following raw material components in the following mass percentages: phage preparation 30%, Bacillus subtilis liquid 23%, allicin 13%, carvacrol extract 13%, berberine 9%, and the remainder of adjuvants.

[0029] The preparation method of bacteriophage preparation is as follows: a. Obtain host bacteria from environmental samples (sewage, soil, leachate from diseased mushroom tissue) or known bacteriophage reservoirs, and place the obtained host bacteria into a culture medium; b. Incubate under shaking at a suitable temperature until the logarithmic growth phase; c. Take sewage / soil suspension or diseased mushroom tissue homogenate, centrifuge to obtain supernatant, filter with a 0.26μm filter membrane to remove bacteria, obtain filtrate that may contain bacteriophages, and directly dilute known bacteriophages for use; d. Infection and culture were carried out using the double-layer plate method. After the phage plaques were generated, the plaques were removed and placed in liquid culture medium for amplification and harvesting. f. Choose the appropriate saving method according to your usage needs.

[0030] The preparation method of Bacillus subtilis liquid is as follows: Strain activation: Bacillus subtilis was inoculated into LB medium and cultured at 27°C with shaking for 22 hours; Fermentation propagation: Transfer to a fermenter with an inoculum of 4% and culture with aeration for 60 hours; Centrifugal concentration: Centrifuge at 8000 rpm for 13 min, then resuspend the bacterial sludge in sterile water to a final concentration of 10¹. 0 CFU / mL.

[0031] The main additives are dispersants, pH adjusters, deionized water, and glycerin.

[0032] A method for preparing the above-described composition includes the following steps: S1. Preparation of the main raw materials of the composition Based on the varieties of edible fungi cultivated and the types of common bacterial diseases in the cultivation areas, bacteriophage preparations were prepared. In step S1, the prepared phage can be a single phage preparation or a mixture of multiple phages. After obtaining the single phage preparation or the mixture of multiple phages, it is stored at 4°C.

[0033] S2, Amount of raw materials Weigh each raw material according to its percentage content by unit mass. S3, Preliminary mixing of raw materials The phage preparation, Bacillus subtilis liquid, allicin, carvacrol extract and berberine were added to the mixing container in sequence and mixed. In step S3, the stirring speed in the mixing container is 225 r / min, and the temperature of the mixing container is 7.5℃.

[0034] S4. Addition of additives Add the dispersant and pH adjuster to deionized water and mix. Add the mixed liquid to a mixing container and bring the volume to 100%. Mix the liquid with the raw materials in the mixing container. In step S4, the pH of the mixed solution is adjusted to 6.25 using a pH adjuster.

[0035] S5, The final composition is formed. Based on the usage requirements of the composition, a suitable storage method is determined to obtain a composition for preventing and controlling bacterial diseases of edible fungi. Example

[0036] Please refer to Figure 1-2 A composition for preventing bacterial diseases of edible fungi, comprising the following raw material components in the following mass percentages: 35% bacteriophage preparation, 25% Bacillus subtilis liquid, 15% allicin, 15% carvacrol extract, 10% berberine, and the remainder of adjuvants.

[0037] The preparation method of bacteriophage preparation is as follows: a. Obtain host bacteria from environmental samples (sewage, soil, leachate from diseased mushroom tissue) or known bacteriophage reservoirs, and place the obtained host bacteria into a culture medium; b. Incubate under shaking at a suitable temperature until the logarithmic growth phase; c. Take sewage / soil suspension or diseased mushroom tissue homogenate, centrifuge to obtain supernatant, filter with a 0.3μm filter membrane to remove bacteria, obtain filtrate that may contain bacteriophages, and directly dilute known bacteriophages for use; d. Infection and culture were carried out using the double-layer plate method. After the phage plaques were generated, the plaques were removed and placed in liquid culture medium for amplification and harvesting. f. Choose the appropriate saving method according to your usage needs.

[0038] The preparation method of Bacillus subtilis liquid is as follows: Strain activation: Bacillus subtilis was inoculated into LB medium and cultured at 30°C with shaking for 24 hours; Fermentation propagation: Transfer 5% inoculum to a fermenter and culture with aeration for 72 hours; Centrifugal concentration: Centrifuge at 8000 rpm for 15 min, then resuspend the bacterial sludge in sterile water to a final concentration of 10¹. 0 CFU / mL.

[0039] The main additives are dispersants, pH adjusters, deionized water, and glycerin.

[0040] A method for preparing the above-described composition includes the following steps: S1. Preparation of the main raw materials of the composition Based on the varieties of edible fungi cultivated and the types of common bacterial diseases in the cultivation areas, bacteriophage preparations were prepared. In step S1, the prepared phage can be a single phage preparation or a mixture of multiple phages. After obtaining the single phage preparation or the mixture of multiple phages, it is stored at 5°C.

[0041] S2, Amount of raw materials Weigh each raw material according to its percentage content by unit mass. S3, Preliminary mixing of raw materials The phage preparation, Bacillus subtilis liquid, allicin, carvacrol extract and berberine were added to the mixing container in sequence and mixed. In step S3, the stirring speed in the mixing container is 250 r / min, and the temperature of the mixing container is 10℃.

[0042] S4. Addition of additives Add the dispersant and pH adjuster to deionized water and mix. Add the mixed liquid to a mixing container and bring the volume to 100%. Mix the liquid with the raw materials in the mixing container. In step S4, the pH of the mixed solution is adjusted to 6.5 using a pH adjuster.

[0043] S5, The final composition is formed. Based on the usage requirements of the composition, a suitable storage method is determined to obtain a composition for preventing and controlling bacterial diseases of edible fungi.

[0044] Application method: Prevention period: After each batch of mushrooms reaches the next flush, spray the mushroom bed and environment with a 1:50 solution of water once a week.

[0045] In the early stage of the disease: dilute with water at a ratio of 1:20 and spray the affected mushrooms and surrounding area, once every other day, for a total of 3 times.

[0046] Postharvest treatment: Immerse fruiting bodies for 10 seconds (1:100 solution) to extend shelf life.

[0047] This technical solution uses a biological method to protect edible fungi from bacterial diseases, which has the following advantages compared to chemical methods:

[0048] Table 1. Comparison of chemical and biological methods for the control of bacterial diseases in edible fungi Biological methods are used to control bacterial diseases of edible fungi. The main materials are bacteriophage preparations and Bacillus subtilis liquid. The bacteriophage preparations are prepared and amplified according to the types of bacterial diseases present in the edible fungi cultivation area, so as to accurately and specifically lyse the target pathogens and ensure the control effect on bacterial diseases of edible fungi. Bacillus subtilis liquid can secrete antimicrobial peptides, competitively inhibiting the colonization of pathogens, thereby further improving the control of bacterial diseases of edible fungi.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for preventing and controlling bacterial diseases of edible fungi, characterized in that, The raw material composition includes the following components by weight percentage: phage preparation 25%-35%, Bacillus subtilis liquid 20%-25%, allicin 10%-15%, carvacrol extract 10%-15%, berberine 8%-10%, and the remainder of adjuvants.

2. The composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, The method for preparing the bacteriophage preparation is as follows: a. Obtain host bacteria from environmental samples (sewage, soil, leachate from diseased mushroom tissue) or known bacteriophage reservoirs, and place the obtained host bacteria into a culture medium; b. Incubate under shaking at a suitable temperature until the logarithmic growth phase; c. Take sewage / soil suspension or diseased mushroom tissue homogenate, centrifuge to obtain supernatant, filter with a 0.22-0.3μm filter membrane to remove bacteria, obtain filtrate that may contain bacteriophages, and directly dilute known bacteriophages for use; d. Infection and culture were carried out using the double-layer plate method. After the phage plaques were generated, the plaques were removed and placed in liquid culture medium for amplification and harvesting. f. Choose the appropriate saving method according to your usage needs.

3. The composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, The preparation method of the Bacillus subtilis liquid is as follows: Strain activation: Bacillus subtilis was inoculated into LB medium and cultured at 25℃-30℃ with shaking for 20-24 hours; Fermentation propagation: Transfer the inoculum to a fermenter at a rate of 3%-5% and culture with aeration for 48-72 hours; Centrifugal concentration: Centrifuge at 8000 rpm for 10-15 minutes, then resuspend the bacterial sludge in sterile water to 10¹. 0 CFU / mL.

4. The composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, The additives mainly consist of dispersants, pH adjusters, deionized water, and glycerin.

5. A method for preparing the composition according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of the main raw materials of the composition Based on the varieties of edible fungi cultivated and the types of common bacterial diseases in the cultivation areas, bacteriophage preparations were prepared. S2, Amount of raw materials Weigh each raw material according to its percentage content by unit mass. S3, Preliminary mixing of raw materials The phage preparation, Bacillus subtilis liquid, allicin, carvacrol extract and berberine were added to the mixing container in sequence and mixed. S4. Addition of additives Add the dispersant and pH adjuster to deionized water and mix. Add the mixed liquid to a mixing container and bring the volume to 100%. Mix the liquid with the raw materials in the mixing container. S5, The final composition is formed. Based on the usage requirements of the composition, a suitable storage method is determined to obtain a composition for preventing and controlling bacterial diseases of edible fungi.

6. The method for preparing a composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, In step S1, the prepared bacteriophage can be a single bacteriophage preparation or a mixture of multiple bacteriophages. After obtaining the single bacteriophage preparation or the mixture of multiple bacteriophages, it is stored at 3℃-5℃.

7. The method for preparing a composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, In step S3, the stirring speed in the mixing container is 200-250 r / min, and the temperature of the mixing container is 5℃-10℃.

8. The method for preparing a composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, In step S4, the pH value of the mixed solution is adjusted to 6-6.5 using a pH adjuster.

9. A method for preparing a composition for preventing and controlling bacterial diseases of edible fungi according to claim 1, characterized in that, In step S5, after the composition is prepared, if it is to be stored for a short period of time, it is to be stored in a low-temperature environment; if it is to be stored for a long period of time, it is to be stored in 50% glycerol.

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