A fungicide with promoting effect on agaricus bisporus, and a preparation method and application thereof

Abstract

The application discloses a bacterial agent with a promoting effect on Agaricus bisporus, a preparation method and application thereof, wherein the bacterial agent is Bacillus velezensis ZJ-1 and / or Bacillus subtilis ZJ-5, the Bacillus velezensis ZJ-1 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M2026796; and the Bacillus subtilis ZJ-5 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:M2026717. The application can effectively promote the mycelium growth of Agaricus bisporus and significantly improve the yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a fungal agent that promotes the growth of button mushrooms, its preparation method, and its application. Background Technology

[0002] button mushroom ( Agaricus bisporus Agaricus bisporus (button mushroom) belongs to the order Agaricales, family Agaricales, and genus Agaricus. It is a saprophytic fungus that cannot produce fruiting without soil covering. Soil covering is a necessary condition for the transition of Agaricus bisporus from vegetative to reproductive growth. Studies have shown that the microbial community in the soil covering layer plays an important role in mycelial knotting and fruiting body development during the growth process of Agaricus bisporus.

[0003] Based on this, this invention analyzes the microbial community interactions in the casing soil of button mushrooms, and screens out the growth-promoting strains of button mushrooms, Bacillus vesicles ZJ-1 and Bacillus subtilis ZJ-5, based on experiments of button mushroom mycelium climbing the soil and fruiting in mushroom houses. On this basis, a green and safe special microbial agent for button mushroom casing soil is developed, providing theoretical support and practical guidance for the sustainable and stable development of button mushroom cultivation. Summary of the Invention

[0004] This invention provides a fungal agent that promotes the growth of button mushrooms, its preparation method, and its application. This fungal agent can effectively promote the mycelial growth of button mushrooms and significantly increase the yield of button mushrooms.

[0005] The technical solution adopted is as follows: This invention discloses a fungal agent that promotes the growth of Agaricus bisporus, comprising Bacillus velezensis ZJ-1 and / or Bacillus subtilis ZJ-5. Bacillus velezensis ZJ-1 was deposited at the China Center for Type Culture Collection (CCTCC) on April 23, 2026, with accession number CCTCC NO:M2026796; Bacillus subtilis ZJ-5 was deposited at the CCTC on April 17, 2026, with accession number CCTCC NO:M2026717.

[0006] Furthermore, it is a compound microbial agent, which includes Bacillus vesiculosus ZJ-1 and Bacillus subtilis ZJ-5.

[0007] Furthermore, the ratio of the effective viable counts of the Bacillus vesiculosus ZJ-1 and the Bacillus subtilis ZJ-5 is 1:2 to 2:1.

[0008] Furthermore, the compound microbial agent is a liquid microbial agent or a suspension.

[0009] Furthermore, the total number of viable bacteria in the liquid bacterial agent or suspension is ≥2.3 × 10⁻⁶. 8 CFU / mL.

[0010] A method for preparing the compound microbial agent of the present invention includes the following steps: S1. The purified ZJ-1 and ZJ-5 were inoculated into liquid fermentation medium and cultured with shaking at 25-30 ℃ and 50-200 r / min to obtain an effective viable count ≥1.5×10⁻⁶. 9 Single-cell fermentation broth at CFU / mL; S2. Mix the fermentation broths of the two strains of bacteria in a ratio of 1:2 to 2:1 to prepare a microbial suspension or liquid inoculum.

[0011] Furthermore, in S2, the fermentation broths of the two strains are mixed, and a dispersant and a stabilizer are added to prepare a stable microbial suspension or liquid inoculum.

[0012] The application of the fungal agent described in this invention in the soil-covered cultivation of button mushrooms.

[0013] Furthermore, during the mushroom casing period, the inoculant is diluted with water and then sprayed or mixed evenly into the casing layer.

[0014] Furthermore, after the button mushrooms are covered with soil, the inoculant is diluted with water 10-50 times and sprayed evenly onto the soil, and mushroom management is carried out according to conventional procedures.

[0015] The beneficial effects of this invention are as follows: This invention provides a strain of Bacillus belyssus ( Bacillus velezensis ZJ-1 and a strain of Bacillus subtilis ( Bacillus subtilis ZJ-5 is provided, along with a compound inoculant formulated from the two strains and its application in button mushroom cultivation. The two strains exhibit excellent coexistence and synergistic colonization capabilities in the casing layer, effectively shortening the cultivation cycle of button mushrooms and significantly increasing yield.

[0016] The strains were obtained through screening. Both Bacillus belye ZJ-1 and Bacillus subtilis ZJ-5 were isolated from the casing layer of high-yield, healthy button mushroom cultivation beds. Through mycelial climbing and fruiting experiments in mushroom houses, it was confirmed that they not only had no antagonistic effect on button mushroom mycelium, but also significantly promoted button mushroom mycelial growth and yield. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of strain ZJ-1.

[0018] Figure 2 This is a colony morphology diagram of strain ZJ-5.

[0019] Figure 3 This is an phylogenetic tree diagram of strain ZJ-1.

[0020] Figure 4 This is a phylogenetic tree diagram of strain ZJ-5.

[0021] Figure 5 A bar chart showing the relative abundance of genera of bacteria used as casing soil at different growth stages of Agaricus bisporus.

[0022] Figure 6 A bar chart showing the relative abundance of fungi in the soil covering mushrooms at different growth stages.

[0023] Figure 7 A network diagram of horizontal bacterial and fungal interactions in the soil covering of Agaricus bisporus at different growth stages.

[0024] Figure 8 Soil-climbing experiment to promote the growth of button mushroom mycelium by the tested bacteria. Figure 9 This is a diagram of a mushroom house fruiting experiment using the fermentation broth of the strain.

[0025] Figure 10 This is a diagram of a mushroom house fruiting experiment after applying inoculant. Detailed Implementation

[0026] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. Example 1

[0027] 1. Test materials Soil samples, button mushroom culture medium, and peat soil were all from Zhejiang Longchen Modern Agriculture Co., Ltd.

[0028] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15~20 g / L, pH 7.0.

[0029] King's B solid medium: tryptone 20 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 1.5 g / L, glycerol 10 ml / L, agar 15~20 g / L, pH 7.0.

[0030] 2. Methods 1) Soil sample collection Soil samples were randomly collected multiple times using the five-point method during the initial inoculation period, mycelial knotting period, fruiting body differentiation period, and fruiting period of Agaricus bisporus. Each group of samples was mixed and divided into four portions for later use.

[0031] 2) High-throughput Illumina sequencing analysis of soil microorganisms Soil 16S V4 and ITS 1-5F regions were amplified and sequenced at Beijing Novogene Technology Co., Ltd. The obtained sequencing fragments were denoised using the DADA2 module in QIIME2 software to obtain the final ASVs and feature tables. Each ASV was annotated using a pre-trained Naive Bayes classifier with the QIIME2 classify-sklearn algorithm, and the annotation database was Silva 138.1. Based on the ASV annotation results and the feature tables of each sample, species abundance tables at the kingdom, phylum, class, order, family, genus, and species levels were obtained.

[0032] 3) Analysis of soil microbial community interactions Python was used to screen the top 100 genera of bacteria and the top 50 genera of fungi in the microbial community, and Spearman correlation coefficients (P<0.01, |r|≥0.6) were used to calculate the interaction relationships between different microbial groups in the cover soil.

[0033] 4) Isolation of beneficial soil microorganisms Isolation of Bacillus: Accurately weigh 1 g of casing soil in the hyphal knotting stage, pour it into an Erlenmeyer flask containing 99 ml of sterile water, shake at 200 r / min for 10 min, and prepare 10 -2 Soil dilution solution; treated with an 80 ℃ constant temperature water bath for 10 min, followed by continuous dilution to prepare 10... -4 10 -5 10 -6 Soil suspensions of 10 concentrations; 50 µL of each 10 concentration was taken. -4 10 -5 10 -6 The soil suspension was spread on LB plates and incubated in a constant temperature incubator at 28 ℃ for 24 h.

[0034] Isolation of Pseudomonas: Accurately weigh 1 g of casing soil in the hyphal knotting stage, pour it into an Erlenmeyer flask containing 99 ml of sterile water, shake at 200 r / min for 10 min, and prepare 10 -2 Soil dilution solution; subsequently continuously diluted to prepare 10 -4 10 -5 10 -6 Soil suspensions of 10 concentrations; 40 µL of each suspension was taken. -4 10 -5 10 -6 The soil suspension was spread on King's B plates and incubated in a constant temperature incubator at 28 ℃ for 24 h.

[0035] 5) Screening of Agaricus bisporus growth-promoting strains Half a glass petri dish was filled with mushroom substrate, and the other half was filled with peat moss. The dish was then incubated at 25°C for 24 hours to enhance mycelial activity. The purified bacterial isolate was then inoculated into LB liquid medium and incubated at 28°C for 16 hours. 1 ml of bacterial culture was collected (CK was taken from LB liquid medium) and diluted with sterile water to 10 ml. This was then inoculated into the substrate layer of the glass petri dish and incubated at 25°C.

[0036] 6) Identification of growth-promoting strains Molecular biological identification: Bacterial DNA was rapidly extracted using a boiling method. 1 mL of bacterial culture in the logarithmic growth phase was collected, centrifuged at 12,000 × g for 1 min to collect the bacterial cells; the bacterial pellet was resuspended in 50 µL of sterile water, and then lysed by heating in a 100 ℃ metal bath for 10 min; after heating, the cells were rapidly cooled on ice for 5 min, centrifuged at 12,000 × g for 5 min, and the supernatant was collected as the DNA template for subsequent PCR.

[0037] PCR amplification was performed using universal primer sequences for bacterial 16S rDNA; The universal primer sequences for bacterial 16S rDNA are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4).

[0038] The PCR amplification products were sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with known sequences in the GenBank database using the BLAST program in NCBI, and a phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 12 software.

[0039] 7) Fruiting test of the strain in the mushroom house The fermentation broth of the strain was diluted 50 times and sprayed into the casing soil of button mushrooms in the mycelial climbing stage. The mushroom production management was carried out according to the conventional process of mushroom house. The experimental site was located at Zhejiang Longchen Modern Agriculture Co., Ltd.

[0040] 3. Results (1) Analysis of soil microbial diversity at different growth stages of Agaricus bisporus See Figure 5 and Figure 6 As shown, the microbial community composition characteristics of Agaricus bisporus at different growth stages were analyzed. The results showed that the community structure of Agaricus bisporus differed significantly at different growth stages.

[0041] The abundance of *Agaricus* species significantly increased during the mycelial climbing stage of *Agaricus bisporus*, consistent with the rapid colonization of mycelia in the casing soil during this period. Simultaneously, the abundance of *Pseudomonas* species significantly increased during the mycelial knotting stage of *Agaricus bisporus*, indicating a close relationship between *Pseudomonas* and mycelial knotting, and a positive correlation with *Agaricus* species. After harvesting *Agaricus bisporus*, the abundance of *Trichoderma* species in the casing soil surged.

[0042] (2) Analysis of the interaction between soil-covering microorganisms in different growth stages of Agaricus bisporus See Figure 7 As shown, the microbial symbiotic network structure of *Agaricus bisporus* at different growth stages was constructed by selecting the top 100 bacterial genera and the top 50 fungal genera in abundance. The results showed that *Agaricus* (the genus to which *Agaricus bisporus* belongs) had direct interactions with 2 bacterial genera and 5 fungal genera, with a positive correlation to *Bacillus* and *Pseudomonas* genera, with correlation coefficients of 0.511762 and 0.534068, respectively. This indicates that *Bacillus* and *Pseudomonas* bacteria may have the potential to promote the growth of *Agaricus bisporus*, and should be a key focus for subsequent strain screening.

[0043] (3) Screening of beneficial microorganisms in the casing layer of Agaricus bisporus Twenty-four bacterial isolates were obtained from the casing soil samples using a gradient dilution method. These isolates were numbered ZJ1-24 and then transferred to LB medium for streak purification. The isolates were then stored at 4°C.

[0044] (4) Soil climbing test of Agaricus bisporus mycelium The aforementioned potential growth-promoting bacteria were added to the soil, and the mycelial growth of *Agaricus bisporus* in the control group and the treatment group were compared. The results are shown in [link to results]. Figure 8 As shown: On day 7 post-inoculation, the mycelium in the control group (CK) was only in the climbing stage at the bottom of the casing layer. In contrast, the treatment groups supplemented with strains ZJ-1 and ZJ-5 showed a significant first-mover advantage in mycelial growth, with the mycelial tips having climbed vertically to more than 50% of the casing layer height, and the mycelial bundles were thicker and more densely branched than the control group. The treatment group supplemented with ZJ-12 was next, while the growth-promoting effect of the treatment group supplemented with ZJ-18 was not significant in the early stages.

[0045] On day 14 after inoculation, the differences between the treatment groups widened further. The hyphae in all four treatment groups had almost completely penetrated the entire casing layer longitudinally, forming dense and uniform hyphae that had begun to twist and knot. The colonization density was significantly higher than that of the CK group. Although the hyphae in the CK group had climbed to the top of the casing layer, they were sparse and grew slowly.

[0046] Table 1. Mycelial climbing rate in different treatment groups Control 15% 70% 1.50 / ZJ-1 35% 100% 2.14 +42.86% ZJ-5 45% 100% 2.14 +42.86% ZJ-12 40% 100% 2.14 +42.86% ZJ-18 30% 80% 1.71 +14.29% (5) Identification of strains that promote the growth of Agaricus bisporus mycelium Based on morphological and molecular biological identification, the growth-promoting bacteria isolated from the casing soil of *Agaricus bisporus* were identified as ZJ-1, ZJ-5, ZJ-12, and ZJ-18, which are *Bacillus belyssus* (…). Bacillus velezensis Bacillus subtilis ( Bacillus subtilis ), Bacillus megaterium ( Bacillus megaterium Now named Priestia megaterium ZJ-18 is *Pseudomonas fluorescens* (…), while ZJ-18 is *Pseudomonas fluorescens* (…). Pseudomonas fluorescens ).

[0047] Among them, Bacillus belesi ( Bacillus velezensis ZJ-1 (see also) Figure 1 and Figure 3 As shown, Bacillus subtilis ( Bacillus subtilis ZJ-5 (see reference) Figure 2 and Figure 4 As shown. The 16S rDNA sequence of Bacillus belyss ZJ-1 is shown in SEQ ID NO:1; the 16S rDNA sequence of Bacillus subtilis ZJ-5 is shown in SEQ ID NO:2.

[0048] (6) Mushroom house test of growth-promoting strains Four bacteria that significantly promoted the mycelial growth of Agaricus bisporus were prepared into 10... 9 The bacterial fermentation broth was prepared at CFU / ml, while the control group was treated with LB medium. See [link to fruiting data] for details. Figure 9 As shown in Table 2, the results revealed that Bacillus vesiculosus ZJ-1 and Bacillus subtilis ZJ-5 significantly increased the yield of Agaricus bisporus, with an average increase of 13.1% and 13.94% in total mushroom buds, respectively, and an average increase of 14.03% and 15.57% in total yield compared to the control group.

[0049] The strains were preserved as bacterial cultures. Bacillus belye ZJ-1 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2026796; Bacillus subtilis ZJ-5 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2026717.

[0050] Table 2. Effects of bacterial fermentation broth on yield increase of button mushrooms. Example 2

[0051] 1. Test materials Dispersants: sodium lignosulfonate, Tween-80, sodium methylene bisnaphthalene sulfonate; Thickeners: xanthan gum, magnesium aluminum silicate, fumed silica; Preservatives: Kathon, sodium benzoate, potassium sorbate.

[0052] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0053] 2. Methods

[0054] 1) Biocompatibility test of adjuvants: Nine test materials were added to LB liquid medium and inoculated with strains ZJ-1 and ZJ-5 respectively. After incubation at 28℃ and 200 rpm for 24 h, OD was measured. 600 .

[0055] 2) Orthogonal test of additive dosage: Design an orthogonal test table for additive dosage with dispersant at 1%, 2%, 3%, thickener at 0.1%, 0.2%, 0.3%, and preservative at 0.1%, 0.2%, 0.3%, and the measured indexes are suspension rate, dispersibility and cell count.

[0056] 3) Single-strain expansion culture: The strains ZJ-1 and ZJ-5 determined in Example 1 were inoculated into LB liquid medium and cultured at 28°C and 200 r / min for 24 h, with bacterial concentrations of 1.5 × 10⁻⁶ and 1.5 × 10⁻⁶, respectively. 9 CFU / mL, 1.7×10 9 CFU / mL 4) Mixed bacterial solution preparation: After centrifuging the bacterial solutions of strains ZJ-1 and ZJ-5, resuspend them and mix them at a volume ratio of 1:1. Mix at 26℃ and 90 r / min for 30 min to obtain the mixed bacterial solution.

[0057] 5) Additives: Based on the mixed bacterial solution obtained after compounding, Tween-80, xanthan gum, and potassium sorbate are added in a certain proportion and mixed evenly to obtain a compound microbial agent.

[0058] Mushroom house experiment with compound microbial inoculants After the button mushroom substrate is placed on the bed, the compound microbial agent is diluted 50 times and sprayed or mixed into the casing soil. The mushroom production is managed according to the conventional mushroom house process, and the number of mushroom buds and yield are statistically analyzed compared with conventionally cultivated button mushrooms.

[0059] 3. Results

[0060] Based on the results of the biocompatibility test of the adjuvants, the strains showed better growth and higher cell counts after the addition of Tween-80, xanthan gum, and potassium sorbate, respectively. Therefore, these three materials were selected as adjuvants for the preparation of the bacterial agent.

[0061] The orthogonal experiment on the dosage of adjuvants showed that the suspension bacterial agent prepared with the addition of 3% Tween-80, 0.2% xanthan gum, and 0.1% potassium sorbate (experiment number 9) had the best overall performance and the highest bacterial count (2.3 × 10⁻⁶). 8 (CFU / ml), meeting the standards for agricultural microbial liquid inoculants, and used for subsequent mushroom house fruiting trials.

[0062] Table 3 Orthogonal Experiment Table for Auxiliary Agent Dosage 1 75.5% good <![CDATA[1.5×10 8 ]]> 2 85.2% good <![CDATA[1.0×10 8 ]]> 3 88.6% good <![CDATA[1.3×10 8 ]]> 4 82.3% excellent <![CDATA[1.4×10 8 ]]> 5 94.5% excellent <![CDATA[9.0×10 7 ]]> 6 91.2% excellent <![CDATA[1.4×10 8 ]]> 7 78.4% good <![CDATA[1.8×10 8 ]]> 8 86.8% excellent <![CDATA[2.0×10 8 ]]> 9 89.1% good <![CDATA[2.3×10 8 ]]>

[0063] See the mushroom production results after inoculant application. Figure 10 As shown in Table 4, the results indicate that the yield of mushrooms per square meter was significantly increased by 8.40% after the application of compound microbial inoculant compared with conventional mushroom house cultivation, indicating that compound microbial inoculant has the effect of increasing the yield of button mushrooms.

[0064] Table 4. Effects of compound microbial inoculants on yield increase of Agaricus bisporus.

[0065] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fungal agent that promotes the growth of Agaricus bisporus, characterized in that, They are Bacillus belyssus ZJ-1 and / or Bacillus subtilis ZJ-5, with Bacillus belyssus ZJ-1 deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO:M2026796; and Bacillus subtilis ZJ-5 deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO:M2026717.

2. The microbial agent according to claim 1, characterized in that, It is a compound microbial agent, which includes Bacillus berberis ZJ-1 and Bacillus subtilis ZJ-5.

3. The microbial agent according to claim 2, characterized in that, The ratio of the effective viable counts of *Bacillus belyssus* ZJ-1 to *Bacillus subtilis* ZJ-5 is 1:2 to 2:

1.

4. The microbial agent according to claim 2, characterized in that, The compound microbial agent is a liquid microbial agent or a suspension.

5. The microbial agent according to claim 4, characterized in that, The total number of viable bacteria in the liquid bacterial agent or suspension is ≥2.3×10⁻⁶. 8 CFU / mL.

6. A method for preparing the microbial agent according to claim 5, characterized in that, Includes the following steps: S1. The purified ZJ-1 and ZJ-5 were inoculated into liquid fermentation medium and cultured with shaking at 25-30 ℃ and 50-200 r / min to obtain an effective viable count ≥1.5×10⁻⁶. 9 Single-cell fermentation broth at CFU / mL; S2. Mix the fermentation broth of the two strains of bacteria in a ratio of 1:2 to 2:1 to prepare a microbial suspension or liquid inoculum.

7. The method for preparing the compound microbial agent according to claim 6, characterized in that, In S2, the fermentation broths of the two strains are mixed, and dispersants and stabilizers are added to prepare a stable microbial suspension or liquid inoculum.

8. The application of the inoculant according to any one of claims 1-5 in the cultivation of button mushrooms.

9. The application according to claim 8, characterized in that, During the casing period of button mushrooms, the fungal agent is diluted with water and then sprayed or mixed evenly into the casing layer.

10. The application according to claim 9, characterized in that, After the button mushrooms are covered with soil, the inoculant is diluted with water 10-50 times and sprayed evenly onto the soil, and mushroom management is carried out according to conventional procedures.

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