Agaricus bisporus endophyte prevotella veridium with antibacterial effect and application thereof

Abstract

This invention discloses a type of endophytic fungus, *Priscilla megaterium*, with antibacterial activity, and its applications. The *Priscilla megaterium* is named... Priestia megaterium The strain, g-2-2, has the accession number CGMCC No. 37094. This invention screened and isolated a strain of *Priscilla gigantea* with antibacterial activity, g-2-2. Identification using morphology and 16S rRNA biotags confirmed g-2-2 as *Priscilla gigantea*. This strain exhibits significant inhibitory activity against harmful *Verticillium* species and can be used as a biocontrol agent.

Description

Technical Field

[0001] This invention relates to the field of mushroom pathogen control technology, and in particular to a fungus with antibacterial properties, *Priestella megaterium*, an endophytic fungus of *Agaricus bisporus*, and its application. Background Technology

[0002] Edible fungi, as an important nutritious food, are widely loved and cultivated worldwide due to their delicious taste and rich nutritional value. However, edible fungi are highly susceptible to infection by pathogens (such as fungi and bacteria) during cultivation, which severely affects their quality. Button mushrooms (…) Agaricus bisporus It is one of the most produced and widely cultivated edible fungi in China. The most serious disease it causes is wet bubble disease, with the most severe pathogen being the harmful *Verticillium*. Hypomyces perniciosus The pathogenic fungus used in this experiment was isolated from the growth stage of *Agaricus bisporus* by the Institute of Biology, College of Agriculture and Biotechnology, Zhejiang University. Molecular identification confirmed it to be the harmful *Verticillium*. Hypomyces perniciosus During the fruiting stage of button mushrooms, *Verticillium* primarily affects the metabolic levels of plant hormones (isopentenyl adenosine, cis-zeatin, and isopentenyl adenine), causing deformed growth and brown lesions, severely impacting the quality and growth of the mushrooms. *Verticillium*, a significant pathogen affecting button mushrooms, can simultaneously harm multiple edible fungi. Infection of button mushroom fruiting bodies with *Verticillium* causes diseases, resulting in losses exceeding 50% and even total crop failure in severe cases. Currently, there are no effective control methods or completely resistant varieties for this disease. In the production process, this pathogen severely damages the fruiting of edible fungi, leading to significant economic losses.

[0003] Although factory cultivation has improved the cultivation management and production efficiency of button mushrooms, significantly reducing their disease incidence, verrucosum disease still occurs frequently. Currently, the main control methods are chemical and biological control.

[0004] Chemical control primarily involves applying pesticides to inhibit the growth of pathogens. Studies have investigated the inhibitory effects of four chemical pesticides at different concentrations on *Verticillium* and their toxicity to several edible fungi. Experiments showed that 0.01 g / L chlorothalonil, 0.005 g / L, and 0.01 g / L sclerotinib exhibited the best inhibitory effects on *Verticillium*. Carbendazim showed poor inhibitory effects on *Verticillium* at various experimental concentrations. Thiophanate-methyl showed good inhibitory effects on *Verticillium* after 48 hours. Chlorothalonil, thiophanate-methyl, and sclerotinib completely inhibited the growth of *Agaricus bisporus*. Among these, carbendazim's inhibitory effects on both *Verticillium* and *Agaricus bisporus* were not particularly significant.

[0005] Biological control of these diseases has raised the bar for people, with a focus on screening beneficial biocontrol bacteria. For example, Wu Xiaoping (2009) screened Bacillus subtilis BS-2 as a counteracting agent of *Neurospora hygroscopica*. Neurospora sitophila It has a strong inhibitory effect; Hao Jie et al. (2011) screened out Bacillus cereus ( Bacillus vallismorti Both the bacterial cells and the fermentation supernatant showed significant antagonistic effects against *Aristolochia debilis*; Huang Fuchang et al. (2013) isolated a strain of *Bacillus amyloliquefaciens* from waste culture medium of *Pleurotus ostreatus*. Bacillus amyloliquefaciens ) for contamination by the fungus Neurospora ( Neurospora sp. Trichoderma () Trichoderma sp. ) and Ghost Umbrella ( Coprinus sp. Both showed significant antibacterial effects when cultured separately in confrontation.

[0006] Traditional pesticide control often pollutes the environment, leaving pesticide residues in button mushrooms and posing a threat to human health. Screening for beneficial bacteria for biological control is an important method to improve the safe production of button mushrooms.

[0007] Bacteria are incredibly diverse. With the development of modern molecular biology, the primary molecular identification of bacteria relies on a type of RNA found in the small subunits of ribosomes in prokaryotes. Bacterial ribosomal RNA is classified into three types based on its sedimentation coefficient: 5S, 16S, and 23S rRNA. 16S rDNA is the DNA sequence on the bacterial chromosome that encodes the rRNA and is present in the genomes of all bacteria. Because of its moderate size (approximately 1.5 kb), 16S rDNA effectively reflects the differences between different bacterial genera and its sequence is relatively easy to obtain using sequencing technology. Therefore, it is accepted by bacteriologists and taxonomists as an important basis for bacterial molecular identification. Summary of the Invention

[0008] This invention isolated a fungus of the genus *Priestella*, which has been shown to have a strong inhibitory effect on harmful *Verticillium*. Identification using DNA biotags confirmed that this fungus is *Priestella megaterium*.

[0009] A type of giant Priestella with antibacterial properties was named Priestia megatherium The strain, g-2-2, has the accession number CGMCC No. 37094. This invention relates to *Priscilla megaterium* strain g-2-2, which exhibits good antibacterial activity among four *Priscilla megaterium* strains (g-2-2, g-2-4, g-5-1, and g-5-3) isolated from *Agaricus bisporus* infected during *Agaricus bisporus* cultivation by the Institute of Biology, College of Agriculture and Biotechnology, Zhejiang University. Sequence alignment revealed that strain g-2-2 is similar to... Priestia megatherium The strain is closely related to the others. The newly screened g-2-2 strain was named... Priestess megalithThe strain number g-2-2 was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) located at the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37094.

[0010] On the other hand, the present invention also provides the application of the aforementioned Priestella megaterium in inhibiting the growth of pathogenic bacteria.

[0011] Preferably, the pathogen is a harmful verrucous fungus (Varicospora rubrum). Hypomyces perniciosus ), which is the pathogen of Agaricus bisporus.

[0012] On the other hand, the present invention also provides the application of the aforementioned Priestella giantiformis in the preparation of biocontrol agents for preventing and controlling pathogenic infections in edible fungi.

[0013] Preferably, the edible fungus is Agaricus bisporus (button mushroom). Agaricus bisporus ); The pathogen is harmful verrucous fungus ( ). Hypomyces perniciosus ).

[0014] On the other hand, the present invention also provides a biocontrol agent for preventing infection by pathogenic fungi in edible fungi, the biocontrol agent comprising an effective amount of the aforementioned Priestella giantiflora.

[0015] On the other hand, the present invention also provides a method for preventing infection by pathogenic fungi in edible fungi, by applying the aforementioned Priestella giantiflora or the aforementioned biocontrol agent to the casing soil of the edible fungi.

[0016] Preferably, the edible fungus is Agaricus bisporus (button mushroom). Agaricus bisporus ); The pathogen is harmful verrucous fungus ( ). Hypomyces perniciosus ).

[0017] Specific prevention and control methods include spraying a pesticide containing the Priestella megaterium strain g-2-2 onto the soil covering the mushroom bed of the edible mushroom, Agaricus bisporus.

[0018] This invention screened and isolated a strain of *Priscilla gigantea* g-2-2 with antibacterial activity. Through morphological and DNA biotag identification, it was confirmed that *Priscilla gigantea* g-2-2 is a species of the genus *Priscilla*. The strain g-2-2 has a significant inhibitory effect on harmful *Verticillium* and can be used as a biocontrol agent. Attached Figure Description

[0019] Figure 1 This is a graph showing the colony morphology of strain g-2-2 on LB medium.

[0020] Figure 2This is a Gram staining result of strain g-2-2. The bacterial cells are rod-shaped and Gram-positive (purple). Scale bar = 10 μm.

[0021] Figure 3 Phylogenetic tree constructed using 16S rRNA fragments (numerical values ​​represent boost values).

[0022] Figure 4 This is a set of images showing the results of the inhibition rate of strain g-2-2 against the pathogenic fungus *Verticillium* and its diameter. A shows the culture image of strain g-2-2 against *Verticillium*; B shows the colony diameter; and C shows the inhibition rate of strain g-2-2 against *Verticillium*.

[0023] Figure 5 The inhibition rate of fermentation filtrate of each strain on the germination of harmful wart spores is shown. Detailed Implementation

[0024] Example 1: Isolation of bacterial strains On September 10, 2025, Agaricus bisporus cultured at the Institute of Biology, College of Agriculture and Biotechnology, Zhejiang University, was used as the isolation sample. The selective medium dilution plate method was used for isolation, and the process is as follows: Select healthy, fruiting button mushrooms, slowly wash away surface dirt with distilled water, allow to drain naturally, and then chop them into small pieces. Separate the mushrooms into two parts: the stem and the cap, taking 1g of each. In a clean bench, disinfect with 75% alcohol for 30 seconds, rinse five times with sterile water, and blot dry with sterile filter paper. Chop the thoroughly disinfected mushroom parts, add an appropriate amount of quartz sand and sterile water to a 1.5ml sterile centrifuge tube, and centrifuge at 10000rpm for 10 minutes. Then, thoroughly mix by shaking at 25℃ and 250rpm for 20-30 minutes. Let stand for 2 minutes after removing from the centrifuge. Add 1 mL of supernatant to 9 mL of sterile water and shake to fully disperse the material, thus preparing a 10-fold dilution. Take 1 mL of this 10-fold dilution and add it to a centrifuge tube containing 9 mL of sterile water, mixing thoroughly. Then take another 1 mL of this dilution and add it to a centrifuge tube containing 9 mL of sterile water, mixing thoroughly to prepare a 1000-fold dilution. Pour 15 mL of the melted LB medium into petri dishes. Spread 50 μL of the filtrate evenly onto LB agar plates containing actinomycete ketone using a sterile spreader. Incubate in the dark at 37°C for 3-4 days. Repeat each concentration three times. After 3 days of incubation at 37°C, observe the colonies. Pick colonies and transfer them to new LB agar plates. Once single colonies have grown, transfer them back to LB agar plates. Four strains were isolated and named g-2-2, g-2-4, g-5-1, and g-5-3.

[0025] The LB medium consisted of: 5g yeast extract, 10g soybean tryptone, 10g sodium chloride (NaCl), pH 7.5, 15g agar, and 1000mL distilled water.

[0026] LB liquid medium: 5g yeast extract; 10g soybean tryptone; 10g sodium chloride (NaCl); pH=7.5, 1000mL distilled water.

[0027] Example 2: Description of Colony Morphology The LB medium was prepared using the formulation in Example 1.

[0028] The strains g-2-2, g-2-4, g-5-1, and g-5-3 isolated in Example 1 were streaked onto 9cm LB plates, repeated three times, and incubated at 37℃ for 2-3 days. Colony morphology was observed and recorded.

[0029] Colony morphology observation results are as follows Figure 1 As shown: Colony g-2-2 was cultured on LB at 37°C for 2 days. Single colonies were relatively obvious, with a white-yellow color, irregular edges, rough surface, and easy removal. The growth rate was relatively fast.

[0030] Colony g-2-4 was cultured on LB at 37°C for 2 days. Single colonies were relatively clear, with a white-yellow color, irregular edges, smooth surface, and easy removal. The growth rate was relatively fast.

[0031] Colony g-5-1 was cultured on LB at 37°C for 2 days. Single colonies were relatively clear, with a white-yellow color, irregular edges, rough surface, and easy removal. The growth rate was relatively fast.

[0032] Colony g-5-3 was cultured on LB at 37°C for 2 days. Single colonies were relatively obvious, with a white-yellow color, irregular edges, smooth surface, and easy removal. The growth rate was relatively fast.

[0033] The colony morphology of g-2-2, g-2-4, g-5-1, and g-5-3 are similar to Priestia megatherium Very similar in appearance.

[0034] Example 3: Gram staining examination of the isolated strains Gram staining solutions include crystal violet stain (crystal violet 2.0g; ammonium oxalate 0.8g; 95% ethanol 20ml; distilled water 80ml), iodine solution (iodine 1.0g; potassium iodide 2.0g; distilled water 300ml), decolorizing solution (95% ethanol), and counterstain (safranin 0.25g; 95% ethanol 10ml; distilled water 90ml).

[0035] Staining process: 1. Pick a bacterial sample from colonies grown on LB plates for 2 days, place it in an EP tube or small beaker, add an appropriate amount of water, and stir to disperse the bacterial sample as much as possible.

[0036] 2. Smear: Take a small drop of the bacterial culture sample prepared above and drop it onto a glass slide. Use a heated inoculation needle to spread the sample into a thin layer and let it air dry naturally, or heat it over a flame to speed up the drying process.

[0037] 3. Fix Quickly pass the glass slide back and forth over the flame once or twice, ensuring the heated side of the slide touches the skin on the back of your hand without feeling too hot. After cooling, pass the slide back and forth over the flame once or twice more, and then cool it again. Repeat this process until the thin film is dry.

[0038] 4. Staining Place a drop of ammonium oxalate crystal violet on the prepared slide, stain for 1 minute, and then wash with water, being careful not to direct the water stream directly onto the thin film.

[0039] 5. Mordling Add about one drop of iodine solution, let it act for 1 minute, then rinse with water. Be careful not to rinse the thin film directly with running water; blot dry with filter paper.

[0040] 6. Decolorization Destaining with 95% ethanol generally takes about 30 seconds. This step is crucial for Gram staining and must be strictly controlled. If the destaining time is too long, Gram-positive bacteria may be mistaken for Gram-negative bacteria; if the destaining time is insufficient, they may be mistaken for Gram-positive bacteria.

[0041] 7. Absorb dry After rinsing with water, blot dry with filter paper.

[0042] 8. Re-dyeing Add one drop of 0.25% safranin staining solution, stain for 10-30 seconds, and then rinse with tap water.

[0043] 9. Observation After drying, observe using an oil immersion microscope and record the results.

[0044] Results: Gram staining revealed that all four isolated bacterial strains were purple and rod-shaped, indicating they were all Gram-positive bacteria (see [link to Gram staining test]). Figure 2 ),and Priestess It matches the morphological characteristics of bacteria.

[0045] Example 4 Molecular identification of the isolated strains (1) PCR amplification of bacterial ribosomal 16S rRNA gene 16S rRNA primers: The upstream primer fD2 sequence is: 5'-AGAGTTTGATCCTGGCTCAG-3'. The downstream primer rP1 sequence is: 5'-ACGGTTACCTTGTTACGACTT-3'; PCR amplification was performed in a 50 μL reaction system containing: 2 μM each of forward and reverse primers, 200 μM of dNTPs, 1.5 mM of MgCl2, 5 μL of 10×PCR buffer, 2 μL of template DNA (i.e., LB liquid culture), and 2 U of Taq enzyme.

[0046] PCR amplification was performed on a Langqi MG96G PCR instrument. Reaction conditions: 94℃ pre-denaturation for 2 min, followed by 35 cycles of: 94℃ denaturation for 30 s, 55℃ annealing for 40 s, and 72℃ extension for 1 min. A final extension at 72℃ for 10 min was also performed.

[0047] (2) Submission of PCR products for testing The purified and recovered target 16S rRNA fragments, after electrophoresis detection, were sent to Shanghai Sangon Biotech for sequencing using an ABIPRIS MA377 automated sequencer. After rigorous verification, the sequencing results yielded 16S rRNA fragment sequences of g-2-2, g-2-4, g-5-1, and g-5-3, as shown in SEQ ID NO. 1~4.

[0048] On the NCBI website, the nucleotide sequences of the 16S rRNA of strains g-2-2, g-2-4, g-5-1, and g-5-3 were obtained and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. The comparison results showed that... The g-2-2 sequence and the accession number OQ825040.1 Priestia megatherium The similarity is 100%, and the accession number is OR647587.1. Priestia megatherium The similarity is 99.93%, and the accession number is MK16863.1. Bacillus aryabhattai The similarity was 99.93%, and the accession number was OR398825.1. Bacillus zanthoxyli The similarity is 99.93%, and the accession number is PQ782484.1. Priestia megatherium The similarity reached 99.93%.

[0049] The g-2-4 sequence and the accession number OQ825040.1 Priestia megatherium The similarity is 100%, and the login ID is PV688267.1. Priestia megatherium The similarity is 99.93%, and the accession number is MK277457.1. Bacillus aryabhattaiThe similarity is 99.93%, and the accession number is OQ786941.1. Bacillus zanthoxyli The similarity is 99.93%, and the accession number is PP111682.1. Priestia megatherium The similarity reached 99.86%.

[0050] The g-5-1 sequence and the accession number MT026569.1 Priestia megatherium The similarity is 100%, and the accession number is MK307785.1. Priestia megatherium The similarity is 100%, and the accession number is KC692200.1. Bacillus megalith The similarity is 100%, and the accession number is MK389288.1. Bacillus zanthoxyli The similarity is 100%, and the accession number is JQ229806.1. Bacillus megaterium The similarity reached 100%.

[0051] The g-5-3 sequence and the accession number MT184834.1 Priestia megatherium The similarity is 100%, and the accession number is OR561998.1. Priestia megatherium With a similarity of 99.3%, accession number CP120609.2 Priestess megalith The similarity is 99.3%, and the accession number is MH889137.1. Priestia megatherium With a similarity of 99.3%, accession number CP109761.1 Priestia megatherium The similarity reached 99.3%.

[0052] The obtained sequences were compared with nucleotide sequences in the GenBank database using BLASTN, and similar sequences were downloaded from the GenBank database (see [link to GenBank database]). Figure 3 The phylogenetic tree was constructed using the nearest neighbor (NJ) method in MEGA.11 software. The stability of the phylogenetic tree branches was evaluated through 1000 bootstrap iterations. Bacillus subtilis As an outgroup, a phylogenetic tree was constructed, and the phylogenetic tree analysis results are as follows: Figure 3 This indicates that the strain is related to Priestia megatherium They are closely related. The newly screened g-2-2, which showed good antibacterial effect, was named... Priestia megatherium The strain number g-2-2 was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) located at the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37094.

[0053] Example 5: Inhibitory effects of g-2-2, g-2-4, g-5-1, and g-5-3 on mushroom pathogens by confrontation culture. The plate confrontation culture method was used. 15 mL of PDA medium was added to a 9 cm diameter Petri dish. After cooling, g-2-2, g-2-4, g-5-1, g-5-3, and a mushroom pathogen were inoculated into the dish, with the two inoculums spaced 4 cm apart. The dishes were then incubated at 25°C for one week to examine the inhibition of pathogens. The pathogen tested was *Verticillium* (a harmful fungus). Hypomyces perniciosus ).

[0054] The results are as follows Figure 4 As shown: Tests revealed that strain g-2-2 exhibited a more significant inhibitory effect on harmful verrucous fungi.

[0055] Example 6: The four tested strains inhibited the germination of conidia of the mushroom pathogen *Verticillium*. Experimental methods: Four tested bacterial strains (g-2-2, g-2-4, g-5-1, and g-5-3) were streaked onto LB agar plates and incubated at 37°C for 24 hours to form single colonies. Single colonies of each strain were picked and inoculated into 10 mL of LB liquid medium and incubated for 24 hours, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was then filtered through a bacterial filter to remove bacteria, yielding fermentation filtrates for each strain. Harmful *Verticillium* spores inoculated on PDA plates were incubated at 25°C for 6 days. Conidia of the harmful *Verticillium* spores were then washed off and adjusted to a concentration of 3 × 10⁻⁶. 4 The spore suspension was prepared by aliquoting 1 ml of spores into five 1.5 ml centrifuge tubes, each containing 1 ml of spore suspension. 0.1 ml of each tested strain was added to one tube of spore suspension and mixed thoroughly, with sterile water as a control. Then, 30 μl of each strain was pipetteed onto a glass slide and placed in a moisture-controlled spore germination incubator. The slides were incubated in the dark at 25°C for 12 h. After incubation, the number of spores germinating was observed under a microscope. At least six fields of view were examined on each slide, and at least 70 conidia were randomly selected from each field to assess germination. The germination rate was calculated. The experiment was repeated three times. Statistical analysis was performed using SPSS 26 software, and the data were analyzed using the unpaired two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

[0056] The inhibition rate of spore germination is calculated using the following formula: Spore germination inhibition rate = (spore germination rate of control - spore germination rate of test strain) / spore germination rate of control * 100%.

[0057] The results are shown in Table 1 and Figure 5As shown.

[0058] Table 1. Effects of fermentation filtrate from various strains on the germination of harmful wart spores. From Table 1 and Figure 5 Compared with the control, all tested strains significantly reduced the germination rate of conidia of the mushroom pathogen *Verticillium*, especially strain g-2-2, whose germination rate was only about 10% of the control (see Table 1). Calculations of the inhibitory effect on spore germination rate showed that strain g-2-2 had the highest inhibition rate, exceeding 86%, while the inhibition rates of other strains did not exceed 50%. Strain g-2-2 showed a significantly higher inhibition rate against *Verticillium* than other strains. Figure 5 ).

Claims

1. A type of endophytic fungus of Agaricus bisporus, *Priscilla megaterium*, with antibacterial properties, characterized in that... The giant Priestella was named Priestiamegaterium The plant number is g-2-2, and the preservation number is CGMCC No.37094.

2. The use of the *Priestella giantiflora* as described in claim 1 in inhibiting the growth of pathogenic bacteria.

3. The application according to claim 2, characterized in that, The pathogen is harmful verrucous fungus ( ). Hypomycesperniciosus ).

4. The use of the *Priestella giantiflora* as described in claim 1 in the preparation of biocontrol agents for preventing and controlling pathogenic infections in edible fungi.

5. The application according to claim 2, characterized in that, The edible fungus is Agaricus bisporus (button mushroom) Agaricus bisporus ); The pathogen is harmful verrucous fungus ( ). Hypomycesperniciosus ).

6. A biocontrol agent for preventing and controlling infection by pathogenic fungi in edible fungi, characterized in that, The biocontrol agent contains an effective amount of the Priestella giantiflora as described in claim 1.

7. A method for preventing and controlling pathogenic fungal infections in edible fungi, characterized in that, Apply the *Priestella giantiflora* of claim 1 or the biocontrol agent of claim 6 to the soil around the roots of edible fungi.

8. The method according to claim 7, characterized in that, The edible fungus is Agaricus bisporus (button mushroom) Agaricus bisporus ); The pathogen is harmful verrucous fungus ( ). Hypomycesperniciosus ).

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