A diaporthe charlesworthii y3-18 endophytic fungus and application thereof

Abstract

The application belongs to the technical field of microorganisms, and relates to a Eupatorium adenophorum endophytic fungus Diaporthe charlesworthii Y3-18 Schizosaccharomyces japonicus and application thereof. The Eupatorium adenophorum endophytic fungus Diaporthe charlesworthii Y3-18 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 42964, and the preservation time is August 10, 2026. The fermentation product of Y3-18 shows inhibitory activity on four kinds of tested bacteria, wherein the minimum inhibitory concentration (MIC) of Staphylococcus aureus, Salmonella and Pseudomonas is 1.875 mg / mL, and the MIC of Escherichia coli is 3.75 mg / mL. The results show that the fermentation product of Y3-18 has inhibitory effect on gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Salmonella, Pseudomonas aeruginosa and Escherichia coli).

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to an endophytic fungus of *Eupatorium adenophorum*. Diaspora charlesworthii Y3-18 and its applications. Background Technology

[0002] Purple-stemmed Eupatorium ( Ageratina adenophora *Ageratum adenophorum* is one of the most serious invasive and noxious weeds in my country, possessing strong environmental adaptability, reproductive and dispersal capabilities, and allelopathic effects. Widely distributed in southwestern China, it causes continuous damage to agricultural and forestry production, ecological security, and livestock development. During its long-term invasion and environmental adaptation, *Ageratum adenophorum* has developed a rich, structurally diverse, and functionally unique endophytic fungal community, representing an important treasure trove for exploring novel microbial resources and natural bioactive substances. In recent years, the endophytic fungi of *Ageratum adenophorum* have become a research hotspot in the fields of microbial resources, natural product chemistry, and agricultural biological control due to their high diversity of metabolites and significant biological activity. Existing literature reports that multiple genera of endophytic fungi, including but not limited to *Alternaria*, can be isolated from different tissues (roots, stems, and leaves) of *Ageratum adenophorum*. (Alternaria) Fusarium (Fusarium), Anthrax (Colletotrichum), Phytophthora (Phoma) Penicillium (Penicillium), Aspergillus (Aspergillus) Anthracnose (Xylaria) And so on. The fermentation products of these endophytic fungi have been shown to possess various pharmacological activities, including antibacterial, antitumor, antioxidant, and anti-inflammatory effects. For example, those isolated from *Eupatorium adenophorum*... Alternaria The metabolites of this genus of bacteria exhibit inhibitory effects against plant pathogens; some Fusarium Strains of this genus can produce compounds with cytotoxic activity. These studies indicate that endophytic fungi of *Ageratum adenophorum* are an important source of naturally occurring active lead compounds.

[0003] genus *Ceratophyllum* ( Diaspora Fungi, as an important group of plant endophytes, exhibit diverse biological activities in their metabolites; however, Diaporthe charlesworthii Whether this specific strain of bacteria exists endophytically in *Ageratum adenophorum*, and whether its fermentation products possess antibacterial activity, are currently unknown and have not been publicly reported. Existing technologies lack methods for isolating and obtaining this strain from *Ageratum adenophorum*. Diaporthe charlesworthii Information regarding the strain and its antibacterial applications is lacking, and no technical solutions have been found regarding the broad-spectrum inhibitory activity of the fermentation products of this strain against various bacteria such as Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, and Escherichia coli.

[0004] Currently, bacterial resistance is becoming an increasingly serious problem, and there is an urgent need for safe, efficient, low-residue, and non-resistant natural antibacterial substances in the food processing, livestock breeding, medical and health, and agricultural production sectors. Novel antibacterial products derived from the endophytic fungus *Eupatorium adenophorum* have dual significance in resource utilization and ecological governance, aligning with the trend of green biomanufacturing.

[0005] Therefore, novel endophytic fungi were screened and isolated from *Eupatorium adenophorum*. Diaporthe charlesworthii Furthermore, developing its application in the field of antibacterial research is of significant scientific and practical value for enriching the species of endophytic fungi of *Aeonium adenophorum* in my country, expanding the sources of natural broad-spectrum antibacterial substances, and promoting the resource utilization of invasive plants. It also fills a gap in the understanding of this field. Diaspora charlesworthii This work fills a technological gap in the research and antibacterial applications of endophytic fungi in *Eupatorium adenophorum*, demonstrating significant innovation and practicality. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an endophytic fungus of *Eupatorium adenophorum*. Diaspora charlesworthii Y3-18 and its applications are detailed below: An endophytic fungus of *Eupatorium adenophorum* Diaporthe charlesworthii Y3-18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42964 on August 10, 2026.

[0007] Furthermore, the colonies are milky white on both sides, with a uniform color and a dense, fluffy texture.

[0008] Furthermore, the hyphae are long and thin, without septate structures; the hyphae are highly branched, uniform in thickness, and without swollen or deformed cells.

[0009] The endophytic fungi of *Eupatorium adenophorum* Diaporthe charlesworthii Application of Y3-18 in antibacterial properties.

[0010] Furthermore, the antibacterial aspect refers to the inhibition of Staphylococcus metalloids, Escherichia coli, Salmonella, and Pseudomonas.

[0011] The endophytic fungi of *Eupatorium adenophorum* Diaporthe charlesworthii Application of fermentation metabolites of Y3-18 in antibacterial properties.

[0012] An antibacterial method using the above-mentioned endophytic fungi of *Eupatorium adenophorum* Diaporthe charlesworthii Strain Y3-18 or its fermentation metabolites were used to inhibit bacterial growth.

[0013] Furthermore, the antibacterial effect is to inhibit Staphylococcus metalloids, Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

[0014] The beneficial effects of this invention are: 1. Endophytic fungi of *Eupatorium adenophorum* provided by this invention Diaporthe charlesworthii Y3-18 grew well in PDA medium, with colonies that were milky white and uniform in color on both sides, exhibiting a dense, fluffy texture. Microscopic observation after lactic acid-carbohydrate-cotton blue staining revealed slender, filamentous hyphae without septa; the hyphae were abundantly branched, of uniform thickness, and without swollen or deformed cells. Amplification of the ITS region using universal primers ITS1 / ITS4 yielded a specific band of approximately 547 bp. Sequencing results were submitted to the NCBI database for BLAST alignment, and strain Y3-18 was found to be compatible with… Diaporthe charlesworthii strain M8-3 (PP421978) formed an independent branch with 100% self-spreading support. Based on morphological characteristics, strain Y3-18 was identified as... Diaporthe charlesworthii It belongs to the endophytic fungi.

[0015] 2. Endophytic fungi of Eupatorium adenophorum Diaporthe charlesworthii The Y3-18 fermentation product exhibited antibacterial activity against all four tested bacteria. The minimum inhibitory concentration (MIC) against Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa was 1.875 mg / mL, while the MIC against Escherichia coli was 3.75 mg / mL. These results indicate that the Y3-18 fermentation product has inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Salmonella, Pseudomonas aeruginosa, and Escherichia coli).

[0016] 3. Endophytic fungi of Eupatorium adenophorum Diaporthe charlesworthii Y3-18 metabolites exert their antibacterial effect by disrupting the integrity of Staphylococcus aureus cell membranes, and this disruptive effect shows a significant dose-dependent increase with increasing treatment concentration.

[0017] 4. Endophytic fungi of Eupatorium adenophorum Diaporthe charlesworthii Y3-18 fermentation metabolites mainly include various structural types such as organic acids, phenolic acids, nitrogen-containing heterocycles, and amides. Among them, compounds such as 4-hydroxybenzaldehyde, cyclic (proline-tyrosine), fenestrated agaricone A, varistocin B, malic acid, 4-hydroxyphenyllactic acid, and succinic acid have antibacterial activity. Attached Figure Description

[0018] Figure 1 The method used is the bacterial cake method to screen for antibacterial active strains.

[0019] Figure 2 It is a Y3-18 colony.

[0020] Figure 3 It is mycelial staining.

[0021] Figure 4Y3-18 phylogenetic tree.

[0022] Figure 5 Effects of different concentrations of Y3-18 fermentation products on the cell membrane integrity of Staphylococcus aureus (fluorescence microscopy images); where: A: negative control group; B: 1 / 4 MIC treatment group; C: 1 / 2 MIC treatment group; D: 1 MIC treatment group.

[0023] Figure 6 Ion flow graphs in positive and negative ion modes, where A: anion mode; B: positive ion mode. Detailed Implementation

[0024] To make the technical solution and beneficial effects of the present invention clearer, the present invention will be described in detail below with reference to typical embodiments. It should be particularly noted that the following embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any reasonable adjustments or improvements made by those skilled in the art based on the core concept of the present invention should be included within the protection scope of the present invention.

[0025] Endophytic fungi of Eupatorium adenophorum Diaporthe charlesworthii Antibacterial activity study of Y3-18 metabolites 1. Experimental Methods 1.1 Isolation of endophytic fungi and screening of strains with antimicrobial activity Endophytic fungi were isolated using a tissue surface disinfection method: Healthy plant tissue was rinsed with tap water, then successively immersed in 75% ethanol for 30–60 s, treated with 2.5% sodium hypochlorite solution for 2–3 min, rinsed three times with sterile water, blotted dry with sterile filter paper, cut into small pieces, and inoculated onto PDA medium for constant temperature incubation. After mycelia grew from the tissue pieces, the pure culture strains were purified by tip picking and multiple streak transfers. Antimicrobial activity was screened using a fungal disc confrontation method: Pathogenic bacteria (Staphylococcus metalloids, Escherichia coli, Salmonella, Pseudomonas) were spread on LB agar plates, and endophytic fungal discs were inoculated onto the plates. After incubation, the inhibition zones were observed and measured, and active strains with significant antagonistic effects were screened.

[0026] 1.2 Identification of Endophytic Fungi The strain Y3-18, which exhibited significant antibacterial effects, was selected and subjected to morphological observation. Colony morphology, color, texture, and growth characteristics were recorded. Microscopic examination using lactic acid-carbohydrate-cotton blue staining clarified structural features such as hyphal septa. Fungal genomic DNA was extracted, and the ITS sequence was amplified using universal primers ITS1 / ITS4. The primer sequence for ITS1 was 5'-TCCGTAGGTGAACCTGCGG- 3', and the primer sequence for ITS4 was 5'-TCCTCCGCTTATTGATATGC- 3'. The ITS sequencing results were analyzed in GeneBank. The highest similarity DNA sequence was found using BLSAT on the NCBI website. Together with the sequencing results, a phylogenetic tree was constructed using the Mega12 software with neighbor-joining (NJ) to identify the genus classification of each endophytic fungus.

[0027] 1.3 Preparation of Fermentation Products Strain Y3-18 was inoculated into potato dextrose liquid medium (PDB) and cultured in a constant-temperature shaker at 25 ℃ and 120 r / min for 15 days. The fermentation broth containing secondary metabolites was collected. The fermentation broth was centrifuged at 4 ℃ and 8000 r / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm sterile filter in a laminar flow hood to obtain sterile fermentation broth. An equal volume of ethyl acetate was added to the fermentation broth, and the mixture was extracted three times, 30 min each time. The organic phases were combined, and the solvent was evaporated under reduced pressure using a rotary evaporator to obtain the crude extract of fermentation metabolites from strain Y3-18.

[0028] 1.4 Minimum inhibitory concentration (MIC) of endophytic fungal fermentation products against bacteria The experiment used a 96-well plate with serial dilution. The specific steps were as follows: 100 μL of culture medium was pre-loaded into wells 2-10. 200 μL of extract solution was added to well 1, and then 100 μL of the sample solution was transferred to well 2, and so on, completing the serial dilution from well 1 to well 10. Subsequently, 10 μL of a 1×10⁻⁶ concentration was added to wells 1-10. 5 CFU / mL bacterial suspension was added. Well 11 was prepared as a control group by adding equal volumes of culture medium, bacterial suspension, and physiological saline, while well 12 was prepared as a blank control by adding only culture medium. After sample addition, the 96-well plate was incubated at 37°C for 24 h. Subsequently, the minimum inhibitory concentration (MIC) was determined by visually observing the clarity of the liquid in each well. Each experimental design included three replicates.

[0029] 1.5 Effects of endophytic fungal fermentation metabolites on the cell membrane integrity of Staphylococcus aureus Staphylococcus aureus was cultured to the logarithmic growth phase, and the bacterial suspension concentration was adjusted to 1×10⁻⁶. 9 CFU / mL, aliquoted into 2 mL centrifuge tubes at 500 μL each. Equal volumes of extract solutions with concentrations of 1 / 2 MIC, 1 MIC, and 2 MIC were added to achieve final concentrations of 1 / 4 MIC, 1 / 2 MIC, and 1 MIC, respectively. Sterile deionized water was used as a blank control instead of the extract. Each treatment group was performed in triplicate. After incubation at 28°C for 4 h, the cells were centrifuged at 4°C and 5000 r / min for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in sterile PBS buffer and washed twice, then centrifuged again and the supernatant was discarded. PI staining solution was added to a final concentration of 10 μg / mL, and the cells were incubated in the dark for 30 min. The cells were then washed twice with PBS to remove free dye, and finally resuspended in PBS. 10 μL of the bacterial suspension was used to prepare slides for observation and image acquisition using a fluorescence microscope with an excitation wavelength of 535 nm and an emission wavelength of 615 nm. When cell membrane integrity is impaired, PI can enter the cell, bind to nucleic acids, and emit red fluorescence. The fluorescence intensity is positively correlated with the number of dead bacteria, thus reflecting the effect of the extract on bacterial cell membrane permeability.

[0030] 1.6 LC-MS determination of fermentation metabolites by endophytic fungi The analysis was performed using an Agilent 1290II-6545 UHPLC-QTOF system. A Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) was used. The mobile phase consisted of 0.1% formic acid-water solution as phase A and 0.1% formic acid-acetonitrile solution as phase B. The flow rate was 0.3 mL / min, the injection volume was 2 μL, and the column temperature was 35℃. The gradient elution program was as follows: 0–0.5 min, 5% B; 0.5–8 min, 5%–40% B; 8–15 min, 40%–100% B; 15–17 min, maintain 100% B; 17.1–20 min, return to 5% B and maintain equilibrium. The sample was prepared as a 1 mg / mL solution with 50% methanol, filtered through a membrane filter, and then injected. The mass spectrometer was used in full scan mode, with the mass-to-charge ratio range for both primary and secondary scans set to 100-3000. The primary scan rate was 4 spectra / sec, and the secondary scan rate was 2 spectra / sec. The ion source parameters were as follows: dry gas temperature 320℃, flow rate 8 L / min, nebulizer gas pressure 35 psig, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, collision energy 0 / 20 V, capillary voltage 3500 V, nozzle voltage 1000 V, fragmentation voltage 175 V, cone voltage 65 V, and octupole RF voltage 750 V.

[0031] 2. Results 2.1 Isolation of endophytic fungi and screening of antimicrobial strains More than 50 pure culture strains of endophytic fungi were isolated and purified from healthy plant tissues using a tissue surface disinfection method. Antibacterial activity against Staphylococcus metalloids, Escherichia coli, Salmonella, and Pseudomonas was screened using a fungal disc confrontation method. The results showed that strain Y3-18 produced a significant inhibition zone against all of the above pathogenic bacteria. Figure 1 It exhibits good antibacterial activity.

[0032] 2.2 Identification of endophytic fungus Y3-18 Combining morphological characteristics and molecular biological methods, the highly antibacterial endophytic fungal strain Y3-18 was classified and identified. Morphological observation results showed that strain Y3-18 grew well in PDA medium, with colonies that were milky white on both sides, uniform in color, and dense, fluffy in texture. Figure 2 After staining with lactic acid, carbolic acid, and cotton blue, microscopic observation revealed that the hyphae were slender and filamentous, without septate structures; the hyphae were abundantly branched, uniform in thickness, and without swollen or deformed cells. Figure 3 The ITS region was amplified using ITS1 / ITS4 universal primers, yielding a specific band of approximately 547 bp. The sequencing results were submitted to the NCBI database for BLAST alignment, and strain Y3-18 was compared with... Diaporthe charlesworthii strain M8-3 (PP421978) forms an independent branch ( Figure 4 The self-expansion support rate was 100%. Based on morphological characteristics, strain Y3-18 was identified as... Diaporthe charlesworthii It belongs to the endophytic fungi.

[0033] 2.3 Minimum inhibitory concentration (MIC) of Y3-18 fermentation product against bacteria The fermentation product of the endophytic fungus Y3-18 showed antibacterial activity against all four tested bacteria. The minimum inhibitory concentration (MIC) against Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa was 1.875 mg / mL, while the MIC against Escherichia coli was 3.75 mg / mL. The results indicate that the Y3-18 fermentation product inhibited both Gram-positive (Staphylococcus aureus) and Gram-negative (Salmonella, Pseudomonas aeruginosa, and Escherichia coli) bacteria, with stronger antibacterial activity against Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa, and relatively weaker antibacterial activity against Escherichia coli.

[0034] Table 1. MIC of fermentation products of endophytic fungus Y3-18 against bacteria

[0035] Note: "-" indicates antibacterial activity, and "+" indicates bacterial growth.

[0036] 2.4 Effect of Y3-18 on the cell membrane integrity of Staphylococcus aureus Figure 5 As shown, the control group showed virtually no fluorescence after PI staining, while the *Staphylococcus aureus* group treated with 1 / 4 MIC metabolite for 4 h emitted sporadic red fluorescence. The red fluorescence gradually increased with increasing extract concentration. At a concentration of 1 MIC, a large amount of red fluorescence was observed, indicating that the *Staphylococcus aureus* cell membrane was disrupted after extract treatment, allowing a large amount of PI to enter the cell and bind to genetic material, emitting red fluorescence. This reveals that the Y3-18 metabolite exerts its antibacterial effect by disrupting the integrity of the *Staphylococcus aureus* cell membrane, and this disruptive effect shows a significant dose-dependent increase with increasing treatment concentration.

[0037] 2.5 LC-MS determination of fermentation metabolites of Y3-18 Untargeted metabolic analysis of fermentation products from strain Y3-18 was performed using UHPLC-Q-TOF-MS. Total ion current information is shown in [link to data]. Figure 6 The strain can produce a variety of secondary metabolites with significant polarity differences. Preliminary analysis using precise relative molecular mass and secondary fragment mass spectrometry data revealed that the fermentation metabolites mainly encompass various structural types, including organic acids, phenolic acids, nitrogen-containing heterocycles, and amides. The main compounds are listed in Table 2. Among them, 4-hydroxybenzaldehyde, a cyclic (proline-tyrosine) compound, arbutin A, varistocin B, malic acid, 4-hydroxyphenyllactic acid, and mesocoumaric acid exhibit antibacterial activity. These results indicate that strain Y3-18 can synthesize structurally diverse active metabolites.

[0038] Table 2. Main material components of Y3-18 metabolites

[0039] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. An endophytic fungus of *Eupatorium adenophorum* Diaporthe charlesworthii Y3-18, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42964, and the deposit date is August 10, 2026.

2. The endophytic fungus of *Eupatorium adenophorum* according to claim 1 Diaporthe charlesworthii Y3-18, characterized in that, The endophytic fungi of *Eupatorium adenophorum* Diaporthe charlesworthii The colonies of Y3-18 are milky white on both sides, with a uniform color and a dense, fluffy texture.

3. The endophytic fungus of *Eupatorium adenophorum* according to claim 1 Diaporthe charlesworthii Y3-18, characterized in that, Endophytic fungi of Eupatorium adenophorum Diaporthe charlesworthii The hyphae of Y3-18 are slender and filamentous, without septate structure; the hyphae are richly branched, uniform in thickness, and without swollen or deformed cells.

4. The endophytic fungus of *Eupatorium adenophorum* as described in claim 1 Diaporthe charlesworthii Application of Y3-18 in antibacterial properties.

5. The application according to claim 4, characterized in that, The antibacterial aspect refers to the inhibition of Staphylococcus metalloproteinases, Escherichia coli, Salmonella, and Pseudomonas.

6. The endophytic fungus of *Eupatorium adenophorum* as described in claim 1 Diaporthe charlesworthii Application of Y3-18 fermentation metabolites in antibacterial properties.

7. A method for inhibiting bacteria, characterized in that, Using the endophytic fungus of *Eupatorium adenophorum* as described in claim 1 Diaporthe charlesworthii Strain Y3-18 or its fermentation metabolites were used to inhibit bacterial growth.

8. The antibacterial method as described in claim 7, characterized in that, The antibacterial effect is to inhibit Staphylococcus metalloids, Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

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