Penicillium sp. from marine source, fermentation method and application thereof

Abstract

The application discloses a marine source penicillium and a fermentation method and application thereof. The marine source penicillium is named Penicillium sp. DSF059, and the preservation number is CCTCC NO: M 20232310. Penicilliu The application optimizes the culture condition of the strain, selects a culture medium and culture condition with high yield of antibacterial active compounds to carry out fermentation, so that more target products can be obtained, the operation is simple, the cost is low, and meanwhile, the marine source penicillium Penicilliu Penicillium sp. DSF059 can provide an excellent strain for new drug development of resisting plant pathogenic fungi such as coffee leaf blight, coffee brown spot disease, grape gray mold disease, Chinese cinnamon brown spot disease, citrus black rot disease, pepper late blight, hickory leaf blight, coffee black spot disease and four pathogenic fungi separated from coffee leaf diseases and Staphylococcus aureus, and has a good application prospect for preventing and treating the plant pathogenic fungi and Staphylococcus aureus.

Description

[0001] This application requests priority to Chinese invention patent application No. 202411773610.5, filed on December 5, 2024, entitled "A marine Penicillium and its fermentation method and application". Technical Field

[0002] This invention relates to the field of microbial technology, and in particular to a marine-derived Penicillium and its fermentation method and application. Background Technology

[0003] The ocean, as one of the largest ecosystems on Earth, covers 71% of the planet's surface. From a geoscience perspective, areas in the ocean deeper than 1000 meters are called the deep sea, characterized by high pressure, low oxygen, high salinity, and extreme temperatures. Researchers both domestically and internationally have obtained some microbial strains and metabolites from this habitat, and have also discovered potential new microbial taxonomic units. The exploration and development of marine fungi, especially deep-sea fungal resources, not only expands our understanding of marine fungal diversity but also provides possibilities for obtaining new metabolites and novel bioactive substances.

[0004] Penicillium ( Penicillium Penicillium is a type of saprophytic fungus. Its colonies are mostly grayish-green, but some varieties are white or bluish-green. Penicillium fungi are found in soil, oceans, glaciers, deserts, swamps, and even inside animals, posing a significant challenge to human life. They can cause plant diseases and frequently lead to mold growth in food and animal feed, resulting in varying degrees of illness in humans and animals, and in severe cases, death. However, it also has enormous economic value, mainly in the following aspects: First, it can decompose organic matter and promote the cycling of various elements such as nitrogen, phosphorus, and potassium in nature; second, it can produce a variety of highly active enzymes, including amylase, chitinase, cellulase, β-glucosidase, alginate lyase, inulinase, ligninase, xylanase, protease, keratinase, lipase, nuclease, and phytase, which are widely used in food processing, industrial raw material preparation, and new energy manufacturing; third, it can produce a variety of active metabolites. More than 1,300 active metabolites, including penicillin and griseofulvin, have been reported in Penicillium fungi, which generally have antibacterial, antifungal, antiviral, antitumor, or anticardiovascular disease effects; fourth, it can degrade a variety of harmful substances in the environment (such as aromatic hydrocarbons), showing great application potential in the field of environmental pollution control.

[0005] Staphylococcus aureus ( Staphylococcus aureusStaphylococcus aureus (S. aureus) is an aerobic or facultative anaerobic Gram-positive cocci, non-spore-forming, non-flagellated, and mostly non-capsulated. It has low nutritional requirements, is relatively resistant to adverse environments, and is widely distributed, found in soil, air, and water. S. aureus is the most common pathogen causing purulent infections in humans, causing localized purulent infections as well as pneumonia, pseudomembranous colitis, pericarditis, and even systemic infections such as sepsis and septicemia. S. aureus is also a significant pathogen causing bacterial food poisoning, primarily contaminating dairy products, eggs and egg products, and various cooked meat products, followed by frozen foods containing dairy products. According to the U.S. Centers for Disease Control and Prevention (CDC), infections caused by S. aureus are currently the leading cause of infection and a primary target for antimicrobial drug development.

[0006] Currently, the conventional methods for controlling Staphylococcus aureus both domestically and internationally are as follows: First, antibiotics are the traditional treatment for Staphylococcus aureus infections, with β-lactam antibiotics (such as penicillin) being the most important and commonly used. However, after long-term use, Staphylococcus aureus has developed high resistance to β-lactam antibiotics. Second, bacteriophages mediate the breakage of peptidoglycan linkages in the bacterial cell wall by encoding lysins, lysozymes, etc., leading to cell wall rupture, bacterial death, and the release of new bacteriophages. Low doses of bacteriophages can inhibit biofilm formation, while high doses can degrade biofilms, thereby killing pathogens. Third, traditional Chinese medicine can inhibit bacterial biofilm formation. Monomers of traditional Chinese medicine, such as eugenol and quercetin, inhibit the bacterial community's sensory system, thereby suppressing biofilm formation, the secretion of virulence factors, and the expression of efflux pumps. Although the above methods can effectively inhibit Staphylococcus aureus, the overuse of antibiotics and other drugs has led to the emergence of drug-resistant Staphylococcus aureus. This has resulted in a continuously increasing demand for new bioactive biological resources and bioactive metabolites, and an increasingly urgent need for the development of new drugs. Therefore, it is imperative to continuously explore unique and novel biological control resources.

[0007] Plant pathogens can infect plants, causing significant plant diseases. Several, even dozens, of pathogens can be found in a single crop, leading to reduced yields and severe damage. Chemical agents are commonly used for control. However, chemical methods often harm the ecological environment, induce drug resistance in pathogens, and, most importantly, leave chemical residues that can affect human health. Therefore, efficient, environmentally friendly, and safe biological control methods are an important option for solving this problem. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention proposes a marine-derived Penicillium, its fermentation method and application, to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] The application of a marine-derived Penicillium in the preparation of anti-pathogenic drugs, wherein the pathogens include coffee leaf blight pathogen, coffee brown spot pathogen, grape gray mold pathogen, cork tree brown spot pathogen, citrus black rot pathogen, pepper phytophthora, hickory leaf blight pathogen, coffee black spot pathogen, coffee leaf disease pathogen, and Staphylococcus aureus.

[0011] Preferably, the pathogen causing coffee leaf blight is a fungus belonging to the genus *Metacarpa* (…). Diaporthe etinsideae The pathogen causing coffee brown spot disease is *Polytrichum neomycetes* (…). Neopestalotiopsis aotearoa The pathogen causing grape gray mold is *Botrytis cinerea* (…). Botrytis cinerea The pathogen causing brown spot disease in Phellodendron amurense is *Ichthyophthirius multifiliis* (Guilin black spore). Black spore from Guilin The pathogen of citrus black rot is Alternaria (Alternaria). Alstroemeria Alternaria The pathogen causing Phytophthora capsici is Phytophthora capsici ( Phytophthora capsicum The pathogen causing pecan leaf blight is *Melastoma* (a type of fungus). Diaporthe pseudophoenicicola The pathogen causing coffee black spot disease is Cladosporium hilkulii ( Collared hilkhuijsenii ), coffee leaf pathogens include brown fusarium ( ), Phlebiopsis brown ), Fomitopsis pinnata ( Fomitopsis submelia e), Coprinus rhombosodium ( Coprinopsis rhombisporoides ), Broom mold ( Spider webs ).

[0012] Preferably, the marine-derived Penicillium is named Penicillium m sp.DSF059 is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 23, 2023; accession number: CCTCC NO: M 20232310.

[0013] Preferably, the ITS gene sequence of the marine Penicillium is the nucleotide sequence shown in SEQ ID NO.1, the BenA gene sequence is the nucleotide sequence shown in SEQ ID NO.2, the CaM gene sequence is the nucleotide sequence shown in SEQ ID NO.3, and the RPB2 gene sequence is the nucleotide sequence shown in SEQ ID NO.4.

[0014] Preferably, the method for preparing the ethyl acetate fermentation broth includes the following steps:

[0015] (1) Penicillium culture and seed culture preparation: marine Penicillium strains were inoculated into Erlenmeyer flasks containing PDA solid medium and cultured at 25-30℃ for 3-8 days to activate the strains. Sterile Tween 80 with a volume fraction of 0.03-0.08% was added and the activated strains were cultured at 90-120 r / min for 8-15 min to prepare spore suspension as seed culture.

[0016] (2) Fermentation products: The seed liquid was inoculated into rice solid fermentation medium at an inoculation rate of 2-7% by volume, and static fermentation was carried out at 25-30℃ and under natural light for 25-35 days to obtain the fermentation products.

[0017] (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) is soaked in ethyl acetate for 8-15 hours, then shaken and cultured at 100-150 r / min for 8-20 minutes. The filtrate is obtained by vacuum rotary evaporation. The filtrate extract obtained by vacuum rotary evaporation is extracted with methanol with a volume concentration of 70-90% for 60-100 minutes to obtain ethyl acetate fermentation broth.

[0018] Preferably, the method for preparing the ethyl acetate fermentation broth includes the following steps:

[0019] (1) Penicillium culture and seed culture preparation: marine Penicillium strains were inoculated into Erlenmeyer flasks containing PDA solid medium and cultured at 28°C for 5 days to activate the strains. Sterile Tween 80 with a volume fraction of 0.05% was added and the activated strains were shaken at 110 r / min for 10 min to prepare a spore suspension as seed culture.

[0020] (2) Fermentation products: The seed liquid was inoculated into rice solid fermentation medium at a volume ratio of 5%, and the fermentation was carried out at 28℃ and under natural light for 30 days to obtain the fermentation products.

[0021] (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 12 hours, then cultured by shaking at 110 r / min for 10 minutes, and the filtrate was obtained by vacuum evaporation. The filtrate was then extracted with methanol at a volume concentration of 80% for 80 minutes to obtain ethyl acetate fermentation broth.

[0022] Preferably, the PDA solid culture medium in step (1) consists of the following components per liter: 150-250g potato, 15-40g glucose, 10-30g agar, 300-500mL aged seawater, and the remainder deionized water. After the PDA solid culture medium is prepared, it is naturally sterilized at pH 5.5-6.5 at 121℃ for 15-30min.

[0023] Preferably, the PDA solid culture medium in step (1) consists of the following components per liter: 200g potato, 20g glucose, 15g agar, 400mL aged seawater, and the remainder deionized water. After preparation, the PDA solid culture medium is sterilized at 121℃ for 20min at natural pH.

[0024] Preferably, the concentration of spores in the spore suspension in step (1) is 1.0 × 10⁻⁶. 6 ~1.0×10 10 cfu / mL.

[0025] Preferably, the concentration of spores in the spore suspension in step (1) is 1.0 × 10⁻⁶. 7 cfu / mL.

[0026] Preferably, the rice solid fermentation culture medium in step (2) consists of the following components: 8-15g of rice, 5-20mL of aged seawater, and 8-30mL of distilled water. After the components are mixed evenly, the mixture is sterilized at 120-130℃ for 25-40min.

[0027] Preferably, the rice solid fermentation culture medium in step (2) consists of the following components: 10g rice, 8mL aged seawater, and 12mL distilled water. After the components are mixed evenly, the mixture is sterilized at 120~130℃ for 25~40min.

[0028] Preferably, the parameters for vacuum rotary evaporation in step (3) are a vacuum degree of 0.02~0.08MPa and a temperature of 30~35℃.

[0029] Preferably, the marine-derived Penicillium or the ethyl acetate fermentation broth prepared by the fermentation method is used in the preparation of anti-Staphylococcus aureus drugs.

[0030] Preferably, the PDA solid culture medium in step (1) can also be replaced by the following culture medium for the culture of Penicillium, and the reagent components and culture medium information involved are as follows:

[0031] Czapek's stock solution: 150g nitric acid, 25g potassium chloride, 25g magnesium sulfate heptahydrate, 0.5g ferrous sulfate heptahydrate, and deionized water to a final volume of 500mL.

[0032] Trace element stock solution: 0.25g anhydrous copper sulfate, 0.5g zinc sulfate heptahydrate, and deionized water to a final volume of 500mL.

[0033] CYA medium: 1g dipotassium hydrogen phosphate, 5g yeast extract, 30g sucrose, 10mL Czapek's stock solution, 20g agar, 400mL deionized water (adjusted to 1L), sterilized at 121℃ for 20min, natural pH.

[0034] G25N medium: 10 mL of Czapek's stock solution, 1 g of dipotassium hydrogen phosphate, 5 g of yeast extract, 30 g of sucrose, 250 g of glycerol, 20 g of agar, 400 mL of aged seawater, and deionized water to a final volume of 1 L. Sterilize at 121 °C for 20 min at natural pH.

[0035] MEA medium: 20g malt extract, 1g peptone, 20g glucose, 10mL trace element stock solution, 20g agar, 400mL deionized water (from aged seawater) to 1L, sterilize at 121℃ for 20min, natural pH.

[0036] YES medium: 20g yeast extract, 150g sucrose, 0.5g magnesium sulfate heptahydrate, 10mL trace element stock solution, 20g agar, 400mL deionized water to a final volume of 1L, sterilized at 121℃ for 20min, natural pH.

[0037] DG18 medium: Dichloran-Glycerol-agar-base 31.5g, glycerol 220g, trace element stock solution 10mL, chloramphenicol 0.05g, agar 20g, and 400mL of aged seawater and deionized water to a final volume of 1L. Sterilize at 121℃ for 20min, at natural pH.

[0038] CREA medium: 30g sucrose, 3g creatine, 1.6g potassium phosphate heptahydrate, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 0.01g ferrous sulfate heptahydrate, 10mL trace element stock solution, 0.05g bromocresol purple, 20g agar, 400mL of aged seawater and deionized water to a final volume of 1L, sterilized at 121℃ for 20min, natural pH.

[0039] OA medium: 30g oats, 10mL trace element stock solution, 20g agar, 400mL of aged seawater, and deionized water to a final volume of 1L. Sterilize at 121℃ for 20min, at natural pH.

[0040] LBA medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15-18g agar, bring the volume to 1L with deionized water, sterilize at 121℃ for 20min, natural pH 7.2-7.4.

[0041] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, deionized water to a final volume of 1L, sterilized at 121℃ for 20min, natural pH 7.2~7.4.

[0042] Preferably, the rice solid fermentation medium in step (2) can also be replaced by the following culture medium for preparing the ethyl acetate fermentation broth of Penicillium, and the relevant culture medium information is as follows:

[0043] Oat solid fermentation medium: 10g oats, 12ml distilled water, 8ml aged seawater, placed in a 250ml Erlenmeyer flask, sterilized at 121℃ for 30min.

[0044] Potato solid fermentation medium: 10g peeled potato chunks, 12ml distilled water, and 8ml aged seawater are placed in a 250ml Erlenmeyer flask and sterilized at 121℃ for 30min.

[0045] Cheriois solid fermentation medium: 10g of General Mills Multi-grain Cheriois, 12ml of distilled water, and 8ml of aged seawater were placed in a 250ml Erlenmeyer flask and sterilized at 121℃ for 30min.

[0046] Compared with the prior art, the beneficial effects of the present invention are: the present invention optimizes the marine-derived Penicillium fungus. Penicillium Fermentation medium and conditions for msp.DSF059, selecting mediums and conditions with high yields of antibacterial active compounds, can yield more target products. The operation is simple and cost-effective. (Note: The last sentence appears to be incomplete and possibly refers to a different topic.) Penicillium msp.DSF059 provides an excellent strain for the development of new drugs against the pathogens of coffee leaf blight (*Mallotus*), coffee brown spot (*Neopteris polychaete*), grape gray mold (*Botrytis cinerea*), phellodendron brown spot (*Ichthyophthirius guilinensis*), citrus black rot (*Alternaria*), pepper blight (*Phytophthora capsici*), hickory leaf blight (*Mallotus*), coffee black spot (*Cladosporium hilckii*), as well as four pathogens isolated from coffee leaf diseases: *Pterocarya stenoptera*, *Fotomyces nephrolepis*, *Coprinus rhombosporus*, *Cladosporium*, and *Staphylococcus*, and a foodborne pathogen. It shows good application prospects for the control of plant pathogens and the foodborne pathogen *Staphylococcus aureus*, and provides a new approach for developing drugs that can control both plant pathogens and foodborne pathogens. Attached Figure Description

[0047] Figure 1 Penicillium Penicillium The growth of m sp. DSF059 on six different solid media; A, top, from left to right, front view on CYA, YES, MEA, OA, DG18 and CREA solid media; A, bottom, from left to right, back view on CYA, YES, MEA, OA, DG18 and CREA solid media; B, optical microstructure of Penicillium conidiophores and conidia; C, scanning electron microstructure of Penicillium conidiophores and conidia.

[0048] Figure 2 Penicillium Penicillium Multigene phylogenetic tree of m sp.DSF059;

[0049] Figure 3 Penicillium Penicillium The inhibition zone of m sp. DSF059 against Staphylococcus aureus;

[0050] Figure 4 Penicillium Penicillium The growth of m sp.DSF059 on four solid fermentation media: rice, oats, potatoes, and Cheriois;

[0051] Figure 5 Penicillium Penicillium m sp.DSF059 is effective against the genus *Ceratophyllum*, the pathogen of coffee leaf blight. Diaporthe etinsideae ), the pathogen of coffee brown spot disease, *Neoproterozoa* ( Neopestalotiopsis aotearoa Grape gray mold pathogen Botrytis cinerea ( Botrytis cinerea ), the pathogen of Phellodendron amurense brown spot disease is *Ichthyophthirius multifiliis* ( Nigrospora guilinensis Alternaria alterniflora, the pathogen of citrus black rot ( Alternaria alstroemeria ), Phytophthora capsici, the pathogen of Phytophthora capsici ( Phytophthora capsicum ), the pathogen of pecan leaf blight, *Phoebe zhennan* ( Diaporthe pseudophoenicicola ), the pathogen of coffee black spot disease, Cladosporium hilkelli ( Collariella hilkhuijsenii ), the pathogen causing coffee leaf diseases ( ), brown fusarium ( Phlebiopsis brown ), Fomitopsis pinnata ( Fomitopsis submelia e), Coprinus rhombosodium ( Coprinopsis rhombisporoid ), Broom mold ( Spider webs (It has an antibacterial effect.) Detailed Implementation

[0052] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0053] A marine-derived Penicillium, named Penicillium m sp.DSF059 is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on November 23, 2023; accession number: CCTCC NO: M 20232310.

[0054] Example 1

[0055] A method for preparing ethyl acetate fermentation broth of marine-derived Penicillium includes the following steps:

[0056] (1) Penicillium culture and seed culture preparation: marine Penicillium strains were inoculated into Erlenmeyer flasks containing PDA solid medium and cultured at 25°C for 3 days to activate the strains. Sterile Tween 80 with a volume fraction of 0.03% was added and the activated strains were shaken at 90 r / min for 8 min to prepare a spore suspension as seed culture.

[0057] (2) Fermentation products: The seed liquid was inoculated into rice solid fermentation medium at a volume ratio of 2%, and the fermentation was carried out at 25°C and under natural light for 25 days to obtain the fermentation products.

[0058] (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 8 hours, then cultured by shaking at 100 r / min for 8 minutes, and the filtrate was obtained by vacuum filtration. The filtrate was subjected to vacuum rotary evaporation at a vacuum degree of 0.02 MPa and a temperature of 30 °C. The extract of the filtrate obtained by vacuum rotary evaporation was extracted with methanol with a volume concentration of 70% for 60 minutes to obtain ethyl acetate fermentation broth.

[0059] The PDA solid culture medium in step (1) consists of the following components: 150g potato, 15g glucose, 10g agar, 300mL aged seawater, deionized water to a final volume of 1L, natural pH 5.5, sterilized at 121℃ for 15min.

[0060] Step (1) The concentration of spores suspended in the spore suspension is 1.0 × 10⁻⁶. 6 cfu / mL.

[0061] The rice solid fermentation culture medium described in step (2) consists of the following components: 8g of rice, 5mL of aged seawater, and 8mL of distilled water. After mixing evenly, it is sterilized at 120℃ for 25min.

[0062] Example 2

[0063] A method for preparing ethyl acetate fermentation broth of marine-derived Penicillium includes the following steps:

[0064] (1) Penicillium culture and seed culture preparation: marine Penicillium strains were inoculated into Erlenmeyer flasks containing PDA solid medium and cultured at 30°C for 8 days to activate the strains. Sterile Tween 80 with a volume fraction of 0.08% was added and the activated strains were shaken at 120 r / min for 15 min to prepare spore suspension as seed culture.

[0065] (2) Fermentation products: The seed liquid was inoculated into rice solid fermentation medium at a volume ratio of 7%, and the fermentation was carried out at 30℃ and under natural light for 35 days to obtain the fermentation products.

[0066] (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 15 h, then cultured with shaking at 150 r / min for 20 min, and filtered to obtain filtrate. The filtrate was subjected to vacuum rotary evaporation at a vacuum degree of 0.08 MPa and a temperature of 35 °C. The filtrate extract obtained by vacuum rotary evaporation was extracted with methanol with a volume concentration of 90% for 100 min to obtain ethyl acetate fermentation broth.

[0067] The PDA solid culture medium in step (1) consists of the following components: 250g potato, 40g glucose, 30g agar, 500mL aged seawater, deionized water to a final volume of 1L, natural pH 6.5, sterilized at 121℃ for 30min.

[0068] Step (1) The concentration of spores suspended in the spore suspension is 1.0 × 10⁻⁶. 10 cfu / mL.

[0069] The rice solid fermentation culture medium described in step (2) consists of the following components: 15g rice, 20mL aged seawater, and 30mL distilled water. After mixing evenly, it is sterilized at 130℃ for 40min.

[0070] Example 3

[0071] A method for preparing ethyl acetate fermentation broth of marine-derived Penicillium includes the following steps:

[0072] (1) Penicillium culture and seed culture preparation: marine Penicillium strains were inoculated into Erlenmeyer flasks containing PDA solid medium and cultured at 28°C for 5 days to activate the strains. Sterile Tween 80 with a volume fraction of 0.05% was added and the activated strains were shaken at 110 r / min for 10 min to prepare spore suspension as seed culture.

[0073] (2) Fermentation products: The seed liquid was inoculated into rice solid fermentation medium at a volume ratio of 5%, and the fermentation was carried out at 28℃ and under natural light for 30 days to obtain the fermentation products.

[0074] (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 12 hours, then cultured with shaking at 110 r / min for 10 minutes, and the filtrate was obtained by vacuum filtration. The filtrate was subjected to vacuum rotary evaporation at a vacuum degree of 0.06 MPa and a temperature of 32℃. The extract of the filtrate obtained by vacuum rotary evaporation was extracted with methanol with a volume concentration of 80% for 80 minutes to obtain ethyl acetate fermentation broth.

[0075] The PDA solid culture medium in step (1) consists of the following components: 200g potato, 20g glucose, 15g agar, 400mL aged seawater, deionized water to a final volume of 1L, natural pH 6.0, sterilized at 121℃ for 20min.

[0076] Step (1) The concentration of spores suspended in the spore suspension is 1.0 × 10⁻⁶. 7 cfu / mL.

[0077] The rice solid fermentation culture medium described in step (2) consists of the following components: 10g rice, 8mL aged seawater, and 12mL distilled water. After mixing evenly, it is sterilized at 121℃ for 30min.

[0078] Isolation, morphological identification, molecular biological identification, physiological and biochemical characterization, and whole genome sequence analysis of strain 1 in Experiment Example 1

[0079] (1) Strains Isolation

[0080] ① A strain of Penicillium was isolated from deep-sea sediments at a depth of 2595m in the South China Sea using Martin medium. Penicillium m sp.DSF059 was inoculated onto Martin medium and cultured at 28°C for 7 days. After incubation, it was stored at 4°C.

[0081] ② Martin medium composition and preparation steps: 10g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 3.3mL 0.1% Bengal red solution, 15-20g agar, 400mL aged seawater; Preparation steps: Distilled water to a final volume of 1L, pH at rest, sterilize at 121℃ for 20min, and wait for the medium temperature to drop below 65℃. In a clean bench, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin, which have been sterilized by ultrafiltration through a 0.22µm membrane, to obtain Martin medium;

[0082] (2) Morphological identification of strains

[0083] ① Colony morphology identification: The three-point inoculation method was used to inoculate strain DSF into CYA, YES, MEA, OA, DG18 and CREA media respectively. After incubation in the dark at 28℃ for 7 days, the growth status of the strain, the color of aerial hyphae, the color of hyphae in the substrate, the presence and color of soluble pigments, etc. were observed and recorded.

[0084] Penicillium Penicillium The colony morphology characteristics of m sp. DSF059 are as follows: After 7 days of incubation at 28℃, the DSF059 strain can grow normally on CYA, YES, MEA, OA, DG18, and CREA media (see details). Figure 1-A). On CYA medium, DSF059 colonies are 33-37 mm in diameter, with a raised center, low and narrow edges, and irregular radial wrinkles. The hyphae are white, velvety or slightly flocculent, with no exudate and no soluble pigment; the reverse side is yellow. On YES medium, colonies are 34-42 mm in diameter, with a distinctly raised center and numerous radial wrinkles. The texture is velvety or slightly flocculent, with white hyphae, abundant dark green conidial structures, no exudate, and green soluble pigment; the reverse side is yellowish-brown to yellow. On MEA medium, colonies are 25-29 mm in diameter, with a slightly raised center and flat edges. The texture is velvety and granular, with abundant conidial structures; the color is grass green, and the hyphae are white. No exudate, soluble pigment is green, yellowish-brown to yellow on the reverse side; on OA medium, colony diameter is 33-47 mm, slightly raised in the middle with concentric lines and slight radial wrinkles, texture is velvety or slightly flocculent, conidia are abundant, dark green, hyphae are white, no exudate, soluble pigment is green, yellowish-brown to yellow on the reverse side; on DG18 medium, colony diameter is 19-20 mm, colony is low, slightly raised in the middle, low edge, entire, hyphae are white, texture is pubescent, conidia are of medium density, conidia are grayish-green overall, no soluble pigment, no exudate, yellowish-brown to yellow on the reverse side; on CREA medium, colony diameter is 28-33 mm, weak growth and moderate acid production.

[0085] Table 1 shows a comparative analysis of the morphological characteristics of Penicillium DSF059 and closely related Penicillium species. These morphological characteristics are different from those of other Penicillium species.

[0086] Table 1. Comparative analysis of morphological characteristics between Penicillium DSF059 and closely related Penicillium fungi.

[0087]

[0088] Note: "—" indicates no data.

[0089] ② Microscopic morphological identification: After culturing the strain at 28℃ for 7 days using the wet slide method, conidiophores and conidia were observed using a Nikon research-grade microscope (Ni-U, NIKON, Tokyo, Japan) and a thermal field emission scanning electron microscope (SEMJSM-7610FPlus, JEOL, Tokyo, Japan). The operation steps of the wet slide method are as follows: ① PDA thin layer pouring: Under aseptic conditions, a PDA thin layer with a thickness of 2-3 mm was poured upside down in a petri dish. ② Preparation of culture chambers: A culture chamber was prepared in another new petri dish. A layer of circular filter paper slightly smaller than the petri dish was placed at the bottom of the chamber. A U-shaped glass rod was placed on the circular filter paper, and a clean glass slide was placed on the glass rod. The petri dish was covered, wrapped in newspaper, and then sterilized. ③ Inoculation: The PDA thin layer was cut into agar blocks with a side length of about 1.0 cm using a scalpel and transferred to both ends of the glass slide in the above chamber. Using a sterile inoculation loop, pick up a small amount of spores or hyphae and inoculate them onto the edge of the agar block. Then, use sterile forceps to cover the agar block with a coverslip. ④ Incubation: Transfer 2-3 mL of sterile 20% glycerol to the filter paper of the petri dish (to maintain humidity). Cover with the culture medium and the dish lid, seal with sealing film, and incubate upright in a 28°C incubator for 5-10 days. Before observing the morphology of the strain under a scanning electron microscope, the coverslips from which the Penicillium morphology has already been observed under an optical microscope must be treated. First, soak the coverslips in 2.5% glutaraldehyde and fix them overnight at 4°C. Rinse the coverslips 3-5 times with phosphate buffer (pH 7.4), then dehydrate them using a gradient of 30%, 50%, 70%, 80%, 90%, and 100% ethanol, treating each gradient for 20 minutes. Afterward, wrap the coverslips in aluminum foil and freeze-dry them for 10 minutes. The dried material was fixed to the aluminum pin with conductive carbon tape, gold was sputtered, and the morphology of the strains and spores was observed using a thermal field emission scanning electron microscope.

[0090] Penicillium Penicillium The microscopic morphological characteristics of m sp.DSF059 are as follows: conidiophores arranged in single whorls, with smooth stem walls; 4-8 phialides per whorl; ampoule-shaped; conidia nearly spherical with distinctly rough walls, 1.9-2.5 μm in diameter; no sclerotia observed (see details). Figure 1 -B、C).

[0091] (3) Molecular biological identification of strains

[0092] ① Pick hyphae from MEA plates, extract DNA using a genomic DNA extraction kit, and use this as a template to amplify the nuclear ribosomal internally transcribed spacer region (ITS) of housekeeping genes 5.8S rDNA and 28S rDNA, β-tubulin gene (BenA), calmodulin gene (CaM), and RNA polymerase II second large subunit (RPB2) gene fragments.

[0093] ②The primer sequences for ITS PCR amplification are ITS1: 5′-tccgtaggtgaacctgcgg-3′ (SEQ ID NO.5) and ITS4: 5′-tcctccgcttattgatatgc-3′ (SEQ ID NO.6); the primer sequences for BenA PCR amplification are Bt2a: 5′-ggtaaccaaatcggtgctgctttc-3′ (SEQ ID NO.7) and Bt2b: ​​5′-accctcagtgtagtgacccttggc-3′ (SEQ ID NO.8); the primer sequences for CaM PCR amplification are CF1: 5′-gccgactctttgacygargar-3′ (SEQ ID NO.9) and CF4: 5′-tttytgcatcatragytggac-3′ (SEQ ID NO.10); RPB2 The PCR amplification primer sequences were 5F: 5′-gaygaymgwgatcayttygg-3′ (SEQ ID NO.11) and 7CR: 5′-cccatrgcttgyttrcccat-3′ (SEQ ID NO.12).

[0094] PCR reaction system: 25uL 2×Taq Master Mix, 19uL ddH2O, 2uL template DNA, 2uL primers each, total volume 50uL.

[0095] PCR amplification conditions for ITS, Ben A and CaM genes: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 60 s, 35 cycles; 72℃ final extension for 7 min.

[0096] RPB2 gene PCR amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 48℃ annealing for 45 s, 72℃ extension for 60 s, 5 cycles; 94℃ denaturation for 45 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, 5 cycles; 94℃ denaturation for 45 s, 52℃ annealing for 45 s, 72℃ extension for 60 s, 30 cycles; 72℃ final extension for 7 min.

[0097] ③ To meet the requirements Penicillium PCR amplification products of ITS, BenA, CaM, and RPB2 from *Penicillium sp. DSF059* strain were sent to BGI Genomics Co., Ltd. for sequencing. Sequencing results for the marine-derived *Penicillium* were as follows: ITS gene sequence (SEQ ID NO. 1), BenA gene sequence (SEQ ID NO. 2), CaM gene sequence (SEQ ID NO. 3), and RPB2 gene sequence (SEQ ID NO. 4).

[0098] ④ Polyphasic taxonomy refers to the process of classifying and identifying phylogenetic studies by integrating phenotypic and genotypic information using multiple methods of modern taxonomy. Polyphasic analysis integrates phenotypie taxonomy, chemotaxonomy, and molecular taxonomy. In chemotaxonomy, Penicillium is limited to a few indicators such as fatty acids, proteins, and carbohydrates. In molecular analysis, Visagie et al. listed four gene sequences as reference marker sequences for Penicillium identification: nuclear ribosomal internal transcribed spacer (nrITS), β-tubulin gene (BenA), calmodulin gene (CaM), and the second largest subunit of RNA polymerase II (RPB2). These sequences have been recognized by the International Commission of Penicillium and Aspergillus (ICPA) (see Ye Ming. Advances in polyphasic taxonomy of fungi [J]. Journal of Anhui University of Engineering Science and Technology (Natural Science Edition), 2003(02):1-7). The ITS sequence serves as a universal DNA barcode for fungi, but it cannot effectively identify certain species of the genus *Penicillium*. BenA, CaM, and RPB2, as secondary identification markers for *Penicillium*, can effectively identify the specific taxonomic position of *Penicillium* fungi. However, RPB2 is difficult to amplify and its database information is incomplete; similarly, CaM also suffers from incomplete database information. BenA, on the other hand, is easy to amplify and has a complete database. For new species, it is necessary to use a combination of the four genes—ITS, BenA, CaM, and RPB2—for taxonomic identification.

[0099] ⑤ Sequencing results were analyzed using BLAST homology alignment on NCBI. ITS, BenA, CaM, and RPB2 sequences of similar strains and one group of exogenous bacteria were individually aligned using MEGA 11.0, and the sequence names were sorted. Finally, Fasta format multiple sequence alignment files were exported (as shown in Table 2). SequenceMatrix software was used to merge the sequences, and then MEGA 11.0 software was used to construct an ITS-BenA-CaM-RPB2 phylogenetic tree using neighbor-joining (NJ) (see details). Figure 2 This strain is quite distant from previously reported Penicillium species, although DSF059 shares similarities with other strains, forming a distinct branch. Based on morphological analysis, the strain was identified as a Penicillium and deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20232310.

[0100] Table 2. ITS, BenA, CaM, and RPB2 sequences of strains used for phylogenetic analysis.

[0101]

[0102] Blast analysis revealed that strain DSF059 was similar to... Penicillium macrosclerotiorum The sequence showed the highest homology, with a maximum similarity of only 98.18%.

[0103] (4) Analysis of the physiological and biochemical characteristics of the strain

[0104] Strain DSF059 can utilize fructose, mannitol, xylose, inositol, sucrose, glucose, and starch as its sole carbon source for growth, with mannitol, xylose, inositol, and fructose being the most suitable carbon sources. Strain DSF059 can utilize sodium nitrate, potassium nitrate, and tyrosine as its sole nitrogen source for growth, with sodium nitrate being the most suitable nitrogen source. The growth temperature of strain DSF059 is 15℃~28℃, and it can grow at 4℃. Strain DSF059 exhibits strong growth on MEA medium containing 3.3% (w / v) sea salt, with growth decreasing sequentially on MEA medium containing 0%~15% NaCl; it shows almost no growth on MEA medium containing 20% ​​NaCl. Halophilic bacteria can be divided into three categories based on their optimal salt requirements: mildly halophilic bacteria (1~5% NaCl), moderately halophilic bacteria (5~20% NaCl), and extremely halophilic bacteria (20~30% NaCl). Based on the growth characteristics of strain DSF059, it should be classified as a mildly halophilic bacterium. The pH tolerance test shows that strain DSF059 can grow on a medium with a pH of 5.0 to 12.0 and has acid and alkali tolerance.

[0105] ① Optimal carbon source utilization experiment

[0106] Experimental Methods: DSF059 cells were inoculated using a three-point inoculation method onto PDA media containing 1% (w / v) glucose, fructose, sucrose, starch, inositol, mannitol, and xylose as the sole carbon source. Three replicates were set up for each medium. After incubation at 28℃ for 7 days, colony diameters were measured and colony morphology was recorded. SPSS 26.0 was used for statistical analysis of the obtained data. Different letters after the data indicate differences at the P < 0.05 level.

[0107] The experimental results are shown in Table 3:

[0108] Table 3. Growth status of DSF059 on different carbon source media

[0109]

[0110] ② Optimal nitrogen source utilization experiment

[0111] Experimental Methods: DSF059 cells were inoculated using a three-point inoculation method onto PDA medium containing 1% (w / v) potassium nitrate, sodium nitrate, urea, and tyrosine as the sole nitrogen source, respectively, with three replicates for each medium. After incubation at 28℃ for 7 days, colony diameters were measured and colony morphology was recorded. SPSS 26.0 was used for statistical analysis of the obtained data. Different letters after the data indicate the difference at the P < 0.05 level.

[0112] The experimental results are shown in Table 4:

[0113] Table 4. Growth status of DSF059 on different nitrogen source media

[0114]

[0115] ③ Temperature tolerance test

[0116] Experimental Methods: DSF059 was inoculated onto PDA medium using a three-point inoculation method and incubated at 4℃, 15℃, 28℃, 37℃, and 45℃, with three replicates at each temperature. After 7 days of incubation, colony diameters were measured and colony morphology was recorded. SPSS 26.0 was used for statistical analysis of the data. Different letters after the data indicate the difference at the P < 0.05 level.

[0117] The experimental results are shown in Table 5:

[0118] Table 5. Growth status of DSF059 at different temperatures

[0119]

[0120] ④ Salinity tolerance test

[0121] Experimental Methods: DSF059 cells were inoculated using a three-point inoculation method onto MEA media containing 3.3% (w / v) sea salt, 0% NaCl, 5% NaCl, 10% NaCl, 15% NaCl, and 20% NaCl, respectively, with three replicates for each medium. After incubation at 28°C for 7 days, colony diameters were measured and colony morphology was recorded. SPSS 26.0 was used for statistical analysis of the data. Different letters after the data indicate differences at the P < 0.05 level.

[0122] The experimental results are shown in Table 6:

[0123] Table 6. Growth status of DSF059 at different salinities

[0124]

[0125] ⑤ Acidity and alkalinity tolerance test

[0126] Experimental Methods: DSF059 was inoculated onto PDA media at pH=5, pH=6, pH=7, pH=8, pH=9, pH=10, pH=11, and pH=12 using a three-point inoculation method, with three replicates for each medium. After incubation at 28℃ for 7 days, colony diameters were measured and colony morphology was recorded. SPSS 26.0 was used for statistical analysis of the obtained data. Different letters after the data indicate the difference at the P<0.05 level.

[0127] The experimental results are shown in Table 7:

[0128] Table 7 Effect of initial pH on the growth of DSF059 strain

[0129]

[0130] ⑥Analysis of the differences in physiological and biochemical characteristics between DSF059 and its closely related Penicillium genus fungi

[0131] Table 8. Analysis of the differences in physiological and biochemical characteristics between DSF059 and its closely related Penicillium fungi.

[0132]

[0133] Note: "+" indicates that it can grow; "-" indicates that it cannot grow; "n" indicates that there is no data.

[0134] In summary, this study determined the taxonomic position of DSF059 based on morphological identification, molecular biological identification, and physiological and biochemical characteristics. According to colony culture and conidial characteristics, and comparative analysis with the *Handbook of Fungal Identification* and *Flora of Chinese Fungi (Volume 35) – Penicillium and Related Sexual Genus (Deluxe Edition)*, strain DSF059 conforms to the growth characteristics of the *Penicillium* genus. However, experimental data indicate that DSF059 differs from currently known *Penicillium* fungi, suggesting that strain DSF059 may be a new species of *Penicillium*.

[0135] (5) Whole genome sequence analysis of the strain

[0136] ① Since prokaryotes provide almost no morphological clues for classification, researchers introduced various genomic distance measures early on as a supplement to the phenotypic identification of pure cultures for classification. The initial judgment was based on the 16S rRNA gene (the international critical value for the 16S rRNA gene sequence similarity of new prokaryotic species is 98.7%). With the development of technology, IJSEM now requires that the publication of new prokaryotic species must provide whole genome data (at least a draft). The gold standard for the identification of new prokaryotic microorganisms is based on the ANI value (average nucleotide identity) of whole genome data being less than 95% and the dDDH value (digital DNA-DNA hybridization) being less than 70% (Chun J, Oren A, Ventosa A, et al. Proposed minimalstandards for the use of genome data for the taxonomy of prokaryotes[J]. International Journal of Systematic and Evolutionary Microbiology, 2018, 68(1):461-466). However, this standard is not entirely applicable to fungi (eukaryotic microorganisms). Compared to bacteria, the greater morphological diversity of fungi underscores the relative importance of morphological characteristics in their taxonomy. With the rise of next-generation sequencing technology and the reduction in data acquisition costs, a large number of fungal genomes are being used in taxonomic research. Among these methods, determining the ANI value is the simplest, as it only requires genome assembly data and does not require precise annotation. This method has been widely used in bacterial species definition and has also been applied to fungal classification in recent years (e.g., Gostinčar, 2020). In 2025, Christophe Lalanne and Philippe Silar developed a software called FungANI (https: / / github.com / podo-gec / fungani) specifically for analyzing the average nucleotide identity (ANI) value between two fungal genomes (Lalanne C, Silar P. FungANI, a BLAST-based program for analyzing average nucleotide identity (ANI) between two fungal genomes, enables easy fungal species delimitation[J]. Fungal Genetics and Biology, 2025, 177.DOI:10.1016 / j.fgb.2025.103969.).According to the software's user guide, the recommended threshold strategy for fungi is that strains of different species have an ANI value <99%, and strains with an ANI value between 99% and 99.5% (a "fuzzy range") need to be assessed for the degree of genetic exchange blockade by combining experimental evidence such as morphological differences and hybridization sterility.

[0137] ②Analysis Method: The applicant uploaded the whole genome sequence data of strain DSF059 to NCBI, obtaining the accession number: JBMNDY000000000. Based on sequence similarity, three Penicillium strains were found to have the highest similarity: Penicillium aquaticum , Penicillium macrosclerotiorum and Penicillium johnpittii Then, when searching for the whole genome data of these three strains on NCBI, only [data missing]. Pencils macrosclerotium Strain IBT 26536 has whole genome data. This data was then downloaded and uploaded to FungANI (https: / / github.com / podo-gec / fungani) for computation and analysis. The results showed... Penicillium msp.DSF059 and recent strains Penicillium macrosclerotiorum The ANI value of strain IBT 26536 (NW 026643081) was 92.978%, which was lower than the 99% threshold, thus identifying strain DSF059 as a new species of Penicillium.

[0138] Experimental Example 1: Marine-derived Penicillium DSF059 inhibits the growth of Staphylococcus aureus.

[0139] 1.1 Test Methods

[0140] Single colonies of Staphylococcus aureus were inoculated into liquid LB medium (lysozyme broth, prepared according to *Molecular Cloning: A Laboratory Manual* (J. Sambrook, DW. Russell). Each liter of medium should contain: 10g tryptone, 5g yeast extract, and 10g NaCl in 950ml deionized water. Shake until the solute dissolves, adjust the pH to 7.0 with 5mol / L NaOH, and bring the volume to 1L with deionized water. Autoclave at 15psi for 21 min). The culture was incubated at 37°C with shaking at 180 rpm for 18 h to obtain the seed culture. The bacterial count was performed using a hemocytometer, and the bacterial concentration was adjusted to 1×10⁻⁶ with sterile water. 7 CFU / mL pathogen suspension. Spread 100 μL of pathogen suspension on LB agar plates, and place 5 mm diameter antagonistic fungal discs at equal intervals on the plates. Incubate at 28 °C for 18 h, and then observe the presence and size of inhibition zones. Each treatment is repeated 3 times.

[0141] 1.2 The results of the flat-plate confrontation show that marine-origin Penicillium Penicillium m sp. DSF059 can inhibit the growth of Staphylococcus aureus, with an inhibition zone of over 2.1 cm (see details). Figure 3 ).

[0142] Experimental Example 2: Screening of fermentation conditions for marine-derived Penicillium DSF059

[0143] 2.1 Penicillium culture at 37℃ and preparation of seed culture: Penicillium The m sp. DSF059 strain was inoculated into an Erlenmeyer flask containing PDA solid medium and cultured at 28°C for 5 days. When spores grew on the PDA solid medium, 50 mL of sterile 0.05% Tween 80 was added, and the mixture was shaken at 110 rpm for 10 min to obtain a spore suspension as seed culture.

[0144] 2.2 Preparation of solid fermentation medium: Prepare four solid fermentation mediums for rice, oats, potatoes and Cheriois according to the above formula, and sterilize them at 121℃ for 30 min.

[0145] 2.3 Fermentation products: Take 2 mL of the above seed liquid and add it to four kinds of solid fermentation medium, namely rice, oats, potatoes and Cheriois, respectively. Then, let it ferment in a static environment at 28℃ and under natural light for 30 days to obtain the fermentation products.

[0146] 2.4 Ethyl acetate fermentation broth: The fermentation product obtained from the fermentation culture was soaked in 60 mL of ethyl acetate for 12 h, then cultured with shaking at 110 rpm for 10 min, and the filtrate was obtained by vacuum filtration. The filtrate was subjected to vacuum rotary evaporation at a vacuum degree of 0.06 MPa and a temperature of 32 °C. The extract of the filtrate obtained by vacuum rotary evaporation was extracted with 5 mL of methanol with a volume concentration of 80% to obtain ethyl acetate fermentation broth.

[0147] 2.5 Antibacterial Activity Test: In a clean bench, sterile 6mm filter paper was placed on a sterile glass slide. 5μL and 10μL of DSF059 ethyl acetate fermentation broth, along with 5μL and 10μL of positive controls, were added to the filter paper, and the slides were allowed to air dry. The positive control was a 4mg / mL chloramphenicol solution prepared with 50% ethanol. Then, 100μL of a 1×10⁻⁶ solution was added. 7 A cfu / mL Staphylococcus aureus bacterial suspension was evenly spread on an LB agar plate. Using sterile forceps, the filter paper discs were placed on the LB agar plate and carefully pressed to ensure they adhered to the surface of the culture medium. The LB agar plates were then inverted and incubated at 37°C for 16-18 hours. The presence and size of inhibition zones were observed. Each treatment was repeated three times.

[0148] 2.6 The results of the fermentation condition screening showed that 5 μL of 4 mg / mL chloramphenicol solution produced an inhibition zone with a diameter of 2.5 cm, and 10 μL of 4 mg / mL chloramphenicol solution produced an inhibition zone with a diameter of 2.7 cm, while marine-derived Penicillium... Penicillium The ethyl acetate fermentation broths of rice, oats, potatoes, and Cheriois solid-state fermentation medium prepared with msp.DSF059 all inhibited the growth of Staphylococcus aureus. The diameter of the inhibition zone produced by 5 μL of ethyl acetate fermentation broth prepared from each solid-state fermentation medium is shown in (see details). Figure 4 The sizes of the samples were 1.9cm, 1.5cm, 1.3cm and 1.6cm, respectively. Among them, the fermentation product of rice solid fermentation medium had the most significant inhibitory effect on Staphylococcus aureus, and could obtain more target products. It was also simple to operate, low in cost, and had good application value.

[0149] Experimental Example 3: Detection of the inhibitory activity of marine-derived Penicillium DSF059 against plant pathogens.

[0150] 3.1 Test Materials

[0151] Plant pathogens: *Coffee leaf blight pathogen*, *Cercospora* spp. ( Diaporthe etinsideae ), the pathogen of coffee brown spot disease, *Neoprothiolane spp.* Neopestalotiopsis aotearoa Grape gray mold pathogen Botrytis cinerea ( Botrytis cinerea ), the pathogen of Phellodendron amurense brown spot disease, *Ichthyophthirius multifiliis* (Guilin black spores) Nigrospora guilinensis s), Alternaria, the pathogen of citrus black rot ( Alstroemeria Alternaria ), Phytophthora capsici, the pathogen of Phytophthora capsici ( Phytophthora capsicum ), the pathogen of pecan leaf blight, *Pseudomonas mesenteroides* ( Diaporthe pseudophoenix ), the pathogen of coffee black spot disease, Cladosporium hilkelli ( Collariella hilkhuijsenii ) and four pathogens isolated from coffee leaf diseases (Bryophyllum urticaria) Phlebiopsis brown , Porphyromonas subulata Fomitopsis submeliae Coprinus rhombosporus Coprinopsis rhombisporoides Broom molds Spider webs The aforementioned plant pathogens were purchased from strain sales platforms or isolated from plant diseases.

[0152] 3.2 Test Methods

[0153] The plate confrontation method was used. The specific steps were as follows: first, the plant pathogen was inoculated onto PDA medium and cultured for 3 days at 28℃ in the dark, then mycelial cakes (φ=5mm) were collected. A cross was marked on the back of the PDA plate, and marine-derived *Penicillium* was inoculated at four points equidistant from each end of the cross using a spot-inoculation method. Penicilliumm sp. DSF059, then inoculate the pathogen disc (φ=5mm) in the center of the culture medium, repeating each treatment 3 times, with no inoculation. Penicillium m sp.DSF059, plates inoculated only with the pathogen were used as controls. The plates were incubated upside down at room temperature for 7 days. The diameter of the pathogen was measured using the cross-hatching method, and the inhibition rate was calculated.

[0154] Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in treatment group) / Coronavirus colony diameter in control group × 100%;

[0155] 3.3 Test Results

[0156] As shown in Table 9:

[0157]

[0158] The results showed that strain DSF059 had an inhibitory effect on all twelve plant pathogens, with inhibition rates all above 30%. Among them, strain DSF059 had the highest inhibition rate against the coffee black spot pathogen, Cladosporium hilkerii.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a marine-derived Penicillium sp. in the preparation of a medicament for the treatment of a pathogenic fungus, characterized in that, The pathogenic bacteria are coffee leaf disease pathogenic bacteria; The coffee leaf disease pathogen is a species of genus Pseudocercospora Coprinopsis rhombisporoides ). The marine source Penicillium is named as Penicilliu m sp. DSF059, which is preserved in the China Center for Type Culture Collection, located at No. 299, Bajiyuan Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is November 23, 2023; the preservation number is CCTCC NO: M 20232310.

2. Use according to claim 1, characterized in that, The preparation method of the ethyl acetate fermentation liquor of the marine Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: inoculate the marine Penicillium strain into PDA solid culture medium, and place it in a temperature of 25-30 DEG C for 3-8 days to activate the strain; add sterile Tween 80 with a volume fraction of 0.05% to the activated strain, and oscillate the strain at 110 r / min for 10 min to prepare a spore suspension as a seed liquid; (2) Fermentation product: take the seed liquid with an inoculation amount of 5% by volume, inoculate it into a rice solid fermentation culture medium, and carry out static fermentation culture at 28 DEG C and natural light for 30 days to obtain a fermentation product; (3) Ethyl acetate fermentation liquor: after the fermentation product obtained in step (2) is soaked with ethyl acetate for 12 h, oscillate the fermentation product at 110 r / min for 10 min, and then carry out suction filtration to obtain a filtrate; the filtrate is subjected to rotary evaporation under reduced pressure, the filtrate extract obtained by rotary evaporation under reduced pressure is extracted with methanol with a volume concentration of 80% for 80 min, and the ethyl acetate fermentation liquor of the marine Penicillium is obtained.

3. Use according to claim 2, characterized in that, The preparation method of the ethyl acetate fermentation liquor of the marine Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: inoculate the marine Penicillium strain into PDA solid culture medium, and place it in a temperature of 25-30 DEG C for 3-8 days to activate the strain; add sterile Tween 80 with a volume fraction of 0.05% to the activated strain, and oscillate the strain at 110 r / min for 10 min to prepare a spore suspension as a seed liquid; (2) Fermentation product: take the seed liquid with an inoculation amount of 5% by volume, inoculate it into a rice solid fermentation culture medium, and carry out static fermentation culture at 28 DEG C and natural light for 30 days to obtain a fermentation product; (3) Ethyl acetate fermentation liquor: after the fermentation product obtained in step (2) is soaked with ethyl acetate for 12 h, oscillate the fermentation product at 110 r / min for 10 min, and then carry out suction filtration to obtain a filtrate; the filtrate is subjected to rotary evaporation under reduced pressure, the filtrate extract obtained by rotary evaporation under reduced pressure is extracted with methanol with a volume concentration of 80% for 80 min, and the ethyl acetate fermentation liquor of the marine Penicillium is obtained.

4. Use according to claim 2, characterized in that, The PDA solid culture medium in step (1) is composed of the following components per liter: potato 150-250 g, glucose 15-40 g, agar 10-30 g, old seawater 300-500 mL, and deionized water in the remainder.

5. Use according to claim 2, characterized in that, Step (1) The concentration of spore suspension in the spore suspension was 1.0 x 10 6 ~1.0 x 10 10 cfu / mL.

6. Use according to claim 2, characterized in that, The rice solid fermentation culture medium in step (2) is composed of the following components: rice 8-15 g, old seawater 5-20 mL, and distilled water 8-30 mL.

7. Use according to claim 2, characterized in that, The parameters for rotary evaporation under reduced pressure in step (3) are a vacuum degree of 0.02-0.08 MPa and a temperature of 30-35 DEG C.

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