Penicillium oxalicum and application thereof

Abstract

The invention provides penicillium oxalicum and application thereof, and relates to the technical field of microorganisms. A new biocontrol bacterium Penicillium oxalicum HN-7 is obtained through screening, and it is proved that the Penicillium oxalicum HN-7 has an inhibition effect on various plant disease pathogenic bacteria such as phytophthora nicotianae, pythium ultimum, botrytis cinerea and ralstonia solanacearum, has broad-spectrum antibacterial performance, can be stably colonized in plant rhizomes, can efficiently prevent and control various diseases such as bacterial wilt, and has good application prospects. A novel biocontrol strain is provided for biological control pesticides.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and particularly to a strain of Penicillium oxalicum and its applications. Background Art

[0002] Globally, yield losses caused by pathogens and pests in crops are a serious problem, posing a major threat to food security and economic development. It is estimated that this loss ranges from 17% to 30%. Plant pathogens include viruses, bacteria, oomycetes, and fungi, etc., which infect plants in different ways, affecting crop growth and yield. Among agricultural disease control measures such as breeding of disease-resistant varieties, biological control, and chemical control, chemical control is still the main means of controlling plant diseases at present, but it also causes problems such as drug resistance, pesticide residues, and environmental pollution. Compared with traditional chemical control, biocontrol microorganisms have the advantages of environmental friendliness, no chemical residues, and high biological safety, and have become the core strategy for the green prevention and control of plant fungal diseases. At present, it is urgent to strengthen the research and development of biocontrol methods and products.

[0003] Using beneficial microorganisms for crop disease control is one of the important development directions of green prevention and control. Trichoderma ( Trichoderma spp. ), Penicillium ( Penicillium spp. ), Bacillus ( Bacillus spp. ), etc. have good effects in inhibiting pathogenic fungi and bacteria. The action mechanisms of biocontrol strains mainly include antibiotic action, competition for colonization sites, nutrients or minerals, parasitism, and bacteriophage action. Among them, exerting antibiotic action against pathogenic fungi by secreting antifungal metabolites (AFMs) is the most common strategy. The antifungal metabolites studied more currently mainly include antibiotics, enzymes, and volatiles.

[0004] Using natural microbial antibacterial extracts is one of the safe means to reduce or replace fungicides. Microbial volatile compounds (VOCs) are small signaling molecules (<C15) produced by organisms such as bacteria and fungi, with characteristics such as low molecular mass (<300 Da), high vapor pressure, and low boiling point. Volatile compounds are usually lipophilic compounds. These molecules are volatile at 25°C and 1 atm pressure and can easily diffuse through the water- and gas-filled pores in the soil and rhizosphere environment, and have the effects of promoting plant growth, inhibiting nematode activity, inducing disease resistance in plants, and inhibiting the growth and spore germination of pathogenic bacteria. Due to the characteristics of high-efficiency antibacterial, rich components, easy volatility, and no residue on the plant surface of volatile substances, they play an important role in agriculture, environment, and healthcare. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide a novel biocontrol bacterium, *Penicillium oxalicum* (…). Penicillium sorrel HN-7 was developed, and its main antibacterial volatile substances were identified as 1-octen-3-ol and linalool. It has broad-spectrum antibacterial activity against a variety of pathogens, and the prepared Penicillium oxalate agent has a significant control effect on Ralstonia solanacearum.

[0006] On the one hand, this application provides a strain of Penicillium oxalate ( Penicillium oxalicum HN-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167.

[0007] Preferably, the Penicillium oxalate ( Penicillium oxalicum HN-7 has antibacterial properties and can be stably colonized in plant roots and stems.

[0008] In a preferred embodiment, the plant is a tomato. This application uses a tomato as an example for experimentation, but those skilled in the art will recognize its applicability to other plants.

[0009] On the other hand, this application also provides a microbial preparation containing the aforementioned Penicillium oxalate.

[0010] Preferably, the *Penicillium oxalate* includes *Penicillium oxalate* and its related products, such as: A1) Penicillium oxalate; A2) Penicillium oxalate inoculum; A3) Penicillium oxalate-containing dead bacterial suspension; A4) Penicillium oxalate hyphae; A5) Penicillium oxalate spores.

[0011] In a preferred embodiment, the viable count of Penicillium oxalate HN-7 in the bacterial agent is ≥5 billion / gram.

[0012] On the other hand, this application also provides the use of the aforementioned Penicillium oxalate or the aforementioned microbial preparation in the preparation of antibacterial agents.

[0013] Preferably, the antimicrobial agent comprises volatile metabolites produced by Penicillium oxalate.

[0014] More preferably, the volatile metabolites include one or more of anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

[0015] Furthermore, the antibacterial agent includes one or more of anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

[0016] Preferably, the antibacterial agent comprises 1-octen-3-ol and / or linalool.

[0017] This application marks the first discovery of Penicillium oxalate ( ). Penicillium oxalicum HN-7 can produce anisole, 1-octen-3-ol, linalool, and m-phenylenediamine, which also confirms that anisole, 1-octen-3-ol, linalool, and m-phenylenediamine are produced by *Penicillium oxalate*. Penicillium oxalicum The main active ingredient responsible for the antibacterial effect of HN-7.

[0018] Preferably, the antimicrobial agent can be used to inhibit the growth activity of pathogenic bacteria, pathogenic oomycetes, and / or pathogenic fungi.

[0019] The pathogenic bacteria include Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum ); The pathogenic oomycetes include Phytophthora nicotineae (… Phytophthora nicotianae ), ultimate phytosis ( Pythium last ), Pythium spp. ( Pythium aphanidermatum One or more of the following; The pathogenic fungi include Botrytis cinerea. (Botrytis cinerea) Fusarium graminearum ( Fusarium grasses ), cucumber anthracnose fungus ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cereal Rice blast fungus ( Magnaporthe oryzae) Alternaria ( Alternaria alternata One or more of the following.

[0020] On the other hand, this application also provides a method for preparing an antibacterial agent, the method comprising: culturing the Penicillium oxalate or the microbial preparation.

[0021] Preferably, those skilled in the art can culture the Penicillium oxalate using conventional methods.

[0022] In a preferred embodiment, the method includes: inoculating the Penicillium oxalate in a culture medium and culturing it at 25°C-30°C for 2-3 days.

[0023] Preferably, the preparation method further includes collecting volatile metabolites of Penicillium oxalate.

[0024] Preferably, the preparation method further includes a purification step.

[0025] Ultimately, one or more antibacterial active substances can be obtained from anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

[0026] In a preferred embodiment, the culture medium may be a PDA culture medium.

[0027] On the other hand, this application also provides an antibacterial agent prepared by the method described above.

[0028] Furthermore, the antibacterial agent includes one or more of anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

[0029] On the other hand, this application also provides the application of the aforementioned Penicillium oxalate, the aforementioned microbial preparation, or the aforementioned antibacterial agent in the prevention and control of plant diseases and / or the inhibition of pathogen growth.

[0030] Furthermore, the pathogen is a pathogenic bacterium, a pathogenic oomycete, and / or a pathogenic fungus.

[0031] Furthermore, the pathogens include Phytophthora tobaccoii (… Phytophthora nicotianae ), ultimate humic acid ( The last Pythium Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Ralstonia solanacearum ( Ralstonia solanacearum ), Pythium spp. ( Pythium aphanidermatum Fusarium graminearum ( ), Fusarium gramineae ), cucumber anthracnose bacteria ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cereale ), rice blast fungus ( Magnaporthe rice Alternaria ( Alternaria alternata One or more of the following.

[0032] Furthermore, the plant disease is a disease caused by pathogens, and the plant disease includes one or more of the following: black shank, damping-off, wilt, gray mold, bacterial wilt, Fusarium head blight, root rot, anthracnose, sheath blight, rice blast, and red spot disease.

[0033] Preferably, the plant disease is a disease caused by pathogens, including one or more of the following: tobacco black shank, crop damping-off and wilt, gray mold of grapes, cucumbers, tomatoes, strawberries, etc., bacterial wilt of solanaceous crops, wheat scab, tobacco root rot, cucumber anthracnose, wheat sheath blight, rice blast, and tobacco red spot disease.

[0034] Among them, Phytophthora indica ( Phytophthora nicotianae ) is the pathogen of tobacco black shank; Pythium cerevisiae ( The last Pythium Botrytis cinerea ( ) is the pathogen of crop damping-off and wilt; Botrytis cinerea ( ) Botrytis cinerea It can infect hundreds of plants, including grapes, strawberries, tomatoes, cucumbers, and flowers, causing rot, soft rot, and the formation of a gray mold layer on the surface; it is the pathogen of gray mold. Ralstonia solanacearum (also known as Bacillus licheniformis) Ralstonia solanacearum This can cause tomatoes, peppers, tobacco, potatoes, peanuts, and other crops to wilt and die rapidly. A milky white bacterial ooze can be seen when the stem base is cut open; this is the pathogen of bacterial wilt. Pythium spp. (…) Pythium aphanidermatumThis fungus causes damping-off and fruit rot (soft rot) in cucurbits (such as cucumbers and watermelons), solanaceous crops, and other crops; Fusarium graminearum ( Fusarium gramineae Anthracnose primarily causes Fusarium head blight in cereal crops such as wheat, barley, and corn, as well as root rot in tobacco. Cucumber anthracnose (… Colletotrichum orbiculare Anthracnose is the pathogen of cucumber anthracnose, and it also damages cucurbitaceous crops such as watermelon, melon, and pumpkin, forming round or oval sunken brown lesions on leaves, stems, and fruits; wheat sheath blight pathogen (… Rhizoctonia cereale It mainly damages the leaf sheaths and stems of crops such as wheat and barley, causing bud rot, seedling blight, flower stalk rot, and whiteheads. It is the pathogen of sheath blight; rice blast fungus (… Magnaporth rice Alternaria alterniflora (Alternaria) is the main pathogen causing rice blast, leading to spindle-shaped spots on leaves, neck blast resulting in whiteheads, and grain blast causing shriveled grains. In severe cases, it can cause the entire field to wither and die, reducing yield. Alternaria alternata It mainly damages tobacco leaves, causing spots, necrosis, and premature aging, leading to a reduction in effective tobacco leaves and a decline in quality. It is the pathogen of tobacco red spot disease.

[0035] Preferably, the inhibition rate of the pathogen can reach 80% or more; optionally, the inhibition rate of the pathogen can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more.

[0036] On the other hand, this application also provides a pesticide, which includes the aforementioned Penicillium oxalate, the aforementioned microbial preparation, or the aforementioned antibacterial agent.

[0037] Those skilled in the art can select the microbial content and antibacterial agent content in pesticide products according to the actual situation.

[0038] In a preferred embodiment, the pesticide contains ≥50 CFU / g of viable Penicillium oxalate HN-7, and the addition amount is greater than or equal to 0.1 g, resulting in a control effect of greater than or equal to 68% against Ralstonia solanacearum.

[0039] Preferably, the amount added can be 0.1 g-10 g, and the amount added can be any value among 0.1 g, 0.2 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, and 10 g.

[0040] Preferably, the prevention and control effect is greater than or equal to any one of the following values: 68%, 70%, 77%, 79% or higher.

[0041] In a preferred embodiment, the concentration of the antibacterial agent in the pesticide is greater than or equal to 0.5 times EC50. 50 .

[0042] Preferably, when controlling Botrytis cinerea, the concentration of the antibacterial agent in the pesticide can be greater than or equal to 0.5 times, 1 time, 1.5 times, 2 times, 2.5 times, or 3 times the EC50 concentration. 50 .

[0043] On the other hand, this application also provides the use of anisole, 1-octen-3-ol, linalool and / or m-phenylenediamine in the prevention and control of plant diseases and / or the inhibition of pathogen growth.

[0044] Furthermore, the pathogen is a pathogenic bacterium, a pathogenic oomycete, and / or a pathogenic fungus.

[0045] Furthermore, the pathogens include Phytophthora tobaccoii (… Phytophthora nicotianae ), ultimate humic acid ( The last Pythium Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Ralstonia solanacearum ( Ralstonia solanacearum ), Pythium spp. ( Pythium aphanidermatum Fusarium graminearum ( ), Fusarium gramineae ), cucumber anthracnose bacteria ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cereale Rice blast fungus ( Magnaporthe rice Alternaria ( Alternaria alternata One or more of the following.

[0046] Furthermore, the plant disease is a disease caused by pathogens, and the plant disease includes one or more of the following: black shank, damping-off, wilt, gray mold, bacterial wilt, Fusarium head blight, root rot, anthracnose, sheath blight, rice blast, and red spot disease.

[0047] The present invention has the following beneficial effects: 1. In this application, a new biocontrol bacterium, *Penicillium oxalicum*, was obtained through screening. Penicillium oxalicum HN-7 was developed and demonstrated to have inhibitory effects on various plant pathogens such as Phytophthora indicum, Pythium cerevisiae, Botrytis cinerea, and Ralstonia solanacearum. It has broad-spectrum antibacterial properties and can stably colonize in plant rhizomes, effectively controlling various diseases such as bacterial wilt. It provides a new biocontrol strain for biological control pesticides. 2. This application further studies the antibacterial properties of the biocontrol bacteria, clarifying that its effective antibacterial components are anisole, 1-octen-3-ol, linalool, and m-phenylenediamine. Among them, 1-octen-3-ol and linalool have inhibitory effects on a variety of common plant pathogens, such as Phytophthora tobaccoii, Botrytis cinerea, Ralstonia solanacearum, Pythium spp., Fusarium graminearum, Anthracnose fungus of cucumber, Rhizoctonia solani of wheat, Bacillus thuringiensis of rice, and Alternaria alternata. It can be widely used in the prevention and control of plant diseases, and is safe and efficient, providing a new effective component for the pesticide field. Attached Figure Description

[0048] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 Image showing the colony morphology of bacterium HN-7 on a PDA plate; Figure 2 This is a phylogenetic tree diagram of bacterium HN-7; Figure 3 The image shows the results of a plate-to-plate experiment of Penicillium oxalicum HN-7 with Phytophthora nicotina, Pythium cerevisiae, Botrytis cinerea, and Ralstonia solanacearum. Figure 4 Statistical chart showing the inhibition rate of Penicillium oxalicum HN-7 against Phytophthora nicotina, Pythium terrestris, and Botrytis cinerea; Figure 5 The diagram shows the antibacterial effects of four volatile compounds on Botrytis cinerea. Figure 6 A statistical chart showing the inhibition rates of four volatile compounds against Botrytis cinerea; Figure 7 The diagram shows the antibacterial effects of linalool and 1-octen-3-ol against common pathogenic fungi. Figure 8 Regression equations for the toxicity of linalool and 1-octen-3-ol against *Pythium spp.* and *Botrytis cinerea*, where (A) is the regression equation for the toxicity of linalool against *Pythium spp.*; (B) is the regression equation for the toxicity of linalool against *Botrytis cinerea*; (C) is the regression equation for the toxicity of 1-octen-3-ol against *Pythium spp.*; and (D) is the regression equation for the toxicity of 1-octen-3-ol against *Botrytis cinerea*. Figure 9 The diagram shows the control effects of linalool and 1-octen-3-ol on Botrytis cinerea infection in grapes. Figure 10 The image shows the effect of Penicillium oxalate HN-7 inoculant on controlling bacterial wilt under potted conditions on day 11. In this image, (A) is a top view of each group of potted plants, and (B) is a front view of each group of potted plants. Figure 11The graph shows the colonization effect of Penicillium oxalate HN-7 in tomato rhizomes on day 11 under potted conditions. (A) is the relative biomass of Penicillium oxalate in the roots, and (B) is the relative biomass of Penicillium oxalate in the stems.

[0049] Preservation of biological materials: A strain of Penicillium oxalicum ( Penicillium oxalicum HN-7 was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, 100101, China. Detailed Implementation

[0050] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0051] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0052] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0053] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0054] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0055] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0056] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0057] The culture media involved in the following examples are as follows: PDA liquid culture medium: 6 g / L potato extract powder, 20 g / L glucose, 1 L distilled water; PDA plate medium: 6 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, 1 L distilled water; NA plate medium: peptone 10 g / L, sodium chloride 5 g / L, beef extract powder 3 g / L, agar 20 g / L, distilled water 1 L; NB liquid culture medium: 10 g / L peptone, 5 g / L sodium chloride, 3 g / L beef extract powder, 1 L distilled water; Preparation method of OA medium: Boil 30 g of oats in distilled water for 15-20 min, filter with gauze and bring the medium volume to 1 L, add 16-18 g of agar and stir well. Phytophthora indicum ( Phytophthora nicotianae ), ultimate humic acid ( Pythium ultimum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Ralstonia solanacearum ( Ralstonia solanacearum ), Pythium spp. ( Pythium aphanidermatum Fusarium graminearum ( ), Fusarium graminearum ), cucumber anthracnose bacteria ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cerealis Rice blast fungus ( Magnaporthe oryzae Alternaria ( Alternaria alternata All of them were purchased from the National Agricultural Environmental Microbial Germplasm Resource Bank (Shandong).

[0058] Example 1: Isolation and Identification of Biocontrol Bacterium HN-7 1. Strains Isolation Strain HN-7 was isolated from saline-alkali soil in mangrove forests of Hainan. The specific isolation method was as follows: 0.1 g of soil sample was placed in a sterile 2 mL centrifuge tube, and approximately 1.5 mL of PDA liquid culture medium was pipetted in and mixed thoroughly to obtain the stock solution. This stock solution was then diluted with sterile water at a dilution factor of 10:10. -2 and 10 -3 Take 100 μL of the stock solution and the diluted sample, and spread them evenly on PDA plates. Repeat the process three times for each gradient. Incubate at 28℃ for 2-3 days until single colonies grow. Pick colonies with different shapes, sizes, colors, etc., number them, and purify them for further culture.

[0059] 2. Morphological observation The strain numbered HN-7 was inoculated into PDA solid medium and cultured at 28°C for 3 days. Figure 1 As shown, HN-7 colonies on PDA medium are bluish-green to dark green in the center and white to grayish-white at the edge, which gradually becomes blurred, forming a radial growth pattern. The colony surface is fluffy and relatively loose, especially the central part where the hyphae are more dense.

[0060] 3. Molecular identification Genomic total DNA was extracted from strain HN-7 using the CTAB method, and the ITS1 / ITS4 genes were amplified and sequenced. The PCR amplification system was 50 μL: 2 μL template DNA (36 ng / μL), 2 μL each of primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') (10 μmol / L) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (10 μmol / L), 25 μL 2×PhantaFlash Mix (Dye Plus), and 19 μL double-distilled water. The PCR amplification program was: 95℃ for 5 min, 95℃ for 15 s, 56℃ for 15 s, 72℃ for 10 s, for 34 cycles; 72℃ for 10 min. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequence of the ITS1 / ITS4 fragment of HN-7 was 562 bp in length, as shown in SEQ ID No. 1.

[0061] Fourteen standard strain sequences were obtained from the NCBI (GenBank) database. MEGA 11 was used to analyze the ITS1 / ITS4 sequences of the isolated strains and reference strains, and a Neighbor-joining phylogenetic tree was constructed. Figure 2 As shown. It can be seen that the HN-7 provided by this invention and Penicillium oxalicum The strain is most closely related to the bacteria. Based on the colony characteristics of this strain on petri dishes, the isolated strain HN-7 of this invention was identified as *Penicillium oxalate*. Penicillium oxalicum The sample was named Penicillium oxalate HN-7 and was deposited on August 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167.

[0062] Example 2: Determination of antibacterial activity of Penicillium oxalate HN-7 1. Plate invert test of Penicillium oxalate HN-7 against Phytophthora nicotine, Pythium cerevisiae, Botrytis cinerea, and Ralstonia solanacearum First, *Penicillium oxalicum* HN-7, which was in a well-activated state as described in Example 1, was selected. A 5mm mycelial disc was taken from the edge of the hyphae using a punch and inoculated into the center of a PDA medium. The medium was then incubated at 28°C for 3 days. *Phytophthora nicotinae*, *Pythium cerevisiae*, or *Botrytis cinerea* were used as pathogens. A 5mm pathogenic mycelial disc was inoculated into the center of a PDA plate. The plates inoculated with *Phytophthora nicotinae*, *Pythium cerevisiae*, or *Botrytis cinerea* were then capped and inverted onto the plate inoculated with *Penicillium oxalicum* HN-7. The plates were sealed with Parafilm. The PDA plate inoculated with *Penicillium oxalicum* HN-7 was placed on top, followed by the plate inoculated with the pathogenic pathogens. A blank PDA plate was used as a control instead of the plate inoculated with *Penicillium oxalicum* HN-7. Each treatment was repeated three times. The plates were then incubated in the dark at 25°C for 2 days. The results were observed as follows: Figure 3 As shown. The antibacterial rate was calculated using the following formula, and the results are as follows. Figure 4 As shown.

[0063] Colony diameter (cm) = Measured colony diameter - 0.5; Inhibition rate (%) = (diameter of blank control colonies - diameter of treated colonies) / diameter of blank control colonies × 100.

[0064] In this embodiment, *Ralstonia solanacearum* was used as the pathogen. Strands were placed on NA plates and cultured at 28°C for 12 h. Single colonies were then picked and cultured in NB liquid medium at 28°C and 180 rpm for 12 h. After centrifugation at 6000 rpm for 3 min, the medium was removed, and the bacterial suspension was diluted to OD using sterile water. 600The value was 0.3. Using a sterile spray bottle, the *Ralstonia solanacearum* suspension was evenly sprayed onto an NA plate. Then, a 5 mm diameter *Penicillium oxalicum* HN-7 mycelial block was placed in the center of the PDA plate. Both plates were inverted and sealed. The plate inoculated with *Penicillium oxalicum* HN-7 was placed on top and incubated at 28°C for 2 days. The results were observed as follows. Figure 3 As shown.

[0065] Figure 3 The results showed that *Penicillium oxalate* HN-7 had inhibitory effects on *Phytophthora nicotine*, *Pythium cerevisiae*, *Botrytis cinerea*, and *Ralstonia solanacearum*. Figure 4 The results showed that *Penicillium oxalate* HN-7 inhibited the growth of *Phytophthora indicum*, *Pythium terrestris*, and *Botrytis cinerea* by 87.32%, 81.87%, and 99.45%, respectively. These results demonstrate that the volatiles produced by *Penicillium oxalate* HN-7 can effectively inhibit the growth of *Phytophthora indicum*, *Pythium terrestris*, *Botrytis cinerea*, and *Ralstonia solanacearum*.

[0066] 2. Determination of volatile components in Penicillium oxalate HN-7 Accurately pipette 10 mL of undried PDA medium into a 20 mL sterile brown headspace vial. Tilt the vial at a 15-degree angle and allow it to cool and solidify. Inoculate a 5 mm mycelial disc of *Penicillium oxalate* HN-7 onto the slant of the medium, quickly cover it, and incubate the headspace vial in the dark at 28°C for 5 days. To eliminate interference from volatiles released from the medium and the loss of the extraction head coating on the identification results of bacterial volatiles, a blank control was set up without coating the bacterial suspension. Each sample was replicated in triplicate. After equilibrating the incubated headspace vial at room temperature for 20 min, insert the aged extraction head (50 / 70 μm DVB / CAR / PDMS, Supelco, USA) into the upper 1 / 3 of the headspace vial and perform headspace sampling at room temperature for 30 min. After sampling, retract the extraction head, immediately insert it into the gas chromatograph injection port, desorb at 250°C for 5 min, and perform GC / MS analysis.

[0067] GC / MS analysis conditions: DB-5MS flexible capillary column, 30 m × 0.25 mm × 0.25 μm; injection port 250℃; splitless mode; carrier gas 99.999% helium, flow rate 1.2 mL / min; column oven: initial temperature 45℃, hold for 1 min, ramp to 180℃ at a rate of 5℃ / min, then ramp to 280℃ at a rate of 20℃ / min, hold for 5 min, total run time 38 min; EI ion source, electron energy 70 eV; ion source temperature 230℃, quadrupole temperature 150℃, transfer line temperature 280℃; full scan mode, scan range 30–400 m / z.

[0068] The volatile components of Penicillium oxalate HN-7 were identified by GC-MS analysis, and four main compounds, namely anisole, 1-octen-3-ol, linalool and m-phenylenediol, were screened out (as shown in Table 1).

[0069] Table 1 Volatile Components

[0070] 3. Antibacterial test of Penicillium oxalate HN-7 volatiles against Botrytis cinerea Purchase volatile organic compounds (anisole, 1-octen-3-ol, linalool, and m-phenylenediamine) identified by GC-MS analysis, and use Botrytis cinerea as an example to test the antibacterial activity of the volatile organic compounds.

[0071] The antibacterial activity against *Botrytis cinerea* strain was evaluated using a fumigation method. *Botrytis cinerea* strains (5 mm mycelial cakes) were inoculated onto PDA plates. Then, a 10 mm × 10 mm sterile filter paper was placed in the center of the inside of the petri dish lid, and 6.5 μL of a volatile compound (final concentration 100 μL / L) was added to the filter paper. The petri dish lid was quickly closed, and the plate was sealed with Parafilm. Filter paper without the volatile compound served as a control. Each treatment was performed in triplicate. The plates were incubated in the dark at 25°C for 3 days, and the diameter (mm) of the bacterial colonies was accurately measured. The inhibition rate was calculated as follows. The antibacterial effect is shown in the figure. Figure 5 Table 2 and Figure 6 As shown.

[0072] Colony diameter (cm) = Measured colony diameter - 0.5; Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100.

[0073] Table 2

[0074] Note: In the table, "a", "b", and "c" indicate that the differences are significant according to Duncan's new multiple range test. P <0.05).

[0075] Depend on Figure 5 The results showed that anisole, 1-octen-3-ol, linalool, and m-phenylenediamine all had inhibitory effects on Botrytis cinerea.

[0076] From Table 2 and Figure 6 The results showed that 1-octen-3-ol and linalool were at 0.107 μL / cm 3 The inhibitory effect against Botrytis cinerea was the highest, with an inhibition rate of 100%, which was significantly better than that of m-phenylenediamine and anisole.

[0077] 4. Effects of Penicillium oxalate HN-7 volatiles 1-octen-3 alcohol and linalool on the growth of common pathogenic fungi. In this embodiment, the inhibitory effects of 1-octen-3 alcohol and linalool on common pathogens were further tested. These common pathogens included *Phytophthora tobaccois* (pathogen of tobacco black shank), *Botrytis cinerea* (pathogen of gray mold in grapes, cucumbers, tomatoes, strawberries, etc.), *Ralstonia solanaceae* (pathogen of bacterial wilt in solanaceous crops), *Pythium spp.* (pathogen of damping-off in solanaceous and cucurbitaceous crops), *Fusarium graminearum* (pathogen of wheat scab and tobacco root rot), *Anthracnose fungus of cucumbers*, *Sheath blight fungus* of wheat, *Blastophytes oryzae* of rice, and *Alternaria alternata* (pathogen of tobacco red spot disease). The experimental methods are as follows: Dissolve 6 μL each of volatiles (1-octen-3 alcohol or linalool) in 9 mL of PDA or OA medium. Pour 9 mL of PDA or OA medium onto the other side of the plate, allow it to cool and solidify, and then inoculate with 5 mm pathogenic fungal discs. *Botrytis cinerea*, *Pythium spp.*, *Fusarium graminearum*, *Anthracis chinensis*, *Rhizoctonia solani*, and *Bacillus oryzae* were cultured on PDA, while *Phytophthora indicum* and *Alternaria alternata* were cultured on OA. Plates without added volatiles served as blank controls. Each treatment was repeated three times. Incubate in the dark at 28℃ for 2-3 days, and statistically analyze the antibacterial effect. Results are shown below. Figure 7 As shown in Table 3, the antibacterial rate was measured. The antibacterial rate was calculated using the same method as above.

[0078] In this embodiment, the antibacterial effects of linalool and 1-octen-3-ol, the volatile compounds of Penicillium oxalicum HN-7, on eight pathogenic fungi, including Phytophthora tobaccoii, Botrytis cinerea, Pythium citrinum, Fusarium graminearum, Anthracnose fungus of cucumber, Rhizoctonia solani, Bacillus oryzae of wheat, Bacillus blast fungus of rice, and Alternaria alternata, were determined. (See Table 3.) Figure 7 It can be known that linalool (0.093 μg / cm) 3 ) and 1-octen-3-ol (0.090 μg / cm) 3 At concentrations of [specific concentration], it exhibits inhibitory effects against a variety of pathogenic fungi, with the best inhibitory effects against Botrytis cinerea and Pythium mellea.

[0079] Table 3. Inhibitory rates of linalool and 1-octen-3-ol against common pathogenic fungi.

[0080] Determination of virulence regression equation: 4 μL, 3 μL, 2 μL, 1.5 μL, and 0.75 μL of the volatile compounds linalool and 1-octen-3-ol, respectively, were dissolved in 9 mL of PDA medium. The plates were poured out, and another 9 mL of PDA medium was poured into the other side of the plate. After cooling and solidification, 5 mm of *Pythium spp.* or *Botrytis cinerea* cakes were inoculated. Plates without added volatile compounds served as blank controls. Each treatment was repeated three times. The plates were incubated in the dark at 28℃ for 2 days. The antibacterial effect was statistically analyzed, and the inhibition rate was calculated using the method described above.

[0081] In this example, the inhibitory concentrations of linalool and 1-octen-3-ol on Pythium moniliforme and Botrytis cinerea were determined, such as... Figure 8 As shown in Figure A, the regression equation for the toxicity of linalool to *Pythium spp.* is y = 4.5966x + 3.7111 (R²). 2 = 0.9894), where "x" is the lg value of linalool concentration and "y" is the bioprobability statistic corresponding to the inhibition rate of linalool against Pythium spp. Substituting the bioprobability statistic corresponding to 50% into the equation, the median inhibitory concentration EC of linalool against Pythium spp. is calculated. 50 =0.030 μg / CM 3 And it increases in a dose-dependent manner. For example... Figure 8 As shown in Figure B, the regression equation for the toxicity of linalool to Botrytis cinerea is y = 1.6877x + 4.6757 (R²). 2 = 0.988), EC 50 =0.024 μg / CM 3 ;like Figure 8 As shown in Figure C, the regression equation for the toxicity of 1-octen-3-ol to *Pythium spp.* is y = 1.6035x + 4.1535 (R²). 2 = 0.9839), EC 50 =0.051 μg / CM 3 ;like Figure 8 As shown in Figure D, the regression equation for the toxicity of 1-octen-3-ol to Botrytis cinerea is y = 1.0291x + 5.2255 (R0). 2 = 0.9948), EC 50 =0.0092 μg / CM 3 .

[0082] 5. Fruit disease prevention test of Penicillium oxalate HN-7 volatiles against Botrytis cinerea. Healthy grapes of equal weight and uniform shape were selected, disinfected with 75% ethanol, washed thoroughly with sterile water, and air-dried. A hole (Ø=2 mm, d=2 mm) was pierced at the maximum circumference of each grape using a sterile toothpick, and then inoculated with *Botrytis cinerea* mycelium at a diameter of Ø=2 mm. All treated samples were then placed in sterile tissue culture flasks, and volatiles (linalool or 1-octen-3-ol) were added to the flasks at amounts corresponding to their EC50 and EC50 values. 50 The fungicides were diluted 0.5, 1.5, and 3 times with the active ingredient and cultured at 25℃ for 3 days, with 5 fruits per group, repeated 3 times. The control group consisted of water. The diameter of the lesions was measured, and the inhibition rate was calculated based on the lesion diameters of the experimental and control groups using the following formula: Lesion diameter (mm) = Measured lesion diameter - 2; Lesion growth inhibition rate (%) = (diameter of lesions in control group - diameter of lesions in treatment group) / (diameter of lesions in control group).

[0083] The results are shown in Table 4 and Figure 9 As shown, in fruit disease control trials, linalool effectively inhibited fruit damage caused by Gracilaria gracilis, and the damage inhibition rate was dose-dependent on concentration (1.5 × EC50). 50 At the specified concentration, it can inhibit 84.8% of the 3×EC3 concentration. 50 At the specified concentration, it can completely inhibit [the disease]. 1-Octen-3-ol can effectively inhibit fruit damage caused by Gracilaria catarrhalis, and the damage inhibition rate is dose-dependent on concentration. At 3×EC [the concentration is not specified in the original text]. 50 It can suppress 90.06%.

[0084] Table 4. Control effects of linalool and 1-octen-3-ol on Botrytis cinerea infection of grapes.

[0085] Note: a / b / c indicates that the differences are significant according to Duncan's new multiple range test. P <0.05).

[0086] Example 3: Potted plant control effect of Penicillium oxalate HN-7 inoculant. 1. Biocontrol pot experiment of Penicillium oxalate HN-7 inoculant Tomato seeds were sown in seedling trays and cultured for approximately 15 days. Three treatment groups were established. 80 g of substrate soil was weighed from each tray, and 0.1 g, 0.2 g, and 0.5 g of Penicillium oxalate inoculant (≥5 billion CFU / g) were added respectively. The mixture was then placed in 9×9×8 cm plastic flowerpots. Tomato seedlings of uniform growth were carefully selected, the soil around the roots was gently shaken off, leaving approximately 5 cm of root. The substrate around the roots was cleaned, and the seedlings were transplanted into the flowerpots. Ten tomato seedlings were placed in each treatment group. A control group (without Penicillium oxalate inoculant) was used. Three days after transplanting, 10 mL of Ralstonia solanacearum (OD200) was inoculated. 600=0.1), and the incidence rate and disease index were counted 11 days after inoculation with Ralstonia solanacearum to calculate the disease prevention effect.

[0087] Grading standards for bacterial wilt: Grade 0: No disease in the whole plant; Grade 1: Occasionally chlorotic spots on the stem, or leaves below 1 / 2 on the diseased side wither; Grade 3: Black streaks on the stem, but not exceeding 1 / 2 of the stem height, or leaves of 1 / 2-2 / 3 on the diseased side wither; Grade 5: Black streaks on the stem exceed 1 / 2 of the stem height, but do not reach the top of the stem, or leaves above 2 / 3 on the diseased side wither; Grade 7: Black streaks on the stem reach the top of the stem, or all leaves of the diseased plant wither; Grade 9: The diseased plant is basically dead.

[0088] Disease index = [ [(Number of diseased plants at each level × Disease severity value) / (Total number of plants surveyed × Highest severity value)] × 100; Disease prevention effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100.

[0089] The results are shown in Table 5 and Figure 10 As shown in Table 5, pot experiments confirmed that the inoculum of Penicillium oxalate HN-7 has a significant control effect on bacterial wilt. Figure 10 As shown, on day 11 after treatment, the control effect of adding 0.2 g of Penicillium oxalate per pot was significantly better than that of adding 0.1 g and 0.5 g of Penicillium oxalate per pot, with a disease index of 10.33 and a control effect of 79.19%.

[0090] Table 5. Disease index and control effect at 11 days in pot experiment.

[0091] Note: a / b / c / d indicate that the differences are significant according to Duncan's new multiple range test. P <0.05).

[0092] 2. Localization of Penicillium oxalate HN-7 in tomato plants Experiments were continued on the tomato plants from step 1. Eleven days after inoculation with bacterial wilt, roots and stems of tomatoes were collected, and total DNA was extracted using the FastDNA™ Spin Kit for Soil (MP). β-actin of *Penicillium oxalicum* HN-7 was used as primers, with β-actin-F (5'-GCCACTCAACAGCCTGATCT-3') and β-actin-R (5'-GCTTGGTGTCTACGGAGCTT-3') as internal reference genes. The tomato actin ACT genes SLACT-F (5'-CGGTGACCACTTTCCGATCT-3') and SLACT-R (5'-TCCTCACCGTCAGCCATTTT-3') were used as internal reference genes. Using DNA from root and stem tissues as templates, the relative biomass of *Penicillium oxalicum* genes in each tissue was detected using 2×SYBR Green PCR Mix reagent. 10 μL qRT-PCR reaction system: 5 μL 2×SYBR Green PCR Mix, 0.4 μL upstream primer, 0.4 μL downstream primer, 1.0 μL cDNA template (200 ng / μL), 3.2 μL ddH2O; reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 56℃ annealing for 45 s, for 40 cycles. -△△Ct The relative expression levels of the *Penicillium oxalicum* gene in different tomato tissues were analyzed using a method. The experimental results are as follows: Figure 11 As shown.

[0093] On day 11 of the pot experiment, *Penicillium oxalate* HN-7 was able to be localized in the roots and stems of tomatoes. Figure 11 As shown in Figure A, the relative biomass of *Penicillium oxalate* HN-7 at treatments of 0.1, 0.2, and 0.5 g / plant were 2.47, 1.93, and 0.30 in the roots, respectively. Figure 11 As shown in Figure B, the relative biomass of Penicillium oxalate HN-7 in the stem was 6.63, 7.51, and 7.85. This indicates that Penicillium oxalate HN-7 is localized in the rhizosphere of tomatoes and gradually migrates into the stem, thus effectively controlling bacterial wilt.

[0094] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A strain of Penicillium oxalicum ( Penicillium oxalicum HN-7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42167.

2. A microbial preparation containing Penicillium oxalate as described in claim 1.

3. The use of Penicillium oxalate as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of antibacterial agents.

4. The application according to claim 3, characterized in that, The antibacterial agent includes volatile metabolites produced by Penicillium oxalate, and the volatile metabolites include one or more of anisole, 1-octen-3-ol, linalool, and m-phenylenediamine.

5. A method for preparing an antibacterial agent, characterized in that, The method includes: culturing Penicillium oxalate as described in claim 1 or a microbial preparation as described in claim 2.

6. The antibacterial agent prepared by the method described in claim 5.

7. The application of Penicillium oxalate as described in claim 1, or the microbial preparation as described in claim 2, or the antibacterial agent as described in claim 6 in the prevention and control of plant diseases and / or the inhibition of pathogen growth.

8. The application according to claim 7, characterized in that, The pathogens include Phytophthora indicum ( Phytophthora nicotianae ), ultimate humic acid ( Pythium ultimum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Ralstonia solanacearum ( Ralstonia solanacearum ), Pythium spp. ( Pythium aphanidermatum Fusarium graminearum ( ), Fusarium graminearum ), cucumber anthracnose fungus ( Colletotrichum orbiculare ), wheat sheath blight fungus ( Rhizoctonia cereals Rice blast fungus ( Magnaporthe oryzae Alternaria ( Alternaria alternata One or more of the following.

9. The application according to claim 7 or 8, characterized in that, The plant diseases mentioned are diseases caused by pathogens, including one or more of the following: black shank, damping-off, wilt, gray mold, bacterial wilt, Fusarium head blight, root rot, anthracnose, sheath blight, rice blast, and red spot disease.

10. A pesticide, characterized in that, The pesticide includes Penicillium oxalicum as described in claim 1, or a microbial preparation as described in claim 2, or an antibacterial agent as described in claim 6.

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