Aspergillus ochraceus AF-P9 and application thereof

Abstract

The application provides an anti-disease promoting Aspergillus ochraceus AF-P9 and an application thereof, and belongs to the technical field of microorganisms.The application provides the Aspergillus ochraceus AF-P9, which has salt tolerance and alkali tolerance, has the ability of producing iron carrier and secreting chitinase, has a high efficient inhibiting effect on multiple pathogenic bacteria such as Fusarium oxysporum, Fusarium acuminatum, Fusarium decemcellulare, Pythium aphanidermatum, Pseudomonas syringae pv., Sclerotinia sclerotiorum and Phytophthora, and can be applied to the prevention and treatment of rice damping-off.Further, metabolites of the Aspergillus ochraceus AF-P9 are analyzed, and it is found that the Aspergillus ochraceus AF-P9 can produce tricyclic cyclohexanone compounds, cyclohexene compounds with hydroxyl, phenanthrene ketone compounds with hydroxyl, benzene ring compounds with two long chain esters and cyclohexene compounds with four annular structures.The Aspergillus ochraceus AF-P9 provided by the application provides a strain resource for the prevention and treatment of disease organisms.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a aspergillus ochraceus AF-P9 and application thereof. BACKGROUND

[0002] Rice seedling stand disease is a common disease in spring seedling. Rice stand disease belongs to soil-borne disease and is caused by infection of multiple pathogenic fungi. The corresponding pathogenic fungi overwinter in the disease residues of multiple hosts and soil, and produce conidia to spread by air current when the environmental conditions are suitable. The occurrence of rice seedling stand disease causes a large number of rice seedlings to lodge, die and rot, and can also directly cause rice seed rot when the rice seedlings have not yet germinated, which seriously affects the growth of rice seedlings and causes great economic losses to rice production. Tomato wilt is a common disease of tomato. The pathogen overwinters in the soil, spreads with rainwater, irrigation water and soil, and enters from the young roots or wounds, spreads in the vascular bundle, blocks the conduit, produces toxic tomato wilt, spreads with the transport tissue, causes the vascular bundle to turn brown and lose transport function, and thus causes the plant to wilt. It can be seen that both rice stand disease and tomato wilt are soil-borne diseases, which seriously affect the yield of rice and tomato.

[0003] At present, the prevention and control of rice stand disease and tomato wilt mainly relies on chemical agents. The commonly used agents for rice stand disease include metalaxyl, diniconazole, oxadixyl, ailmiao, manzao and dali peak. Tomato wilt is also mainly controlled by chemical pesticides to kill pathogenic fungi. However, although chemical control can control the development of rice stand disease and tomato wilt in the field to a certain extent, it easily causes secondary pollution to the ecological environment and crop production, is prone to drug resistance in long-term repeated use, leads to increased dosage and increasing production cost, pollutes the environment and causes pesticide residue problems.

[0004] In recent years, biological control of the two plant diseases has attracted people's attention. For example, Pseudomonas aeruginosa and Fusarium can be used as antagonistic microorganisms to prevent and control tomato wilt, and actinomycetes can produce active substances against pathogenic fungi, which have the potential to be applied to tomato wilt. For example, Bacillus velezensis can be used to prepare bacillus agent to prevent and control rice stand disease. For example, a microbial preparation composed of Bacillus thuringiensis, Trichoderma longibrachiatum and Saccharomyces cerevisiae has a strong inhibitory effect on the pathogen of rice stand disease and shows good control effect on rice stand disease. However, the currently reported microbial agents for tomato wilt or rice stand disease often have limited control effect, and there is no report on microbial agents with control effect on both diseases. SUMMARY

[0005] In view of the defects in the prior art, the object of the present invention is to provide an Aspergillus ochraceus that can be used to prevent and control rice damping-off disease and tomato wilt disease, and promote the growth of rice and tomatoes.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for treating Aspergillus ochraceus ( Aspergillus ochraceus ) AF-P9, the preservation number of the Aspergillus ochraceus AF-P9 is CGMCC No.41804.

[0008] The present invention provides a microbial preparation comprising the Aspergillus ochraceus AF-P9 and / or its fermentation product described in the above technical solution.

[0009] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0010] The Aspergillus ochraceus AF-P9 is cultured in a culture medium to obtain a microbial preparation.

[0011] Preferably, the culture temperature is 25-28° C., and the culture time is 5-7 days.

[0012] The present invention provides an application of the Aspergillus ochraceus AF-P9 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution in the preparation of any one or more of the following products (1) to (5):

[0013] (1) tricyclic cyclohexanone compounds; (2) cyclohexene compounds with hydroxyl groups; (3) phenanthronene compounds with hydroxyl groups; (4) benzene ring compounds with two long-chain esters; (5) cyclohexene compounds with four parallel ring structures.

[0014] The present invention provides use of the Aspergillus ochraceus AF-P9 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution in preparing a preparation for inhibiting pathogens.

[0015] Preferably, the pathogen includes Fusarium oxysporum Fusarium oxysporum , Fusarium acuminate Fusarium acuminatum , Fusarium trilineatum Fusarium tricinctum , Pythium spp. Pythium aristosporum , Microbacteria Didymella bryoniae , Phomopsis spp. Phomopsis longicolla , Sclerotinia sclerotiorum Sclerotinia sclerotiorum and Phytophthora Phytophthora sojae Any one or more of the following.

[0016] The application provides application of the Aspergillus ochraceus AF-P9, the microbial preparation or the microbial preparation prepared by the preparation method in promoting plant growth and / or disease control.

[0017] Preferably, the plant comprises tomatoes and / or rice; and the disease comprises rice damping-off and / or tomato wilt.

[0018] The application provides a method for promoting plant growth and / or disease control, comprising:

[0019] The microbial preparation is applied during plant growth.

[0020] The application provides an Aspergillus ochraceus (AF-P9). Aspergillus ochraceus The Aspergillus ochraceus AF-P9 has a preservation number of CGMCC No.41804. The Aspergillus ochraceus AF-P9 has high salt tolerance and alkali tolerance, has the ability to produce siderophores and secrete chitinase, thereby being beneficial to promoting plant growth and improving the stress resistance of plants. The Aspergillus ochraceus AF-P9 has a high efficient inhibiting effect on a plurality of pathogenic bacteria, for example, Fusarium oxysporum, Fusarium acuminatum, Fusarium decemcellulare, Pythium aphanidermatum, Pseudonectria pannicola, Phoma exigua, Sclerotinia sclerotiorum and Phytophthora, and is beneficial to the prevention and treatment of diseases caused by the pathogenic bacteria. The Aspergillus ochraceus AF-P9 can prevent and treat rice damping-off and promote rice growth according to the results of the examples. The Aspergillus ochraceus AF-P9 provides a strain resource for biological disease control.

[0021] Biological preservation instructions

[0022] The Aspergillus ochraceus AF-P9 is classified and named as: Aspergillus ochraceus and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 22, 2025, and has a preservation number of CGMCC No.41804. DRAWINGS

[0023] Figure 1 It is a plate confrontation test result diagram of the AF-P9 biocontrol fungus on Fusarium oxysporum;

[0024] Figure 2 It is a colony morphology diagram and a morphology diagram under an optical microscope of the AF-P9;

[0025] Figure 3 It is a phylogenetic analysis result diagram of the AF-P9 biocontrol fungus;

[0026] Figure 4Figure for the result of detection of siderophore production ability of AF-P9 and the result of detection of chitinase production ability of AF-P9;

[0027] Figure 5 Figure for the comparison of normal mycelium of Fusarium oxysporum and mycelium of Fusarium oxysporum confronted with biocontrol fungus AF-P9;

[0028] Figure 6 Figure for the hyperparasitism of biocontrol fungus AF-P9 on mycelium of Fusarium oxysporum;

[0029] Figure 7 Figure for the result of TLC analysis of secondary metabolites produced by AF-P9 in different culture media;

[0030] Figure 8 Figure for the inhibitory effect of five compounds on Fusarium oxysporum;

[0031] Figure 9 Figure for the structure of compound 1; 1 H NMR (600 MHz, DMSO- d 6) figure;

[0032] Figure 10 Figure for the structure of compound 1; 13 C NMR (151 MHz, DMSO-d6) figure;

[0033] Figure 11 Figure for the structure of compound 1;

[0034] Figure 12 Figure for the structure of compound 2; 1 H NMR (600 MHz, DMSO- d 6) figure;

[0035] Figure 13 Figure for the structure of compound 2; 13 C NMR (151 MHz, DMSO-d6) figure;

[0036] Figure 14 Figure for the structure of compound 2;

[0037] Figure 15 Figure for the structure of compound 3; 1 H NMR (600 MHz, DMSO- d 6) figure;

[0038] Figure 16 Figure for the structure of compound 3; 13 C NMR (151 MHz, DMSO-d6) figure;

[0039] Figure 17 Figure for the structure of compound 4;1 H NMR (600 MHz, DMSO- d 6) Figure;

[0040] Figure 18 For compound 4 13 C NMR (151 MHz, DMSO-d6) pattern;

[0041] Figure 19 is a structural diagram of a structural analogue of compound 4;

[0042] Figure 20 For compound 5 1 H NMR (600 MHz, DMSO- d 6) Figure;

[0043] Figure 21 For compound 5 13 C NMR (151 MHz, DMSO-d6) pattern;

[0044] Figure 22 Schematic diagram of the structural analogs of compound 3 and compound 5. DETAILED DESCRIPTION

[0045] The present invention provides a method for treating Aspergillus ochraceus ( Aspergillus ochraceus ) AF-P9, the Aspergillus ochraceus AF-P9 has a deposit number of CGMCC No. 41804. The Aspergillus ochraceus AF-P9 provided by the present invention was isolated from soil collected from a tomato plantation in Qibaojia alkaline land in Dawa District, Panjin City, Liaoning Province. The Aspergillus ochraceus AF-P9 can grow in a variety of culture media, with PDB medium being its most suitable medium. The results of the present invention, as demonstrated by the examples, demonstrate that the extraction efficiency, product richness and diversity, and inhibitory effect on pathogens of fermentation products of Aspergillus ochraceus AF-P9 in PDB medium are superior to those of other culture media.

[0046] The Aspergillus ochraceus AF-P9 provided by the present invention has a colony diameter of 40 mm after being cultured on PDA culture medium for 5 days (28°C). The front of the colony is yellow and the back is white. Yellow conidia are visible on the surface of the colony. The conidiophores of the Aspergillus ochraceus AF-P9 are mostly upright. Under a microscope, the conidiophores of the Aspergillus ochraceus AF-P9 are large and long, but the conidiophores vary in size, with spherical swells at the top and radial stalks attached. The conidia are colorless to light yellow spherical. In the present invention, the ITS sequencing analysis results of the Aspergillus ochraceus AF-P9 are shown in SEQ ID NO.1. The taxonomic status of the strain AF-P9 was further confirmed by the morphological and molecular biological information of the strain.

[0047] The Aspergillus ochraceus AF-P9 has high salt resistance and alkali resistance, has the ability to produce iron carrier and to secrete chitinase, thereby facilitating plant growth, improving plant stress resistance and the like.

[0048] The Aspergillus ochraceus AF-P9 has the inhibiting ability to Fusarium oxysporum, Fusarium acuminatum, Fusarium decemcellulare, Pythium aphanidermatum, Pseudoperonospora cubensis, Sclerotinia sclerotiorum and Phytophthora.

[0049] The Aspergillus ochraceus AF-P9 can produce any one of the following two or more of the three-ring cyclohexanone compound, the cyclohexene compound with a hydroxyl group, the phenanthrene compound with a hydroxyl group, the benzene compound with two long-chain esters and the cyclohexene compound with four annular structures. The metabolites of the Aspergillus ochraceus AF-P9 have high efficient prevention and control effect on pathogenic fungi such as Fusarium oxysporum. The present application tracks and separates and identifies the metabolites of the biocontrol fungi with high activity, and studies the activity, and reveals the bacteriostatic mechanism of the Aspergillus ochraceus AF-P9, thereby providing a theoretical basis for optimizing the biological control strategy.

[0050] The Aspergillus ochraceus AF-P9 can be applied to the prevention and control of rice damping-off and / or tomato wilt, and can also promote the growth of rice and / or tomato, thereby facilitating the improvement of the yield of rice and / or tomato and the like. The fermentation supernatant diluent of the Aspergillus ochraceus AF-P9 can enhance the germination potential of tomato and improve the germination rate of tomato.

[0051] The present application provides a microbial preparation comprising the Aspergillus ochraceus AF-P9 and / or fermentation products thereof. According to an optional embodiment of the present application, the viable cell count of the Aspergillus ochraceus AF-P9 in the microbial preparation is greater than or equal to 1x10 7 CFU / mL, which can be 1x10 7 ~1x10 9 CFU / mL, which can be 1x10 8 CFU / mL.

[0052] The present application provides a preparation method of the microbial preparation, comprising: culturing the Aspergillus ochraceus AF-P9 in a culture medium to obtain a microbial preparation. According to an optional embodiment of the present application, the culture medium can be PDA medium and / or PDB medium; the culture temperature can be 25-28 DEG C, or 28 DEG C; the culture is accompanied by rotation; the rotation speed is 150 r / min; the culture time can be 5-7 days, or 7 days. After the culture is completed, the obtained culture solution can be directly used as a microbial preparation; the obtained culture solution can also be collected to resuspend the bacterial cells to obtain a bacterial suspension, and the bacterial suspension is used as a microbial preparation; the obtained culture solution can also be collected to obtain a supernatant, and the supernatant is used as a microbial preparation.

[0053] The present application provides an application of the Aspergillus ochraceus AF-P9, the microbial preparation or the microbial preparation prepared by the preparation method in preparing any one or two or more of the following (1)-(5) products: (1) a tricyclic cyclohexanone compound; (2) a cyclohexene compound with a hydroxyl group; (3) a phenanthrene ketone compound with a hydroxyl group; (4) a benzene compound with two long-chain esters; (5) a cyclohexene compound with four annular structures. In the present application, the tricyclic cyclohexanone compound is a structural analogue of a sterol compound; the sterol compound includes ergosterol and / or 24-epibrassilactone. In the present application, the cyclohexene compound with a hydroxyl group is a phenol derivative, a structural analogue of Trieffusol D and sporogen AO-1. In the present application, the phenanthrene ketone compound with a hydroxyl group belongs to a terpene derivative, a diterpenoid compound, a structural analogue of Aspewentin M and 18-hydroxypodocarpen-8(14)-en-13-one. In the present application, the benzene compound with two long-chain esters belongs to a p-benzoic acid ester derivative, a structural analogue of auriculatum A and some plasticizers. In the present application, the cyclohexene compound with four annular structures belongs to a terpene derivative, a tetracyclic diterpene compound, containing four annular structures, a structural analogue of (6-strobilactone-B) ester of (E,E)-6-carbonyl-7-hydroxy-2,4-octadienoic acid. The present application shows by the example results that the tricyclic cyclohexanone compound has a 30.51% inhibition rate on Fusarium oxysporum, the cyclohexene compound with a hydroxyl group has a 35.22% inhibition rate on Fusarium oxysporum; the phenanthrene ketone compound with a hydroxyl group has a 39.2% inhibition rate on Fusarium oxysporum; the benzene compound with two long-chain esters has a 28.34% inhibition rate on Fusarium oxysporum; and the cyclohexene compound with four annular structures has a 27.25% inhibition rate on Fusarium oxysporum.

[0054] The present application provides an application of the Aspergillus ochraceus AF-P9, the microbial preparation or the microbial preparation prepared by the preparation method in preparing a pathogenic bacteria inhibiting preparation. As an optional embodiment of the present application, the pathogenic bacteria include any one or two or more of Fusarium oxysporum, Fusarium acuminatum, Fusarium decemcellulare, Pythium aphanidermatum, Leptosphaeria korrae, Phomopsis sojae, Sclerotinia sclerotiorum and Phytophthora.

[0055] The application provides application of the Aspergillus ochraceus AF-P9, the microbial preparation or the microbial preparation prepared by the preparation method in promoting plant growth and / or disease control. As an optional embodiment of the application, the plant includes tomatoes and / or rice; and the disease includes rice damping-off and / or tomato wilt. The application shows through the embodiment results that the Aspergillus ochraceus AF-P9 or the microbial preparation can be applied to the prevention and control of rice damping-off and / or tomato wilt, and can promote the growth of rice and / or tomatoes while preventing and controlling rice damping-off and / or tomato wilt, thereby facilitating the increase of the yield of rice and / or tomatoes. The fermentation supernatant diluent of the Aspergillus ochraceus AF-P9 can also improve the germination potential of tomato seeds, improve the germination rate of tomato seeds, thereby facilitating the growth of tomatoes; and the dilution multiple of the fermentation supernatant can be 50-100 times.

[0056] The application provides a method for promoting plant growth and / or disease control, which comprises: applying the microbial preparation in the growth process of the plant. As an optional embodiment of the application, the plant includes rice; and the microbial preparation can be applied when the rice seedlings grow to 1-2 leaf stages. The application does not have special limitation on the application mode, and the conventional application methods in the art can be used. The preferred application method of the application includes root irrigation. As an optional embodiment of the application, the plant includes tomatoes; and the microbial preparation can be applied when the tomatoes grow to 2-4 true leaves, or can be applied when the tomatoes grow to 3-4 true leaves. The application does not have special limitation on the application mode, and the conventional application methods in the art can be used. The preferred application method of the application includes root irrigation.

[0057] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and embodiments, but they should not be understood as limiting the protection scope of the application.

[0058] The main reagents and culture media used in the following embodiments

[0059] Table 1 Fermentation culture medium

[0060]

[0061] In Table 1, the first three culture media of PDB culture medium, malt culture medium and rice culture medium are configured into liquid-state culture media for subsequent use, and the potato No. 1 culture medium, the potato No. 2 culture medium and the potato No. 3 culture medium are configured into solid-state culture media for subsequent use.

[0062] Embodiment 1

[0063] 1. Isolation and purification of biocontrol fungi: Soil was collected from a tomato plantation in Qibaojia, Dawa District, Panjin City, Liaoning Province, and a persimmon plantation in Daqing City, Heilongjiang Province. The microorganisms in the soil were isolated using the dilution plate method. 10 g of fresh rhizosphere soil sample was added to 90 mL of sterilized water containing glass beads and shaken for 10 minutes. After standing, the concentration was 10 -1 The soil stock solution was serially diluted with sterile water to obtain 10 -2 -10 -6 Then pipette 10 -4 , 10 -5 , 10 -6 200 μL of each soil suspension from three gradients was plated onto PDA medium containing 50 μg / mL chloramphenicol. The PDA was set at two salinity levels: 0.1% and 0.15% Na2CO3. Three replicates were set for each dilution gradient. The cultures were inverted at 30°C for 3 days, and the dominant colonies were selected for further purification and culture.

[0064] 2. Screening of biocontrol fungi: Fusarium oxysporum, the pathogen of wilt Fusarium oxysporum (Strain No.: CICC 41029) was used as the target bacteria, and the plate confrontation method was used to preliminarily screen strains with inhibitory effects on the target bacteria on PDA plates. The strains obtained in the initial screening were further screened using the plate confrontation culture method, and a strain with stable growth and good antibacterial effect was obtained, named AF-P9. AF-P9 was isolated from soil collected from a tomato plantation in Qibaojia alkaline land in Dawa District, Panjin City, Liaoning Province. Its antibacterial effect is as follows: Figure 1 and as shown in Table 2. Figure 1 Figure A shows the results of a plate confrontation test of AF-P9 biocontrol fungus against Fusarium oxysporum; Figure 1 Figure B shows the natural growth of Fusarium oxysporum on a plate.

[0065] Table 2 Inhibition rate of AF-P9 on mycelial growth of Fusarium oxysporum

[0066]

[0067] Depend on Figure 1 As shown in Table 2, compared with the control group CK, AF-P9 can significantly inhibit the mycelial growth of pathogens, with an inhibition rate of 63.33%.

[0068] 3. Morphological and molecular biological identification of biocontrol fungi

[0069] (1) Colony and microscopic morphology of AF-P9 biocontrol fungus: The colony morphology of AF-P9 and its morphology under an optical microscope are as follows: Figure 2 As shown, Figure 2a is the colony morphology of AF-P9, b is the mycelium morphology of AF-P9, c is the sporulation structure of AF-P9, and d is the spore morphology of AF-P9. The colony diameter of AF-P9 is 40 mm after being cultured on PDA medium for 5 days (28°C), the front surface of the colony is yellow, and the back surface is white. Yellow conidium can be observed on the surface of the colony, and most of the conidium stalks are upright. Under a microscope, it is observed that the conidium stalk of AF-P9 is large and long, but the size of the spore stalk is not uniform, the top is swollen into a spherical shape, and the radial small stalks are attached. The conidium is colorless to light yellow spherical.

[0070] (2) Molecular biological identification of the biocontrol fungus AF-P9: the purified AF-P9 was subjected to ITS sequencing analysis by Shanghai Meiji Biological Technology Co., Ltd. The sequencing results were subjected to library construction by using the BLAST tool, and sequence alignment and phylogenetic tree construction were performed by using the MEGA software.

[0071] The ITS sequencing results of the biocontrol fungus AF-P9 are shown in SEQ ID NO. 1, and are specifically as follows:

[0072] GGTCCTCGGGGCCCAACCTCCCACCCGTGTATACCGTACCTTGTTGCTTCGGCGAGCCCGCCCCCTtTTTCTTTTAGGGGGCACAGCGCTCGCCGGAGACACCAACGTGAACACTGTCTGAAGTTTTGTCGTCTGAGTCGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAATTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCACCCCCTGGTATTCCGGGGGGTATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCCCCCAGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTTGTAGGCCCGGCCGGCTGCTGGCCGACGCTGAAAAGCAACCAACTATTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAAGGCGGGAGGAAAAATTGGGGGGGGGGAGA.

[0073] The ITS sequence of the AF-P9 biocontrol fungus obtained by sequencing was compared with the GenBank database in the BLAST tool, and a phylogenetic tree was constructed using MEGA software, and the results are shown in Figure 3 According to its morphological identification, it is determined that AF-P9 is Aspergillus ochraceus Aspergillus ochraceus .

[0074] 4. Salt tolerance of the AF-P9 biocontrol fungus: The screened AF-P9 biocontrol fungus was inoculated on PDA plates containing 0.05%, 0.15%, 0.25%, 0.35%, 0.45% and 0.55% Na2CO3, respectively, and incubated at 28°C for 5 days. The no-salt treatment group was set as the control, and the growth of the strain was observed.

[0075] The growth of the AF-P9 biocontrol fungus on different salt concentration media is shown in Table 3.

[0076] Table 3 Growth of AF-P9 biocontrol fungus on different salt concentration media (cm)

[0077]

[0078] Note: Different letters in the table indicate significant differences, the same below.

[0079] Table 3 shows that AF-P9 fungus can grow on medium with salt concentration as high as 0.55%, and has salt tolerance. The colony diameter of AF-P9 changes little with the increase of salt concentration, showing relatively good salt tolerance.

[0080] 5. Alkali tolerance of the AF-P9 biocontrol fungus: The screened AF-P9 biocontrol fungus was inoculated on PDA plates with pH of 7, 8, 9, 10, 11 and 12, respectively, and incubated at 28°C for 5 days. The growth of the strain was observed as shown in Table 4.

[0081] Table 4 Growth of AF-P9 biocontrol fungus on different pH media (cm)

[0082]

[0083] The results of Table 4 show that AF-P9 fungus can grow on medium with pH of 12, and has good alkali tolerance. With the increase of pH, the colony diameter of AF-P9 also shows a trend of first decreasing and then increasing, and at pH 12, the colony diameter reaches a maximum of 4.50 cm, which is 8.43% higher than that at pH 7.

[0084] Example 2 Study on biocontrol properties of AF-P9 biocontrol fungus

[0085] 1. Siderophore Production Capacity: Inoculate a 5 mm diameter cake of AF-P9 biocontrol fungus at the center of a CAS culture medium and incubate inverted at 28°C for 7 days. If a yellow or purple-red transparent ring forms around the colony, it indicates the strain's ability to produce siderophores. Measure and calculate the ratio of the transparent ring to the colony diameter (HC) to estimate the strain's siderophore production capacity. HC = transparent ring diameter / colony diameter.

[0086] like Figure 4 As shown in Figure A, the colony of AF-P9 can produce a purple-red transparent circle on the CAS culture medium, indicating that the bacteria has the ability to produce siderophores. The diameter of the AF-P9 transparent circle is 14 mm, and the HC value is 2.33.

[0087] 2. Chitinase activity: A 5-mm-diameter AF-P9 biocontrol fungus cake was inoculated in the center of a colloidal chitin culture medium and incubated upside down at 28°C for 7 days. If a transparent zone formed around the colony, it indicated that the colony had the ability to secrete chitinase. The ratio of the transparent zone to the colony diameter (HC) was measured and calculated to reflect the chitinase activity secreted by the strain.

[0088] like Figure 4 As shown in Figure 1B, AF-P9 has the ability to secrete chitinase. The diameter of the transparent zone of AF-P9 is 55 mm and the HC value is 1.08. This strain and its chitinase preparation have the potential for biological control of pathogenic fungi and agricultural pests.

[0089] 3. Inhibitory spectrum of biocontrol fungi

[0090] Fusarium oxysporum Fusarium oxysporum QLJ1 (accession number MT180464), Fusarium solani Fusarium solani WF7 (2) (accession number MT180477), Fusarium spp. Fusarium acuminatum CGMCC 3.6865, Fusarium trilineatum Fusarium tricinctum DJWZ3(3) (accession number MT180474), Rhizoctonia solani Rhizoctonia solani XLZ7 (1) (Accession No. MT180476) [Liu Jinxin. (2020). Population structure and genetic diversity analysis of rice blast pathogen in Heilongjiang Province (Master's thesis, Northeast Agricultural University)], Pythium spp. Pythium aristosporumJS22 [Liu Jinxin, Zhang Ruisi, Xu Chuzhen, Liu Chunlai, Zheng Yanyan, Zhang Xue & Li Yonggang. (2022). Characterisation of Pythium aristosporum Oomycete—A Novel Pathogen Causing Rice Seedling Blight in China. Journal of Fungi, 8(9), 890-890.],Pythium aristosporum Didymella bryoniae CFCC88122, Phymatotrichopsis parasitica Phomopsis longicolla MP4PL11PS (GenBank accession number HQ130441.1), [Geng Xiaobing, Wang Chunling, Huang Minghui & Li Yonggang. (2015). Identification of Phymatotrichopsis parasitica causing soybean seedling root rot. Plant Protection, 41(05), 127-129+144.],Sclerotinia sclerotiorum Sclerotinia sclerotiorum CCTCC KF2008457, Phytophthora sojae Phytophthora sojae Ps-H2 [Yang Mingxiu et al. Establishment of a single-oospore strain of Phytophthora sojae. Journal of Northeast Agricultural University, (06), 702-706. doi:10.19720 / j.cnki.issn.1005-9369.2005.06.003.] as the target strain, the inhibition effect of AF-Q5 biocontrol bacteria on 10 kinds of soil-borne pathogenic fungi was determined by plate confrontation method and cross method.

[0091] The inhibition activity of AF-P9 on 10 kinds of pathogenic fungi was determined by plate confrontation method, and the results are shown in Table 5.

[0092] Table 5 Inhibition spectrum of AF-P9 biocontrol fungi on tested pathogenic fungi

[0093]

[0094] Note: -: no inhibition activity detected; +: inhibits the growth of pathogenic fungi

[0095] From the results in Table 5, AF-P9 has inhibition ability on 8 kinds of pathogenic fungi, including Fusarium oxysporum, F. acuminatum, F. tricinctum, Pythium aphanidermatum, Pythium aristosporum, Phymatotrichopsis parasitica, Sclerotinia sclerotiorum and Phytophthora sojae. Further, AF-P9 with wide inhibition spectrum was subjected to plate confrontation with the tested pathogenic fungi, and the inhibition rate was calculated, and the results are shown in Table 6.

[0096] Table 6 Inhibition rate of AF-P9 on different pathogenic fungi

[0097]

[0098] As can be seen from Table 6, AF-P9 showed the highest inhibition rate of 78.33% against F. acuminatum, which is the pathogen of wheat scab and other plant diseases, thereby obtaining the potential of AF-P9 for preventing and treating wheat scab and other plant diseases; AF-P9 also showed a strong inhibitory effect on S. sclerotiorum, with an inhibition rate of 75.00%, which is the pathogen causing a variety of plant sclerotial diseases, thereby obtaining the potential of AF-P9 for preventing and treating a variety of plant sclerotial diseases; the inhibition rates of AF-P9 against F. sojae, F. oxysporum and D. bicolor were all 66.7%; the inhibition rates of AF-P9 against P. infestans and P. nicotianae were 64.18% and 63.33%, respectively; and the inhibition rate of AF-P9 against F. tricinctum was relatively low, with an inhibition rate of 48.28%.

[0099] Example 3: Study on the inhibitory activity of AF-P9 biocontrol fungus

[0100] 1. Direct inhibitory effect of AF-P9 biocontrol fungus on pathogenic fungal mycelium

[0101] The inhibitory effect of AF-P9 biocontrol fungus on the mycelium of Fusarium oxysporum f. sp. glycines (F. oxysporum) (strain number: CICC 41029) was observed by the plate confrontation method combined with the insertion method and staining method. Pathogenic fungus and AF-P9 biocontrol fungus were inoculated at a distance of 2 cm from the center of the PDA plate medium, each with a diameter of 5 mm. Two pieces of sterile glass slides were symmetrically inserted on the vertical bisector of the two strains, and inverted culture was carried out at 28°C until the mycelium of the two strains climbed or intersected on the glass slide. After slicing, cotton blue staining was used to observe the morphology of aerial mycelium. The results are shown in Fusarium oxysporum , wherein Figures 5 and 6 B is the normal growth mycelium of F. oxysporum, and the scale in the figure is 36 µm. Figure 5 A in the figure is the mycelium of F. oxysporum confronted with AF-P9 biocontrol fungus, and the scale in the figure is 50 µm; Figure 5 is the parasitic effect of AF-P9 biocontrol fungus on the mycelium of F. oxysporum. Figure 6

[0102] As can be seen from Figures 5 and 6 , the mycelium of the control group (CK) naturally grows smoothly and naturally stretches, with uniform thickness. Under the interaction with AF-P9 biocontrol fungus, the mycelium of F. oxysporum shows obvious distortion, accompanied by thinning. The mycelium of AF-P9 biocontrol fungus grows around the mycelium of F. oxysporum, spirally wraps the mycelium of F. oxysporum after recognizing and contacting the mycelium of F. oxysporum, and this wrapping and further penetration and parasitism of the cell wall can interfere with the nutrient absorption of the pathogenic fungus, eventually leading to the stop of mycelium growth. ​

[0103] Example 4: Test of the control effect of the biocontrol fungus AF-P9 on rice pythium damping-off

[0104] 1. Test design Test materials: rice variety: Zhongkefa No. 5. Pathogenic fungus: Fusarium oxysporum (strain number: CICC 41029). Biocontrol fungus: Aspergillus ochraceus AF-P9. Medium: PDA medium for culture of pathogenic fungus and biocontrol fungus. Test treatments: Blank control group: no inoculation of pathogenic fungus and no addition of biocontrol fungus. Control group: inoculation of Fusarium oxysporum and no addition of biocontrol fungus. Treatment group 1: inoculation of Fusarium oxysporum + Aspergillus ochraceus AF-P9. Three parallel tests were set for each group.

[0105] 2. Culture of pathogenic fungus and biocontrol fungus Culture of Fusarium oxysporum: Fusarium oxysporum was inoculated on PDA medium and cultured in a 25-28°C incubator for 5-7 days. Spores were collected and a spore suspension (concentration about 1×10 6 spores / mL) was prepared. Culture of biocontrol fungus: Aspergillus ochraceus AF-P9 biocontrol fungus was inoculated on PDA medium and cultured at 28°C for 5-7 days. After completion of the culture, the fungus was collected and a fungus suspension (concentration about 1×10 7 CFU / mL) was prepared.

[0106] 3. Rice seedling culture and inoculation Seedling culture: Healthy rice seeds were selected. The rice seeds were first sterilized by soaking in 75% alcohol for 30 s. After taking out the seeds, the second sterilization was performed by soaking in 5% sodium hypochlorite for 1 min. The seeds were rinsed with sterile water for 3 times to eliminate residual chlorine. Finally, the surface water of the seeds was absorbed and the seeds were ready for use. The seeds were placed on double-layer absorbent filter paper in a 10 cm culture dish and moistened with 10 mL distilled water. When the radicle was 2 mm or longer, the seed was considered to be germinated. Sterilized nutrient soil was added to the seedling culture plate. The germinated seeds were sown in the seedling culture plate. Appropriate temperature and humidity were maintained for seedling culture.

[0107] 4. Inoculation of pathogenic fungus: When the rice seedlings grew to 1-2 leaf stage, Fusarium oxysporum spore suspension was used for root irrigation inoculation. The inoculation amount of rice Fusarium oxysporum was 50 mL per plate, and the concentration of the fungus liquid was 1×10 6 spores / mL. Inoculation of biocontrol fungus: Three days before the inoculation of pathogenic fungus, the biocontrol fungus suspension was used for root irrigation. That is, the biocontrol fungus suspension was irrigated for 3 days, and then Fusarium oxysporum was inoculated. The inoculation amount of biocontrol fungus was 50 mL per plate, and the concentration of the fungus liquid was 1×10 7 CFU / mL. The control group was set at the same time, which was not irrigated with biocontrol fungus suspension, but with the same amount of sterile water. The blank control group was not irrigated with Fusarium oxysporum spore suspension, nor with biocontrol fungus suspension, but with the same amount of sterile water.

[0108] 5. Seedling stage management Environmental control: Maintain temperature at 25-30°C and humidity at 70-80%, simulating suitable conditions for disease occurrence. Water management: Keep the soil moist, avoiding excessive dryness or wetness.

[0109] 6. Data collection Randomly select 100 rice plants at the three-leaf-one-heart growth stage from each treatment group, and measure their stem base width, root number, root length, and plant height. In addition, determine the fresh weight and dry weight of 100 rice plants in each treatment group. Through comprehensive analysis of the above indicators, evaluate the incidence of rice and the control effect of the biocontrol fungus.

[0110] 7. Notes Aseptic operation: Ensure a sterile operating environment when inoculating pathogenic fungi and biocontrol fungi to avoid cross-contamination. Consistency of tests: Keep the test conditions consistent across groups to reduce errors.

[0111] 8. The results of the detection of rice growth indicators are shown in Table 7.

[0112] Table 7 Detection results of rice plant growth indicators in different test groups

[0113]

[0114] As shown in Table 7, the inoculation of Fusarium oxysporum on rice seedlings had a significant adverse effect on the seedlings, significantly reducing the stem base width and root number. However, the inoculation of biocontrol fungus AF-P9 after the inoculation of Fusarium oxysporum significantly alleviated the inhibition of Fusarium oxysporum on the growth of rice plants. Compared with the blank control group, the inoculation of AF-P9 biocontrol fungus also significantly promoted the growth of rice plants, increasing the plant height, root length, stem base width, root number, and fresh weight of the plants. Therefore, the AF-P9 biocontrol fungus can significantly improve the incidence of Fusarium oxysporum and promote the growth of rice.

[0115] 9. The incidence of rice seedling blight in the three-leaf-one-heart growth stage of rice plants in each treatment group was calculated, and the incidence rate (%) = (number of diseased plants / total number of plants surveyed) x 100%. The statistical results are shown in Table 8.

[0116] Table 8 Effect of AF-P9 on the incidence of rice seedling blight

[0117]

[0118] As shown in Table 8, the AF-P9 biocontrol fungus can significantly improve the incidence of Fusarium oxysporum and reduce the incidence of rice seedling blight.

[0119] Example 5 Screening of fermentation methods of AF-P9 biocontrol fungus and determination of antibacterial activity of crude extract

[0120] Six kinds of fermentation medium shown in Table 1 were selected respectively for fermentation of the biocontrol fungus strain AF-P9.

[0121] (1) AF-P9 seed liquid was inoculated into the six kinds of fermentation medium respectively with an inoculation amount of 5%, and each medium was repeated twice. The solid medium was placed in a constant temperature incubator at 28°C, and the liquid medium was placed in a shaker at 28°C, under the condition of 150 r / min, and was taken out after 15 days of fermentation. The mycelium and fermentation broth were separated by filtering with 300 mesh gauze. (2) Preparation of crude extract: two kinds of extractants, ethyl acetate and n-hexane, were selected. The fermentation broth was extracted with equal volume of organic phase for three times, and the mycelium and solid medium were soaked in 95% ethanol. The ratio of the mycelium and solid medium to the ethanol aqueous solution was 1 g:1 mL, and the mycelium and solid medium were treated with ultrasound for 3 h at a frequency of 40 kHz to obtain the ethanol extract. The crude extract was obtained by rotary evaporation, dissolved in distilled water, and extracted with water phase and organic phase in a volume ratio of 1:1 for three times. The organic phase was concentrated by rotary evaporation instrument to obtain the crude extract. (3) Determination of the mass of the crude extract: the mass of the crude extract was obtained by the difference method. 2 g of silica gel was weighed in a culture dish, dried, and the total mass was m0. The crude extract was dissolved in a small amount of methanol and dropped on the silica gel. After drying again, the mass was m1, and the mass of the crude extract was m=m1-m0.

[0122] The AF-P9 strain was fermented with six different media, and the fermentation products were extracted with ethyl acetate and n-hexane. The mass of the crude extract of the fermentation products is shown in Table 9.

[0123] Table 9 Mass of crude extract of different extractants after fermentation with different fermentation media

[0124]

[0125] From Table 9, it can be seen that the fermentation crude extract yield of PDB medium is high, and it is more suitable for fermentation of AF-P9 strain.

[0126] (4) Thin layer chromatography analysis: the crude extract was completely dissolved in methanol. Methanol and dichloromethane were mixed in a ratio of 1:10 to make the developing agent, and ammonia water was added dropwise as the tailing agent to reduce the tailing phenomenon of the strip. A 0.3 mm capillary spotting tube was used to spot at a distance of 1 cm from the edge of the chromatographic plate, and the origin line of the sample was drawn with a pencil. The thin layer plate was placed in the chromatographic lever, and when the solvent front approached the other end of the plate, the thin layer plate was taken out, immediately marked with a pencil, and developed at 254 nm. Then, the plate was immersed in 5% vanillin sulfuric acid ethanol solution, dried with a hairdryer, and developed. The fermentation of AF-P9 strain was carried out with six different media, and the thin layer chromatography analysis results of the crude extract obtained by extraction of the fermentation products are shown in Table 10. Figure 7 Table 10 Thin layer chromatography analysis results of the crude extract of different extractants after fermentation with different fermentation media Figure 7 Figure 1 is a TLC analysis result of secondary metabolites in different media. Figure 7 The left graph in FIG. 1 is a TLC of ethyl acetate phase metabolites of fermentation products obtained from different culture media; Figure 7 The right graph in FIG. 1 is a TLC of n-hexane phase metabolites of fermentation products obtained from different culture media. Figure 7 a corresponds to the TLC of metabolites obtained from PDB culture medium; b corresponds to the TLC of metabolites obtained from malt culture medium; c corresponds to the TLC of metabolites obtained from rice culture medium; d corresponds to the TLC of metabolites obtained from potato No. 1 culture medium; e corresponds to the TLC of metabolites obtained from potato No. 2 culture medium; f corresponds to the TLC of metabolites of fermentation products obtained from potato No. 3 culture medium.

[0127] (5) Bacteriostatic activity determination of fermentation broth

[0128] The bacteriostatic activity of each fermentation broth was determined by drug-containing plate method. Dimethyl sulfoxide (DMSO) was used as a cosolvent, and the crude extract was configured into a 0.5% solution with sterile water, and a drug-containing PDA plate was prepared at a concentration of 100 μg / mL, and sterile water was used as a control. The pathogenic bacteria were prepared into 5 mm bacterial cakes by a puncher and placed in the center of the plate. The treatment group and the control group were placed in a constant temperature incubator at 28°C for 5 days, and the bacteriostatic activity of each crude extract was recorded, with 3 repeats for each group.

[0129] The crude extracts extracted from 6 different fermentation media were prepared into drug-containing plates (concentration of 100 μg / mL), and the inhibition effect on the growth of Fusarium oxysporum (strain number: CICC 41029) was shown in Table 10 (P<0.05).

[0130] Table 10 Influence of crude extracts of different fermentation methods on the growth of Fusarium oxysporum

[0131]

[0132] From the results in Table 10, it can be seen that the crude extracts of the two extraction phases of PDB culture medium both showed good inhibition effect, and it was speculated that the fermentation products might contain a high concentration of antifungal active ingredients. In summary, PDB medium was determined as the best fermentation medium for AF-P9 active bacteriostatic product.

[0133] Example 6 Isolation and purification of AF-P9 biocontrol fungal bacteriostatic active substance and preliminary identification

[0134] 1. Isolation and purification of AF-P9 biocontrol fungal bacteriostatic active substance

[0135] (1) Preparation of fermentation crude extract According to the results of screening of fermentation methods, the most suitable medium PDB medium was determined, and AF-P9 biocontrol fungus was subjected to liquid fermentation (fermentation volume was 2000 mL) at 28°C constant temperature incubator for 28 d. Ethyl acetate was used to extract the fermentation broth and mycelium, respectively, and the two were combined and concentrated in a rotary evaporator to obtain the crude extract. Hexane was used to extract the fermentation broth and mycelium, respectively, and the two were combined and concentrated in a rotary evaporator to obtain the crude extract. The method for obtaining the crude extract is shown in Example 5.

[0136] (2) Silica gel column chromatography Wet column packing: 200-300 mesh silica gel powder was soaked in petroleum ether for 24 h, and then poured into the chromatographic column after saturation. Dry sample loading: the crude extract was completely dissolved in a small amount of methanol, mixed with 2 g of silica gel and dried to make the sample evenly adsorbed on the silica gel. The silica gel powder containing the sample was added to the column filled with silica gel, and 10 mm thick quartz sand was added to protect the sample layer from being damaged by the eluent. The eluent was petroleum ether: ethyl acetate gradient (V:V=1:0, 4:1, 2:1) and dichloromethane: methanol gradient (V:V=1:0, 98:2, 95:5, 92:8, 90:10, 85:15, 80:20, 70:30, 60:40, 1:1), respectively. When the eluent containing the sample flowed out of the bottom of the column was detected by TLC, the eluent was collected according to the volume. At the same time, TLC was used to monitor each fraction, and similar eluents were combined and concentrated for the next step.

[0137] (3) HPLC semi-preparative purification of active substances The eluent components with antibacterial activity were completely dissolved in methanol, and the dissolved mixture was filtered through a 0.22 μm filter membrane to remove impurity particles in the sample. The test selected methanol: water systems of 90:10, 80:20 and 70:30, respectively. The eluent flowed through a C18 reverse phase chromatographic column at a flow rate of 5 mL / min, and the retention time and peak shape were recorded. The eluents with the same components were combined and concentrated.

[0138] The mycelium and fermentation broth of AF-P9 strain after PDB fermentation were ultrasonically extracted with 95% ethanol, concentrated, dissolved in water, and extracted with an equal volume of an organic phase to obtain 761.1 mg of an ethyl acetate phase. After silica gel column chromatography, 152 components were obtained, which were combined to obtain 16 crude components (Fb1a-Fb16a) by TLC detection. Among them, Fb1a and Fb2a were oily substances, and the next step of separation and purification was not performed. Components Fb5a (methanol: water = 80:20) were separated and purified by high-performance liquid chromatograph to obtain compound 1, components Fb9a (methanol: water = 90:10) to obtain compound 2, and components Fb10a (methanol: water = 90:10) to obtain no effective compound. Similarly, 226.2 mg of n-hexane phase was obtained, which was subjected to silica gel column chromatography to obtain 104 components, which were combined to obtain 15 components (Fb1b-Fb15b) by TLC detection. Among them, Fb1b was an oily substance, and the next step of separation and purification was not performed. Components Fb5b (methanol: water = 80:20) were separated and purified by high-performance liquid chromatograph to obtain compound 3, and components Fb6b and Fb9b (methanol: water = 90:10) to obtain compound 4 and compound 5.

[0139] 2. Tracking of active substances (1) The active components in the metabolites were tracked and located by adopting the method of inhibiting mycelial growth rate of drug-containing plates. The eluent components of the ethyl acetate phase and the n-hexane phase were prepared into drug-containing plates at a concentration of 150 µg / mL, and the pathogenic fungi were inoculated in the middle of the plates. After constant temperature culture at 28℃ for 5d, the pathogenic fungi radius was recorded. Taking water as a control, the inhibition rate was calculated. (2) The inhibition effect of the compounds after HPLC semi-preparative purification on the pathogenic fungi was evaluated by filter paper disc method. The pathogenic fungus cake was inoculated in the middle of the plate. A filter paper disc with a diameter of 5 mm was prepared, soaked with a solution of the compound, and placed at a distance of 2 cm from the pathogenic fungus cake after removing the excess solution. After constant temperature culture at 28℃ for 3d, the inhibition zone diameter of the pathogenic fungi was observed. (3) The 28 components separated by silica gel column chromatography were subjected to activity tracking, including 14 components (Fb3a-Fb16a) of the ethyl acetate phase and 14 components (Fb2b-Fb15b) of the n-hexane phase. The antifungal activity of each component on Fusarium oxysporum was determined, and the results are shown in Table 11.

[0140] Table 11 Antifungal activity of each component of the fermentation crude extract

[0141]

[0142] Note: The last letter a in the sample name indicates the ethyl acetate phase, and the last letter b in the sample name indicates the n-hexane phase.

[0143] The inhibition rates of Fb5a, Fb9a and Fb10a in ethyl acetate phase on Fusarium oxysporum were all above 30%, and the inhibition effect of Fb10a was the best, with an inhibition rate of 36.40%. The Fb5b component in n-hexane phase had the strongest antibacterial activity, with an inhibition rate of 39.20%. The inhibition rates of Fb6b and Fb9b were both above 25%. According to the antibacterial activity results, it was speculated that the above six crude extract components might contain compounds with high antibacterial activity.

[0144] (4) The inhibition effects of five compounds obtained by HPLC semi-preparative purification on Fusarium oxysporum were evaluated by filter paper method. After constant temperature culture at 28℃ for 3d, the compound treatment group showed different antibacterial activities compared with the control group. The stronger the antibacterial activity, the smaller the radius of the corresponding pathogenic fungus mycelium. The results are shown in Table 3. Figure 8 Figure 8 The inhibition effects of five compounds on Fusarium oxysporum are shown in Table 3. Figure 8 It can be seen from Table 3 that the antibacterial activity of compound 3 was the strongest among the five compounds, with an inhibition rate of 32.14%; followed by compound 4 and compound 5, both with an inhibition rate of 20.83%; the inhibition rate of compound 2 was 17.86%; and the antibacterial activity of compound 1 was the weakest, with an inhibition rate of 10.71%.

[0145] 3. Structure identification of active substances The nuclear magnetic resonance (NMR) spectrum of the target compound was determined by a fully digitized superconducting nuclear magnetic resonance spectrometer (Avance NEO 600M). In the test, the compound was dissolved in deuterated dimethyl sulfoxide (DMSO-d6), and the chemical shift standard of the solvent was δ H 2.50 ppm and δ C 39.5 ppm. The obtained 1H NMR and 13C NMR data were analyzed by Mest Renova software to analyze the structure of the compound. In addition, ultraviolet spectrum (UV) data provided information about the electronic properties of the functional groups of the compound, which, combined with NMR data, further verified the structure of the compound.

[0146] (1) In order to further understand the molecular structure and chemical properties of these compounds, the nuclear magnetic resonance spectrum of compounds 1-5 was determined by a fully digitized superconducting nuclear magnetic resonance spectrometer. The determination results of compound 1 are shown in Table 4. Figures 9 and 10 Figure 9 Table 4 is the 1H NMR data of compound 1. 1 H NMR (600 MHz, DMSO- d 6); Figure 10 Table 4 is the 1H NMR data of compound 1. 13 C NMR (151 MHz, DMSO-d6). The determination results of compound 2 are shown in Table 5. Figures 12-13 Figure 12 Table 5 is the 1H NMR data of compound 2.​​​1 H NMR (600 MHz, DMSO- d 6); Figure 13 For compound 2 13 C NMR (151 MHz, DMSO-d6). The determination results of compound 3 are as follows Figures 15 and 16 shown. Figure 15 For compound 3 1 H NMR (600 MHz, DMSO- d 6); Figure 16 For compound 3 13 C NMR (151 MHz, DMSO-d6). The determination results of compound 4 are shown in Figures 17 and 18 shown. Figure 17 For compound 4 1 H NMR (600 MHz, DMSO- d 6); Figure 18 For compound 4 13 C NMR (151 MHz, DMSO-d6). The determination results of compound 5 are as follows Figures 20-21 shown. Figure 20 For compound 5 1 H NMR (600 MHz, DMSO- d 6); Figure 21 For compound 5 13 C NMR (151 MHz, DMSO-d6).

[0147] (2) Structural analysis of active substances The structure of the compound was inferred by combining the physical characteristics of the compound and the chemical properties analysis results such as the Rf value and color reaction of TLC. The results are as follows:

[0148] Compound 1: Pale yellow solid, TLC spot plate showed brown fluorescence under 254 nm UV light, 5% vanillin sulfate ethanol color development was yellow-brown, Rf value was 0.80 in dichloromethane-methanol (V / V=15:1) system. 1 H NMR data showed that the compound had three olefinic hydrogens (δ 7.60 (t, J = 7.9 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.94 (d, J =8.4 Hz, 1H)), two methine hydrogens of cyclohexene (δ 4.54 (t, J = 4.3 Hz, 2H)), two methine hydrogen atoms of cyclopentane (δ 1.06-0.98 (m, 2H)), 18 methylene hydrogen atoms (δ 2.16 (t,J = 7.3 Hz, 2H), 1.57-1.43 (m,4H), 1.42-1.37 (m, 2H), 1.24 (q, J = 8.4, 7.3 Hz, 10H)), and finally includes four long chains of methine and methyl hydrogen (δ 0.86 (ddt, J = 9.2, 6.5, 3.8 Hz, 4H)). 13 The C NMR spectrum showed that the compound had one carbonyl carbon (δ 1194.04) and four double bond carbons (δ 171.88, 171.28, 143.62, 137.06). Combined with the above data, it was speculated that the compound might be a tricyclic cyclohexanone compound.

[0149] Compound 2: brown solid, TLC spot plate showed brown fluorescence under 254 nm ultraviolet light, 5% vanillin sulfate ethanol color development to purple, Rf value in dichloromethane-methanol (V / V=10:1) system was 0.28. 1 H NMR data showed that the compound had one double bond hydrogen (δ 7.80-7.00 (m, 1H)), one hydroxyl hydrogen (δ 5.66-4.91 (m, 1H)), and one methine hydrogen attached to the hydroxyl group (δ 3.87 (t, J = 6.0 Hz, 1H)), and finally includes 13 methylene hydrogens and methine hydrogens (δ 0.22-1.89 (m, 2H), 1.50-1.35 (m, 3H), 1.23 (s, 4H), 1.07-0.75 (m, 4H)), 13 C NMR data showed that the compound had two aromatic carbons (δ 29.65, 98.48) and eight alkyl carbons (δ 3.56, 33.49, 31.77, 30.24, 29.49, 23.76, 22.57, 19.95). Combined with the above data, it was speculated that the compound might be a cyclohexene compound with a hydroxyl group.

[0150] Compound 3: pale yellow solid, TLC spot plate has fluorescent spots under 254 nm UV light, 5% vanillin sulfate ethanol color development is light purple, Rf value is 0.51 in dichloromethane-methanol (V / V=10:1) system. 1H NMR data showed that the compound had two olefinic hydrogens (δ 7.74-7.70 (m, 1H), 7.69-7.65 (m, 1H)), one hydroxyl hydrogen (δ 5.36-5.29 (m, 1H)), two methylene hydrogens attached to the hydroxyl group (δ 4.22 (t, J = 6.6 Hz, 2H)), two methylene hydrogens attached to the carbonyl group (δ 1.64 (dq, J = 8.8, 6.7 Hz, 2H)), two methine hydrogens (δ 1.40-1.36 (m, 2H)), and ten methylene hydrogens (1.06-0.95 (m, 3H), 0.94-0.89 (m, 3H), 0.89-0.82 (m, 4H)). 13 C NMR data showed that the compound had one carbonyl carbon (δ 167.44) and one carbon atom connected to a hydroxyl group (δ 65.50). Combined with the above data, it was speculated that compound 3 might be a phenanthronine-like compound with a hydroxyl group.

[0151] Compound 4: yellow solid, TLC spot plate has fluorescent spots under 254 nm UV light, 5% vanillin sulfate ethanol color development is yellow-brown, Rf value is 0.90 in dichloromethane-methanol (V / V=10:1) system. 1 H NMR data showed that the compound had four aromatic hydrogens (δ 8.07 (s, 1H), 7.78-7.65 (m, 2H), 7.29-7.23 (m, 1H)), four methylene hydrogens near the ester group (δ 5.32 (q, J = 7.3, 6.7 Hz, 2H), 4.22 (t, J = 6.6 Hz, 2H)), and one methine hydrogen near the ester group (δ 2.74 (t, J = 6.7 Hz, 1H)). 13 C NMR data showed that the compound had two ester carbon atoms (δ 213.68, 210.30) and six benzene ring carbon atoms (δ 198.31, 194.46, 193.97, 192.54, 192.21, 178.06). Combined with the above data, it was speculated that compound 4 might be a benzene ring structure with two long-chain esters.

[0152] Compound 5: brown solid, TLC spot plate has fluorescent spots under 254 nm UV light, 5% vanillin sulfate ethanol color development is purple, Rf value is 0.15 in dichloromethane-methanol (V / V=10:1) system. 1H NMR data showed that the compound had three olefinic hydrogens (δ 7.52-7.23 (m, 2H), 7.23-6.91 (m, 1H)), three methine hydrogens (δ 3.01-2.63 (m, 2H), 2.17-1.86 (m, 1H)), and finally a series of methylene hydrogens and two methine hydrogens (δ 1.24 (d, J = 15.1 Hz, 12H), 0.92-0.79 (m, 8H)). 13 C NMR data showed that the compound had four aromatic carbon atoms (δ130.12, 129.94, 129.02, 127.24). Combined with the above data, it was speculated that the compound might be a cyclohexene compound with four paracyclic structures.

[0153] Among them, the structural analogs of compound 1 are Figure 11 As shown. Among them, ergosterol, which is similar in structure to compound 1, is a type of sterol unique to fungi. Sterols are a large class of compounds with diverse biological activities, including naturally occurring hormones and artificially synthesized compounds, which can interfere with the structure and function of fungal cell membranes and have important application value in preventing and controlling fungal diseases. In current research practice, the production of ergosterol mainly depends on yeast strains. Song Haoyue et al. applied ergosterol at a concentration of 0.1 mg / L ( Figure 11 In a), the activity of the defense enzyme PPO in wheat leaves increased by 58.42%, the activity of POD increased by 73.68%, and the expression levels of the disease resistance genes LTP-1 and Glu1 increased by 2.07 times and 1.89 times, respectively. This indicates that ergosterol has significant disease prevention potential and can enhance wheat's resistance to ergot. In addition, sterol hormones can also alleviate abiotic stress in plants. The sterol compound 24-epibrassinolide (EBL) ( Figure 11 b) can promote K through polyamine-mediated + absorption and transport, effectively reducing Na + The accumulation of ions and the maintenance of ion homeostasis in tomatoes are crucial for maintaining the physiological functions of plants under salt stress.

[0154] Structural analogs of compound 2 such as Figure 14 As shown in FIG. 1 , compound 2 separated from the ethyl acetate phase has two six-membered rings and is a phenol derivative.

[0155] Structural analogs of compounds 3 and 5 such as Figure 22The structures of compounds 3 and 5 separated by n-hexane phase separation are both terpenoid derivatives. The structure of compound 3 is inferred to be a diterpenoid compound. Studies have shown that some diterpenoid compounds can exhibit strong inhibitory effects on specific bacteria and fungi. Li et al. (Li X D, Li X, Li XM, et al. 20-Nor-Isopimarane epimers produced by Aspergillus wentii SD310, a fungal strain obtained from deep sea sediment[J]. Marine Drugs, 2018, 16(11):440.) isolated a new methyl derivative of diterpenoid compound Aspewentin M from the deep sea fungus Aspergillus wentii (A. wentii) SD-310, which had an inhibitory effect on Fusarium graminearum (F. graminearum) comparable to that of the positive control amphotericin B. Aspergillus wentii Fusarium Graminearum

[0156] The structural analog of compound 4 is shown in Figure 19

[0157] Compound 4 separated by n-hexane phase separation belongs to p-benzoic acid ester derivatives, which are structurally similar to some plasticizers.

[0158] In summary, in combination with the inhibitory effect of compounds 3 and 5 on F. oxysporum, it is speculated that these two terpenoid compounds are the key metabolites of the biocontrol fungus AF-P9 that play an antagonistic role, effectively limiting the growth of pathogenic fungi and demonstrating their potential mechanism in biological control.

[0159] Example 7: Test of the control effect of the biocontrol fungus AF-P9 on tomato fusarium wilt

[0160] 1. Test design Test materials: Tomato variety: Gao Yang Da Hong. Pathogenic fungus, biocontrol fungus, and culture medium are the same as in Example 4. Test treatments: Blank control group: no inoculation of pathogenic fungus and no addition of biocontrol fungus. Control group: inoculation of F. oxysporum and no addition of biocontrol fungus. Treatment group 1: inoculation of F. oxysporum + A. wentii AF-P9. Each group was set up 3 times.

[0161] 2. Cultivation of pathogenic fungus and biocontrol fungus Same as in Example 4.

[0162] ​​​3. Tomato seedling and inoculation Seedling: Select healthy tomato seeds, immerse in 75% alcohol for 30s for the first sterilization, then immerse in 5% sodium hypochlorite for 1 min for the second sterilization, rinse with sterile water for 3 times to eliminate residual chlorine, and finally dry the surface of the seeds. The culture dishes, filter paper, tweezers and pipette tips used in the experiment are sterilized at 121℃ for 20 min. Place two filter papers in each dish, put the sterilized tomato seeds (50 seeds per dish) in the middle of the two filter papers, add 15 mL of fermentation supernatant to each dish, and repeat each dilution gradient for 3 times. Incubate at room temperature. The emergence of tomato seedlings is recorded when the radicle of the seed is white. Add sterilized nutrient soil to the seedling tray, sow the germinated seeds in the seedling tray, and maintain appropriate temperature and humidity for seedling.

[0163] 4. Inoculation of pathogenic bacteria: When the tomato seedlings grow to 3-4 true leaves, inoculate with Fusarium oxysporum spore suspension. The inoculation amount of F. oxysporum is 50 mL per dish, and the concentration of the bacterial solution is 1×10^6 spores / mL. Inoculation of biocontrol fungi: Inoculate biocontrol fungi 3 days before inoculation of pathogenic bacteria, and irrigate with biocontrol fungus suspension. That is, irrigate with biocontrol fungus suspension for 3 days, then inoculate F. oxysporum. The inoculation amount of biocontrol fungi is 50 mL per dish, and the concentration of the bacterial solution is 1×10^7 CFU / mL. The control group is irrigated with the same amount of sterile water instead of biocontrol fungus suspension. The blank control group is irrigated with the same amount of sterile water instead of F. oxysporum spore suspension and biocontrol fungus suspension.

[0164] 5. Seedling stage management Environmental control: maintain temperature at 25-30℃ and humidity at 70%-80% to simulate suitable conditions for disease occurrence. Water management: keep the soil moist to avoid excessive dryness or wetness.

[0165] 6. Data collection Measure the growth indicators of the plants 30 days after inoculation of biocontrol fungi, and count the incidence of tomato, using the same method as in Example 4.

[0166] 7. Matters needing attention Same as Example 4. The test results are shown in Tables 12-13.

[0167] Table 12 Detection results of tomato plant growth indicators in different test groups

[0168]

[0169] As shown in Table 12, F. oxysporum significantly inhibited the growth of tomato. The aboveground fresh weight and underground fresh weight of tomato in the treatment group inoculated with AF-P9 strain and F. oxysporum were significantly improved compared with the treatment group inoculated with F. oxysporum only. AF-P9 strain can improve the incidence of F. oxysporum to some extent.

[0170] Table 13 Disease incidence of tomato plants in different test groups

[0171]

[0172] As shown in Table 13, the AF-P9 biocontrol fungus can significantly improve the Fusarium oxysporum disease incidence and reduce the incidence of tomato wilt.

[0173] Although the above embodiments have been described in detail, they are only some embodiments of the present application, not all embodiments, and other embodiments can be obtained according to the above embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A species of Aspergillus ochraceus ( Aspergillus ochraceus ) AF-P9, characterized in that, The deposit number of the Aspergillus ochraceus AF-P9 is CGMCC No.41804.

2. A microbial preparation, characterized in that: The method comprises the Aspergillus ochraceus AF-P9 according to claim 1.

3. A method for preparing the microbial preparation according to claim 2, characterized in that: include: The Aspergillus ochraceus AF-P9 is cultured in a culture medium to obtain a microbial preparation.

4. The preparation method according to claim 3, characterized in that The culture temperature is 25-28° C., and the culture time is 5-7 days.

5. Use of the Aspergillus ochraceus AF-P9 according to claim 1, the microbial preparation according to claim 2, or the microbial preparation prepared by the preparation method according to claim 3 or 4 in the preparation of a preparation for inhibiting pathogens; the pathogens are any one or more of Fusarium oxysporum, Fusarium acuminate, Fusarium trilineata, Pythium iris, Micrococcus micrococcus, Phomopsis pseudomorpha, Sclerotinia sclerotiorum, and Phytophthora.

6. Use of the Aspergillus ochraceus AF-P9 of claim 1, the microbial preparation of claim 2, or the microbial preparation prepared by the preparation method of claim 3 or 4 in promoting plant growth and / or preventing and controlling diseases; the plants are tomatoes and / or rice; and the diseases are rice damping-off and / or tomato wilt caused by Fusarium oxysporum.

7. A method for promoting plant growth and / or preventing and controlling diseases, characterized in that: include: Applying the microbial preparation according to claim 2 during plant growth; The plant is tomato and / or rice; The disease is rice damping-off and / or tomato wilt.

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