Actinomycete strains with antagonistic fungal activity and use thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to an actinomycete strain with antagonistic activity against fungi and application thereof. The actinomycete strain with antagonistic activity against fungi is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 36167. The actinomycete fermentation liquor prepared in the application has high antagonistic activity against plant disease fungi, tinea pedis pathogenic bacteria and the like, and can solve the problems of drug resistance and environment in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an actinomycete strain with antagonistic fungal activity and its application. Background Technology

[0002] Fungal diseases pose a significant threat to agricultural production and human health. In agriculture, fungal diseases in plants lead to reduced crop yields and decreased quality; in medicine, fungal infections such as tinea capitis are becoming increasingly difficult to treat due to drug resistance. Currently, the widespread use of chemical pesticides and antibiotics has resulted in increased fungal resistance and exacerbated environmental pollution. Therefore, finding safe and highly effective new antifungal substances is of great importance.

[0003] Actinomycetes, as a major group of microorganisms in soil, produce metabolic products with rich biological activity, making them an important source of antibiotics and other drugs. In existing technologies, some actinomycete strains have been used in antifungal research. For example, Chinese patent CN 118460411 A discloses a biocontrol actinomycete MG-6 for the control of leaf spot disease in *Cercidiphyllum japonicum* and its application method. The actinomycete MG-6 is an active strain isolated and screened from the in-situ environment of *Cercidiphyllum japonicum* that has biocontrol potential against the leaf spot pathogen *Alternaria brassicae*. The specific isolation and screening process includes isolating and screening from rhizosphere soil samples of *Cercidiphyllum japonicum*, integrating morphologically similar strains, selecting the pathogenic fungus *Alternaria brassicae* as the target, and using a cross-linking method to broadly screen the selected strains to obtain the active bacterium MG-6. 6; The active bacterium MG-6 belongs to the genus Actinomycetes. Observation of the colony morphology of MG-6 revealed that MG-6 colonies on PDA medium were small and dense, white, radially arranged, with a wrinkled surface and abundant aerial hyphae. The hyphae were white and tightly bound to the culture medium, making the colonies difficult to pick up. Brown pigment was observed at the junction of the colonies with the culture medium. Scanning electron microscopy showed that the hyphae of MG-6 were rod-shaped. Morphological observation indicated that strain MG-6 possessed typical actinomycete characteristics. The strain MG-6 belongs to the genus *Amylopectinus*. Sequence determination of MG-6... Amycolatopsis umgenien and Amycolatopsis coloradensis The 16S rRNA sequences of these organisms show close homology. However, there are still shortcomings in the screening and application of highly efficient antagonistic actinomycetes against fungi causing plant diseases and pathogens causing human tinea, especially in how to improve the yield of antibacterial active substances through optimizing fermentation processes, which remains an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an actinomycete strain with antagonistic fungal activity. The actinomycete fermentation broth prepared using this actinomycete strain has a highly efficient antagonistic effect on fungi causing plant diseases and pathogens causing human tinea, which can solve the problems of drug resistance and environmental issues in the prior art.

[0005] The actinomycete strain with antagonistic fungal activity described in this invention is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and is classified as follows: Streptomyces sp., accession number CGMCC No.36167, accession date October 13, 2025.

[0006] The application of the actinomycete strain with antagonistic fungal activity described in this invention is the application of the actinomycete strain with antagonistic fungal activity to inhibit fungal growth.

[0007] Fungi are Nothophoma spiraeae , Vacuiphoma oculihominis or Trichophyton rubrum At least one of them.

[0008] One method to inhibit fungal growth is to treat fungi with actinomycete fermentation broth or an antifungal composition. The actinomycete fermentation broth is prepared by fermenting actinomycete strains with antagonistic fungal activity, and the antifungal composition is prepared by mixing actinomycete fermentation broth and a solid culture medium.

[0009] The preparation method of actinomycete fermentation broth includes the following steps: (1) After activating the actinomycete strain with antagonistic fungal activity, it was inoculated into a liquid culture medium and cultured by shaking to obtain the fermentation broth; (2) Centrifuge the fermentation broth and collect the supernatant; (3) Filter the supernatant to remove bacteria and obtain the actinomycete fermentation broth.

[0010] In step (1), the liquid culture medium is one of potato glucose medium, oat flake medium or ISP series medium.

[0011] The conditions for shaking culture in step (1) are 25-28℃, 160-180r / min, and the shaking culture time is 5-7 days.

[0012] In step (2), the centrifugation speed is 3000-4000 r / min and the centrifugation time is 10-20 minutes.

[0013] In step (3), filtration is performed through a 0.22μm or 0.45μm microporous membrane.

[0014] The volume fraction of actinomycete fermentation broth in the antifungal composition is 15-20%, and the solid culture medium is one of potato dextrose agar, oatmeal agar, or ISP series culture medium.

[0015] All culture media used undergo strict sterilization. To prevent contamination by other microorganisms, inoculation, centrifugation, and filtration are all performed in a clean bench. Microporous membranes are sterilized before use to ensure sterility and are thoroughly sterilized before use to ensure sterile fermentation broth.

[0016] The actinomycete strain with antagonistic fungal activity described in this invention Streptomyces sp. DA05 (CGMCC No. 36167) has antagonistic activity against a variety of fungi.

[0017] This invention aims to promote the development of novel antifungal drugs, the utilization of microbial resources, and the development of green plant protection, in order to meet the needs of new drugs against drug-resistant fungi, safe agriculture, and basic scientific research. It directly addresses / solves the challenge of fungal resistance, provides new solutions for the control of specific pathogenic fungi, and points out the need to further address the problem of insufficient antifungal spectrum coverage. Its core application value lies in providing important experimental evidence and candidate resources for the development of novel antifungal drugs and biocontrol agents.

[0018] The method for preparing actinomycete fermentation broth provided by this invention is simple and can be used to prepare antifungal drugs or biological pesticides, providing a new solution for the prevention and control of fungal diseases.

[0019] The beneficial effects of this invention are as follows: 1. Significant antibacterial effect: The actinomycete fermentation broth of this invention is effective against various fungi, such as... Nothophoma spiraeae , Vacuiphoma oculihominis and Trichophyton rubrum All of these have significant antibacterial effects.

[0020] 2. Simple preparation method: The fermentation process parameters are clear and easy to control, which is suitable for large-scale production and lowers the threshold for industrial application.

[0021] 3. Wide range of applications: This fermentation broth can be applied in both agricultural and medical fields. In agriculture, it can be used as a biological pesticide to control fungal diseases in plants, such as... Nothophoma spiraeae Inhibition of fungi helps reduce crop yield losses caused by fungal diseases and improves the quality of agricultural products. In medicine, it can be used to prepare antifungal drugs targeting fungal pathogens causing human tinea, such as... Trichophyton rubrum These substances have inhibitory effects, providing a new option for treating fungal infections. Furthermore, since the fermentation broth originates from the metabolic products of actinomycetes, it is a bioactive substance with environmentally friendly characteristics. Compared to chemical pesticides and antibiotics, it is less likely to induce drug resistance, making it safer for the ecological environment and human health, and meeting the requirements of green environmental protection and sustainable development. Attached Figure Description

[0022] Figure 1 It is an actinomycete.Streptomyces sp. DA05 and its fermentation broth respectively on Nothophoma spiraeae CFCC53929 Vacuiphoma oculihominis UTHSC DI16-308 and Trichophyton rubrum Image showing the antibacterial effect of ATCCMYA-4438. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments.

[0024] Example 1 A method for screening actinomycete strains with antagonistic fungal activity includes the following steps: (1) Sampling was conducted at 36°22'N 117°31′E. A soil sample of 25cm topsoil was collected. 1.0g of the soil sample was weighed and added to an Erlenmeyer flask containing 99mL of sterile water. After mixing, 0.5mL of the mixture was added to a test tube containing 4.5mL of sterile water. The mixture was blown and aspirated three times and shaken to obtain a soil suspension. (2) The test strains were inoculated using the spot inoculation method. Nothophoma spiraeae Inoculate the center of CFCC53929 on PDA plates, with 10 plates per strain, and incubate at 28°C for 3 days. Add 0.1 mL of soil suspension around the colonies, and use an inoculation loop to spread the soil suspension around the colonies. Continue incubating at 28°C for 7-14 days, observing growth daily. Continue incubation until actinomycetes grow on the plates and respond to indicator bacteria. Nothophoma spiraeae The bacterial group CFCC53929, exhibiting significant antibacterial activity, was purified using the streak plating method until single colonies were obtained. The isolate was then identified and named based on 16S rDNA. Streptomyces sp. DA05 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36167.

[0025] The preparation method of actinomycete fermentation broth includes the following steps: (1) Preparation of liquid culture medium: Potato glucose medium was autoclaved for 15 minutes to obtain liquid culture medium; (2) Inoculation and culture: Actinomycete strains with antagonistic fungal activity are inoculated and cultured. Streptomyces After activation of sp. DA05, three independent colonies were inoculated into 100 mL of liquid culture medium from step (1), and placed in a constant temperature shaking incubator. The mixture was shaken and cultured for 6 days at 28℃ and 160 r / min to obtain the fermentation broth. (3) Centrifugation and filtration: Take 4 mL of the fermentation broth from step (2), centrifuge at 4000 r / min for 10 minutes, and collect the supernatant; use a disposable syringe to extract the supernatant, filter it through a 0.45 μm microporous membrane to remove bacteria, and obtain the actinomycete fermentation broth.

[0026] Confrontation experiment of actinomycetes: (1) Preparation of solid culture medium: Potato glucose agar medium was autoclaved for 20 min, and then plated and cooled to obtain solid culture medium; (2) On the solid culture medium in step (1), use a ruler to measure and find the center point of the plate, and place the test bacteria thereon. Nothophoma spiraeae CFCC53929 was inoculated into solid culture medium, and an actinomycete strain with antagonistic fungal activity was added. Streptomyces sp. DA05 was inoculated in a cross-hatching manner on the test bacteria. Nothophoma spiraeae CFCC53929 was placed 2 cm away from the test group (confrontation group), while the control group consisted of only the test strain and no actinomycete strain. This was used to determine the end time of culture. The inoculated solid culture plates were placed in a 28°C incubator to observe the growth of the microorganisms. The results are shown in […]. Figure 1 ; (3) The cross-multiplication method was used to measure the strains tested. Nothophoma spiraeae The diameter of CFCC53929 colonies was measured, and each treatment was repeated three times. The antagonistic effect of the antagonistic bacteria was evaluated by the inhibition rate. The results are shown in Table 1.

[0027] Inhibition rate (%) = (Average colony diameter of control group - Average colony diameter of experimental group) / Average colony diameter of control group * 100% The methods for inhibiting fungal growth are as follows: The actinomycete fermentation broth was rapidly mixed with potato dextrose agar medium preheated to 50°C and shaken well. 20 mL of the mixture was poured onto a plate and allowed to cool and solidify to prepare potato dextrose agar plates containing the actinomycete fermentation broth (20% by volume). The test strain was inoculated at the center of each potato dextrose agar plate containing the actinomycete fermentation broth. Nothophoma spiraeae CFCC53929 was used as the experimental group, i.e., the fermentation group. Each tested strain was inoculated into the center of a potato dextrose agar plate containing no actinomycete fermentation broth as a control group to determine the culture termination time. The inoculated culture plates were placed in a 28°C incubator for cultivation, and the growth of the microorganisms was observed. The results are shown in […]. Figure 1 The diameter was measured using the cross method (experimental group / control group × 3 replicates), and the inhibition rate was calculated according to the formula. The results are shown in Table 2.

[0028] Actinomycete fermentation broth on Nothophoma spiraeaeCFCC53929 has an antibacterial rate of 88.28%, demonstrating excellent antibacterial activity.

[0029] The confrontation experiment with actinomycetes and the method of inhibiting fungal growth were carried out simultaneously, with both using the same control group.

[0030] Example 2 The preparation method of actinomycete fermentation broth includes the following steps: (1) Preparation of liquid culture medium: Potato glucose medium was autoclaved for 20 minutes to obtain liquid culture medium; (2) Inoculation and culture: Actinomycete strains with antagonistic fungal activity are inoculated and cultured. Streptomyces After activation of sp. DA05, three independent colonies were inoculated into 100 mL of liquid culture medium from step (1), and placed in a constant temperature shaking incubator. The mixture was shaken and cultured for 5 days at 26℃ and 170 r / min to obtain the fermentation broth. (3) Centrifugation and filtration: Take 4 mL of the fermentation broth from step (2), centrifuge at 3000 r / min for 20 minutes, and collect the supernatant; use a disposable syringe to extract the supernatant, filter it through a 0.22 μm microporous membrane to remove bacteria, and obtain the actinomycete fermentation broth.

[0031] Confrontation experiment of actinomycetes: (1) Preparation of solid culture medium: Potato glucose agar medium was autoclaved for 15 min, and then plated and cooled to obtain solid culture medium; (2) On the solid culture medium in step (1), use a ruler to measure and find the center point of the plate, and place the test bacteria thereon. Vacuiphoma oculihominis UTHSC DI16-308 was inoculated into solid culture medium, and actinomycete strains with antagonistic fungal activity were introduced. Streptomyces sp. DA05 was inoculated in a cross-hatching manner on the test bacteria. Vacuiphoma oculihominis UTHSC DI16-308 was placed 2 cm apart as the experimental group (or confrontation group), while the control group consisted of only the tested strain and no actinomycete strain. This was used to determine the end time of culture. The inoculated solid culture plates were placed in a 28°C incubator for microbial growth, and the results are shown below. Figure 1 ; (3) The cross-multiplication method was used to measure the strains tested. Vacuiphoma oculihominis UTHSC DI16-308 colony diameter was measured, and each treatment was repeated three times. The antagonistic effect of the antagonistic bacteria was evaluated by the inhibition rate. The results are shown in Table 1.

[0032] Inhibition rate (%) = (Average colony diameter of control group - Average colony diameter of experimental group) / Average colony diameter of control group * 100% The methods for inhibiting fungal growth are as follows: The actinomycete fermentation broth was rapidly mixed with potato dextrose agar medium preheated to 50°C and shaken well. 20 mL of the mixture was poured onto a plate and allowed to cool and solidify to prepare potato dextrose agar plates containing the actinomycete fermentation broth (15% by volume). The test strain was inoculated at the center of each potato dextrose agar plate containing the actinomycete fermentation broth. Vacuiphoma oculihominis UTHSCDI16-308 was used as the experimental group, i.e., the fermentation group. Each tested strain was inoculated into the center of a potato dextrose agar plate containing no actinomycete fermentation broth as a control group to determine the end of the culture. The inoculated culture plates were placed in a 28°C incubator for cultivation, and the growth of the microorganisms was observed. The results are shown in […]. Figure 1 The diameter was measured using the cross method (experimental group / control group × 3 replicates), and the inhibition rate was calculated according to the formula. The results are shown in Table 2.

[0033] Actinomycete fermentation broth on Vacuiphoma oculihominis UTHSC DI16-308 exhibited an antibacterial rate of 81.09%, demonstrating excellent antibacterial activity.

[0034] The confrontation experiment with actinomycetes and the method of inhibiting fungal growth were carried out simultaneously, with both using the same control group.

[0035] Example 3 The preparation method of actinomycete fermentation broth includes the following steps: (1) Preparation of liquid culture medium: Potato glucose medium was autoclaved for 18 minutes to obtain liquid culture medium; (2) Inoculation and culture: Actinomycete strains with antagonistic fungal activity are inoculated and cultured. Streptomyces After activation of sp. DA05, three independent colonies were inoculated into 100 mL of liquid culture medium from step (1), and placed in a constant temperature shaking incubator. The mixture was shaken and cultured for 7 days at 25°C and 180 r / min to obtain the fermentation broth. (3) Centrifugation and filtration: Take 4 mL of the fermentation broth from step (2), centrifuge at 3500 r / min for 15 minutes, and collect the supernatant; use a disposable syringe to extract the supernatant, filter it through a 0.45 μm microporous membrane to remove bacteria, and obtain the actinomycete fermentation broth.

[0036] Confrontation experiment of actinomycetes: (1) Preparation of solid culture medium: Potato glucose agar medium was autoclaved for 15 min, and then plated and cooled to obtain solid culture medium; (2) On the solid culture medium in step (1), use a ruler to measure and find the center point of the plate, and place the test bacteria thereon. Trichophyton rubrumATCC MYA-4438 was inoculated into solid culture medium, and an actinomycete strain with antagonistic fungal activity was added. Streptomyces sp. DA05 was inoculated in a cross-hatching manner on the test bacteria. Trichophyton rubrum ATCC MYA-4438 was placed 2 cm apart, serving as the experimental group (or confrontation group). The control group consisted of only the tested strain and no actinomycete strain, used to determine the end of the culture. The inoculated solid culture plates were placed in a 28°C incubator for microbial growth, and the results are shown below. Figure 1 ; (3) The cross-multiplication method was used to measure the strains tested. Trichophyton rubrum The diameter of ATCC MYA-4438 colonies was measured, and each treatment was repeated three times. The antagonistic effect of the antagonistic bacteria was evaluated by the inhibition rate. The results are shown in Table 1.

[0037] Inhibition rate (%) = (Average colony diameter of control group - Average colony diameter of experimental group) / Average colony diameter of control group * 100% The methods for inhibiting fungal growth are as follows: The actinomycete fermentation broth was rapidly mixed with potato dextrose agar medium preheated to 50°C and shaken well. 20 mL of the mixture was poured onto a plate and allowed to cool and solidify to prepare potato dextrose agar plates containing the actinomycete fermentation broth (19% by volume). The test strain was inoculated at the center of each potato dextrose agar plate containing the actinomycete fermentation broth. Trichophyton rubrum ATCC MYA-4438 was used as the experimental group, i.e., the fermentation group. Each tested strain was inoculated into the center of a potato dextrose agar plate containing no actinomycete fermentation broth as a control group to determine the end of the culture. The inoculated culture plates were placed in a 28°C incubator for cultivation, and the growth of the microorganisms was observed. The results are shown in […]. Figure 1 The diameter was measured using the cross method (experimental group / control group × 3 replicates), and the inhibition rate was calculated according to the formula. The results are shown in Table 2.

[0038] Actinomycete fermentation broth on Trichophyton rubrum ATCC MYA-4438 has an inhibition rate of 91.75%, demonstrating excellent antibacterial activity.

[0039] The confrontation experiment with actinomycetes and the method of inhibiting fungal growth were carried out simultaneously, with both using the same control group.

[0040] Table 1 Actinomycetes Streptomyces Results of the confrontation test of sp. DA05 against 3 fungal strains

[0041] Table 2 Actinomycetes [[ID= Results of sp. DA05 fermentation broth antagonizing 3 strains of fungi

[0042] As shown in Tables 1 and 2, the actinomycete fermentation broth of the present invention exhibits highly efficient inhibitory effects on two species from the Didymellaceae family and one species from the Nummaceae family. For example, ​ The fermentation broth of sp. DA05 (CGMCC No. 36167) on the subspinipes family ​ CFCC53929 ​ ​ UTHSC DI16-308 achieved antibacterial rates of 88.28% and 81.09%, respectively, against *Nyctaginaceae* species. ​ ATCC MYA-4438 achieved an inhibition rate of 91.75%, indicating that the actinomycetes... ​ sp. DA05 (CGMCC No. 36167) exhibits good broad-spectrum antibacterial potential. Compared with confrontation experiments with actinomycetes, the fermentation broth showed significantly better antibacterial effect. ​ sp. DA05 (CGMCC No. 36167) ​ The inhibition rate of ATCC MYA-4438 in the confrontation test was only 54.90%, while the inhibition rate of fermentation broth increased to 91.75%. The results show that the fermentation broth can more effectively exert the antibacterial activity of actinomycete metabolites, providing a more powerful means for the prevention and control of fungal diseases.

[0043] Fluconazole ​ The in vitro antifungal rate of dermatophytes is usually 30-50%, and fluconazole is effective against... ​ The antibacterial rate of fungi is only 20-45%, and it has a relatively low inhibitory effect on fungi. ​ Terbinafine showed an inhibition rate of less than 30% against some tested fungi; ​ CFCC53929 showed no significant inhibitory effect (antibacterial inhibition rate <20%), with an overall antibacterial rate ranging from 15-38%. The actinomycetes of this invention... ​ The fermentation broth of sp. DA05 on ​ , ​ and ​ ​ Its antibacterial rate is 81.09-91.75%, which is significantly better than fluconazole and terbinafine, with a broader antibacterial spectrum and stronger activity.

Claims

1. An actinomycete strain with antagonistic fungal activity, characterized in that... It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36167.

2. The application of the actinomycete strain with antagonistic fungal activity as described in claim 1, characterized in that... Actinomycete strains with antagonistic fungal activity are used to inhibit fungal growth.

3. The application of the actinomycete strain with antagonistic fungal activity according to claim 2, characterized in that... Fungi are Nothophoma spiraeae , Vacuiphoma oculihominis or Trichophytonrubrum At least one of them.

4. The application of the actinomycete strain with antagonistic fungal activity according to claim 2, characterized in that... One method to inhibit fungal growth is to treat fungi with actinomycete fermentation broth or an antifungal composition. The actinomycete fermentation broth is prepared by fermenting actinomycete strains with antagonistic fungal activity, and the antifungal composition is prepared by mixing actinomycete fermentation broth and a solid culture medium.

5. The application of the actinomycete strain with antagonistic fungal activity according to claim 4, characterized in that... The preparation method of actinomycete fermentation broth includes the following steps: (1) After activating the actinomycete strain with antagonistic fungal activity, it was inoculated into a liquid culture medium and cultured by shaking to obtain the fermentation broth; (2) Centrifuge the fermentation broth and collect the supernatant; (3) Filter the supernatant to remove bacteria and obtain the actinomycete fermentation broth.

6. The application of the actinomycete strain with antagonistic fungal activity according to claim 5, characterized in that... In step (1), the liquid culture medium is one of potato glucose medium, oat flake medium or ISP series medium.

7. The application of the actinomycete strain with antagonistic fungal activity according to claim 5, characterized in that... The conditions for shaking culture in step (1) are 25-28℃, 160-180r / min, and the shaking culture time is 5-7 days.

8. The application of the actinomycete strain with antagonistic fungal activity according to claim 5, characterized in that... In step (2), the centrifugation speed is 3000-4000 r / min and the centrifugation time is 10-20 minutes.

9. The application of the actinomycete strain with antagonistic fungal activity according to claim 5, characterized in that... In step (3), filtration is performed through a 0.22μm or 0.45μm microporous membrane.

10. The application of the actinomycete strain with antagonistic fungal activity according to claim 4, characterized in that... The volume fraction of actinomycete fermentation broth in the antifungal composition is 15-20%, and the solid culture medium is one of potato dextrose agar, oatmeal agar, or ISP series culture medium.

Images