A new streptomyces species capable of producing volatile bacteriostatic substances and its use

Abstract

The present application relates to the field of microorganism, especially to a new Streptomyces species capable of producing volatile bacteriostatic substances and application thereof.The Streptomyces strain AMJ-169 identified in the present application is a new Streptomyces species, which also has significant inhibitory activity on strawberry anthracnose fungus, cucumber fusarium wilt fungus, tomato gray mold fungus and the like.The volatile organic compounds produced by the strain AMJ-169 have significant inhibitory activity on papaya anthracnose fungus, mango anthracnose fungus, banana anthracnose fungus and the like;the volatile organic compounds and the volatile compounds thereof also have inhibitory activity on various plant pathogenic fungi.Among them, the levorotatory-alpha-pinene can effectively reduce the strawberry anthracnose lesion diameter on strawberry fruit, and has significant inhibitory activity on strawberry anthracnose.The new Streptomyces species and the volatile bacteriostatic substances produced thereby provide a new way for the biological control of strawberry anthracnose, and have broad development space and development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to a new species of Streptomyces capable of producing volatile antibacterial substances and its applications. Background Technology

[0002] Strawberry anthracnose primarily affects the leaves, petioles, runners, rhizomes, flowers, and fruits of strawberries, and has become one of the major diseases threatening the development of the strawberry industry, following strawberry gray mold and powdery mildew. Once infected, strawberry anthracnose can cause fruit rot and plant wilting; in severe years, it can lead to extensive fruit decay. After infection, timely control with chemical agents such as difenoconazole, cyproconazole, and mancozeb is necessary. However, the use of chemical agents can easily lead to increased drug resistance in pathogens, resulting in increased pesticide residues in strawberries.

[0003] Biological control utilizes the interrelationships between biological species to suppress one or more species. Its greatest advantage is that it does not pollute the environment and does not lead to increased drug resistance in pathogens. Compared with chemical control, it has the outstanding characteristics of being green and environmentally friendly, providing a new approach to the control of strawberry anthracnose. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new species of Streptomyces capable of producing volatile antibacterial substances and its applications.

[0005] The first aspect of the present invention is to provide a Streptomyces AMJ-169 capable of producing volatile antibacterial substances. Streptomyces sp. AMJ-169), classified as Streptomyces sp It is registered and preserved at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67083, and the preservation date is October 13, 2025.

[0006] A second aspect of the invention is to provide a fermentation broth of Streptomyces AMJ-169 as described in the first aspect of the invention.

[0007] A third aspect of the invention is to provide volatile gases and / or volatile organic compounds produced by *Streptomyces AMJ-169* as described in the first aspect of the invention.

[0008] Furthermore, the volatile organic compounds are ethyl isobutyrate, L-α-pinene, β-pinene, and / or isooctyl acetate.

[0009] A fourth aspect of the present invention is to provide a method for preparing volatile gases from Streptomyces AMJ-169 as described in the first aspect of the present invention, comprising the following steps:

[0010] The Streptomyces AMJ-169 strain was inoculated onto YE solid medium, sealed, and cultured. Volatile organic matter was then collected.

[0011] The fifth aspect of the present invention is to provide the use of Streptomyces AMJ-169 as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, in the preparation of preparations that inhibit plant pathogens and / or diseases caused by said plant pathogens, said plant pathogens being: strawberry anthracnose fungus, banana wilt pathotype 4, cucumber wilt fungus, banana wilt pathotype 1, tomato gray mold fungus, and / or tomato wilt fungus.

[0012] A sixth aspect of the present invention is to provide the use of the volatile organic compound as described in the third aspect of the present invention in the preparation of an antimicrobial agent and / or an antimicrobial agent for plant diseases caused by said plant pathogens, said plant pathogens being: strawberry anthracnose, banana anthracnose, mango anthracnose and / or papaya anthracnose.

[0013] A seventh aspect of the present invention is to provide the use of the volatile organic compound as described in the third aspect of the present invention in the preparation of an agent for inhibiting plant pathogens and / or diseases caused by said plant pathogens, said plant pathogens being: *Anthracnose spp.* strawberry and / or *Anthracnose spp.* mango; said volatile organic compound being ethyl isobutyrate, L-α-pinene, β-pinene, and / or isooctyl acetate.

[0014] The eighth aspect of the present invention is to provide the use of the volatile organic compound as described in the third aspect of the present invention in the preparation of formulations for inhibiting plant pathogens and / or diseases caused by said plant pathogens, wherein the plant pathogens are: Fusarium wilt race 4 of banana, Fusarium wilt of cucumber, Botrytis cinerea of ​​tomato, Fusarium wilt of tomato and / or Fusarium graminearum of wheat; wherein the volatile organic compound is L-α-pinene.

[0015] A ninth aspect of the present invention is to provide a method for preventing and controlling strawberry anthracnose, comprising the following steps:

[0016] The volatile organic compound described in the third aspect of this invention is simultaneously sealed and stored with strawberry fruit; the volatile organic compound is levorotatory-α-pinene.

[0017] The tenth aspect of the present invention is to provide an antibacterial agent containing Streptomyces AMJ-169 as described in the first aspect of the present invention, fermentation broth as described in the second aspect, volatile gases as described in the third aspect, and / or volatile organic compounds produced by the Streptomyces.

[0018] The *Streptomyces* strain AMJ-169 involved in this invention has been identified as a new species of *Streptomyces*, exhibiting significant inhibitory activity against *Streptomyces* var. *stella*, the causal agent of strawberry anthracnose, and also showing significant inhibitory activity against *Fusarium wilt* race 4, *Fusarium wilt* var. *stella*, *Fusarium wilt* var. *stella* race 1, *Botrytis cinerea*, and *Fusarium wilt* var. *stella*. The volatile organic compounds produced by strain AMJ-169 have certain inhibitory activity against *Streptomyces* var. *stella*, *Fusarium wilt* var. *stella*, *Fusarium wilt* var. *stella*, and *Fusarium wilt* var. *stella*. The volatile organic compounds identified from the volatile organic compounds, including ethyl isobutyrate, L-α-pinene, β-pinene, and / or isooctyl acetate, all exhibit varying degrees of inhibitory activity against *Streptomyces* var. *stella* and *Fusarium wilt* var. *stella*. The volatile organic compound L-α-pinene exhibits the best inhibitory activity against strawberry anthracnose fungus. L-α-pinene also significantly reduces the diameter of anthracnose lesions on strawberry fruits, achieving an inhibition rate of 88.21%, demonstrating a significant control effect. The new Streptomyces species and its produced volatile antibacterial substances described in this invention provide a new approach for the biological control of strawberry anthracnose, with broad development potential and application prospects. Attached Figure Description

[0019] Figure 1 The culture characteristics of isolated soil actinomycetes.

[0020] Figure 2 The initial screening results for antagonistic actinomycetes.

[0021] Figure 3 The results of the secondary screening for antagonistic actinomycetes.

[0022] Figure 4 Phylogenetic tree of strain AMJ-169 constructed based on 16S rRNA sequence.

[0023] Figure 5 Calculation of ANI and DDH for strain AMJ-169.

[0024] Figure 6 The characteristics of strain AMJ-169 on different culture media.

[0025] Figure 7 The results show the screening results for antibacterial active ingredients.

[0026] Figure 8 This is a gas chromatogram of strain AMJ-169.

[0027] Figure 9 The results show the inhibition rate of volatile organic compounds in strain AMJ-169.

[0028] Figure 10 The results show the broad-spectrum antibacterial activity of strain AMJ-169 and levorotatory-α-pinene.

[0029] Figure 11 Results of antibacterial rate determination for different concentrations of levorotatory-α-pinene.

[0030] Figure 12 Images of strawberry fruit tested with levorotatory α-pinene.

[0031] Figure 13 Statistics on the diameter of plaques from the levorotatory-α-pinene strawberry anthracnose fungus.

[0032] The Streptomyces AMJ-169 described in this invention ( Streptomyces sp. AMJ-169), classified as Streptomyces sp It is registered and deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67083, deposit date October 13, 2025, and deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0034] Example 1

[0035] I. Isolation of Actinomycetes from Soil

[0036] Soil samples were collected from the forests of Mojiang Hani Autonomous County, Pu'er City, Yunnan Province, China. Larger impurities and lumps were manually removed from the collected soil, which was then dried at room temperature in a fume hood for 3 days. Subsequently, the soil was ground in a mortar and pestle and passed through a sieve (10-20 mesh) three times. 1 g of soil was weighed into a 10 ml centrifuge tube using an electronic balance and diluted with 9 ml of sterile water to a 10 ml suspension. This process was repeated to achieve a 10 ml suspension. -2 and 10 -3 Suspension. Then, 100 μl of each of the three dilutions was pipetted onto Gao's No. 1 medium and spread using a spreader until the surface of the petri dish was free of water stains. The plates were then incubated upside down in a 28°C biochemical incubator for one week. Based on color, growth time, hyphal morphology, and colony size, different strains were selected and transferred to fresh YE medium for strain isolation and purification. Figure 1 Store in 30% glycerol and in an ultra-low temperature freezer at -80°C.

[0037] II. Screening for actinomycetes (primary screening, secondary screening)

[0038] Initial screening: The isolated strains were screened for antagonistic activity using the four-point confrontation method. First, four isolated actinomycetes were placed at four points 2.5 cm from the center of the PDA medium. After incubation at 28°C for 3 days to allow the actinomycetes to grow, a 5 mm diameter mycelial disc of *Colletotrichum fragariae* (ATCC 58718), the causal agent of strawberry anthracnose, was inoculated at the center of the medium. The culture was then kept at a constant temperature of 28°C for 7 days in a biochemical incubator. The experiment was repeated three times.

[0039] Antibacterial rate = [(Control group colony diameter - Treated group colony diameter) / Control group colony diameter] × 100%

[0040] Secondary screening: Actinomycetes were streaked onto YE medium and incubated at 28°C for 1–2 days. Strawberry anthracnose pathogens (5 mm in diameter) were inoculated onto PDA medium. The actinomycete-inoculated dish was placed upside down on the pathogen-inoculated dish, with the bottoms facing each other. The gaps between the two dishes were sealed with sealing film to create a closed space for volatile organic compounds (VOCs). The control group consisted of actinomycete dishes containing only sterile medium or no inoculation, and were incubated upside down at 28°C for 7 days. Smaller colony diameters indicated stronger VOCs-inhibiting activity. The experiment was repeated in triplicate. The colony diameter was measured using the cross-sectional method, and the inhibition rate was calculated.

[0041] Among them, strain AMJ-169 had an inhibition rate of 63% ( Figure 2 The VOCs inhibition rate of strain AMJ-169 was 75%. Figure 3 ).

[0042] Example 2

[0043] I. Identification of strain AMJ-169

[0044] Phylogenetic tree constructed based on 16S rRNA sequence

[0045] a. Rapid extraction of actinomycete DNA: Total DNA from actinomycetes was extracted using the Bioteke Bacterial Genomic DNA Rapid Extraction Kit.

[0046] b. 16S rRNA sequence amplification: Using the genome of the target actinomycete as a template, PCR amplification of the actinomycete was performed using the Weietal 2020 method. The universal primers for 16S rRNA were: upstream: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream: 5'TACGGCTACCTTGTTACGACTT-3'. The 50 μL PCR reaction volume is shown in Table 4. PCR amplification program: pre-denaturation 95℃ (5 min), denaturation 94℃ (1 min), annealing 55℃ (1 min), extension 72℃ (2 min), final extension 72℃ (10 min), storage at 4℃. There were 32 cycles from denaturation to annealing to extension, followed by the subsequent steps.

[0047] Table 1 PCR reaction system

[0048]

[0049] c. Electrophoretic detection and delivery of PCR products: Prepare an agarose gel (1%), wait 15 min for solidification, then take 5 μL of PCR product and 5 μL of marker and place the gel in an electrophoresis tank containing 1×TAE electrophoresis buffer. Run at 150V for 25 min. After electrophoresis, observe the results at 254 nm using a UV-Vis spectrophotometer. If there is a PCR band at 1400 bp, send the PCR product to Shanghai Sangon Biotech Co., Ltd. for 16S rRNA sequencing.

[0050] d. Phylogenetic Tree Construction: After receiving the sequencing data, the obtained sequences were uploaded to the EzBioCloud and GenBank databases for homology comparison and to find and download highly similar 16S rRNA gene sequences. Multiple comparison analyses were performed on selected strains, and a phylogenetic tree was constructed using MEGA 7.0 software with a neighbor-joining method and a Bootstrap check value set to 1000. Results are as follows: Figure 4 As shown, strain AMJ-169 has the highest similarity (98.44%) to the type strain Streptomyces roseirectus CRXT-G-22 and is most closely related. Preliminary identification suggests that this strain belongs to the genus Streptomyces. Figure 4 ).

[0051] (2) Genome alignment of antagonistic actinomycetes with closely related species: The genome sequences of the standard strain were downloaded from the NCBI database using the Nucleotide algorithm and the EzBioCloud database. The genomes of the standard strain and the test strain were uploaded to EzBicloud to calculate the average nucleotide identity (ANI) (Yun Tianyan 2020a) to determine the taxonomic position of the test strain. The genome data of related strains were downloaded from NCBI or EzBicloud, and the genomes of related strains and strain AMJ-169 were uploaded to EzBicloud for ANI calculation and uploaded to (https: / / ggdc.mz.de) for DNA-DNA hybridization (DDH) calculation. The results showed that the ANI value of strain AMJ-169 and its closely related species was less than the 95% new species threshold, and the DDH value was less than the 70% new species threshold (…). Figure 5 Based on a comparison of physiological and biochemical characteristics, strain AMJ-169 was identified as a new species of Streptomyces and classified as such. Streptomyces sp It is registered and deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67083, deposit date October 13, 2025, and deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0052] II. Culture characteristics of strain AMJ-169

[0053] Prepare ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, and Gao's No. 1 medium, and pour the plates for later use. Inoculate strain AMJ-169 into the above plates and incubate at 28℃ for 7–10 days. Observe the culture characteristics of antagonistic strains according to "Rapid Identification and Systematic Classification of Actinomycetes" and "Identification Methods of Streptomyces" (Shirlinetal 1966); compare colony colors using the ISCC-NBS (Cao Hong 2013) color spectrum (Table 2 and...). Figure 6 ).

[0054] The physiological and biochemical characteristics of the actinomycetes to be tested were identified in accordance with the guidelines in "Systematics of Actinomycetes - Principles, Methods and Practice", mainly including carbon and nitrogen source utilization tests, pH and NaCl tolerance tests, and metabolite tests (Xu Lihua et al., 2007). Specific results are shown in Tables 3-6.

[0055] (1) Carbon source utilization experiment: Using Pugol's medium as the base medium, 1% of different carbon sources were added to prepare corresponding carbon source mediums, and the plates were sterilized and poured. The strain AMJ-169 was inoculated on different carbon source medium plates, with a negative control. The plates were incubated at 28℃ for 7 days, and the growth was observed and recorded.

[0056] (2) Nitrogen source utilization experiment: Mix 1% of different types of nitrogen sources in the basic culture medium to prepare the corresponding nitrogen source culture medium, inoculate strain AMJ-169 into it, set up a negative control, and incubate at 28℃ for 7 days. Then observe and record its growth.

[0057] (3) pH tolerance test: Liquid Bennett medium was used as the base medium, and pH gradient medium ranging from 4.0 to 10.0 was prepared. The strain AMJ-169 was inoculated and cultured on a shaker (28℃, 180r / min) for 7 to 14 days. The growth status was observed and recorded.

[0058] (4) NaCl tolerance test: Different amounts of NaCl were added to liquid Bennett medium to prepare medium with NaCl concentrations of 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15%. The strain AMJ-169 was inoculated into each medium and cultured in an incubator at 28°C for 7 days to observe the growth status.

[0059] (5) Temperature tolerance test: Prepare YE medium, inoculate strain AMJ-169 into it, and place it in a constant temperature incubator with different temperature settings (15℃, 20℃, 45℃) to test whether it has the characteristics of low temperature resistance and high temperature resistance.

[0060] (6) Phosphorus solubilization and nitrogen fixation test: Prepare phosphate-solubilizing bacterial culture medium and nitrogen-fixing Assabbey medium, sterilize and pour the plates, inoculate strain AMJ-169 in the plates, and the control is an uninoculated bacterial plate. After incubation at 28℃ for 1 week, observe the bacterial plates in the culture medium. If there is a transparent zone, it has the ability to solubilize phosphorus and fix nitrogen. If not, it means that it does not have the ability.

[0061] (7) Metabolite test

[0062] a. Hydrogen sulfide detection: Inoculate strain AMJ-169 onto hydrogen sulfide detection medium and incubate at 28°C for 2 weeks. Observe the medium. If the medium turns black, the result is positive; if the medium does not turn black, the result is negative.

[0063] b. IAA production detection: Inoculate strain AMJ-169 into King culture and culture on a shaker (28℃, 180r / min) for one week. Take an appropriate amount of bacterial culture into a 2ml centrifuge tube, centrifuge at 8000r / min for 3min, take 60µL of supernatant and mix with 60µL Lalkowski colorimetric solution, react in the dark for 15min. The negative control is the supernatant without colorimetric reagent, and the positive control is 60µg / mL IAA mixed with 60µL Lalkowski colorimetric solution. Observe whether the treatment group turns red. If it turns red, it means that IAA is produced.

[0064] (8) Enzyme metabolite test

[0065] a. Urease test: This test determines whether the strain has the ability to produce urease. Strain AMJ-169 is inoculated onto urease medium, with an uninoculated urease medium as the control. After incubation at 28°C for 4 days, the color change of the medium is observed. If the medium turns pink, it indicates a positive result, meaning the strain has the ability to produce urease; otherwise, no color change indicates no urease production.

[0066] b. Esterase (Tween 40, Tween 60, Tween 80) test: This test determines whether the strain has the ability to produce esterase. Each Tween reagent is sterilized separately, then mixed thoroughly with the esterase medium and poured onto a plate. Strain AMJ-169 is inoculated onto the plate and incubated for one to two weeks. Observe whether there is a halo around the growth of the strain on the plate; the presence of a halo indicates a positive result, while the absence of a halo indicates a negative result.

[0067] c. Starch hydrolysis: This test determines whether the strain possesses amylase activity. Strain AMJ-169 is inoculated onto starch hydrolysis medium and incubated at 28°C for 5–7 days. Iodine solution (without mycelia) is then added around the colonies for testing. A clear zone indicates a positive result; the absence of a clear zone indicates a negative result.

[0068] d. Gelatin liquefaction: This test assesses the ability of the strain to produce proteases. Strain AMJ-169 was inoculated onto the surface of a gelatin liquefaction medium and cultured at 28°C for 3–4 days, then placed in a 4°C refrigerator for 2 hours. The control was a medium treated similarly but without inoculation. The degree of liquefaction was observed upon removal; fluidity indicated a positive result, while solidification indicated a negative result.

[0069] e. Nitrate Reduction: Inoculate strain AMJ-169 into nitrate-reducing medium and incubate at 28°C for one week. Take a small amount of the liquid in a test tube and add one drop each of indicator solution A and B. The control is a medium without inoculation. A red color in the test tube indicates a positive result, while no change indicates a negative result.

[0070] f. Cellulose Dissociation Test: Prepare sodium carboxymethyl cellulose medium, sterilize and pour the culture medium onto a plate. Inoculate strain AMJ-169 onto the plate and incubate at 28℃ for 7 days. Remove the plate and pour 1 g / L Congo red solution into it to immerse the bacterial cells. Stain for 30 min, then pour off the stain. Destain the plate with 1 mol / L NaCl solution for 30 min. Observe whether a clear zone is formed on the plaque. If a clear zone appears, the test is positive, indicating the presence of cellulase production; otherwise, it is negative.

[0071] (9) Antibiotic susceptibility test: Select a single colony of well-grown strain AMJ-169 and inoculate it into YE liquid medium. Incubate at 28℃ and 180 r / min for 3 days with shaking. Take a small amount of bacterial solution and count it under an optical microscope. Then dilute it with sterile water to 1×106 CFU / mL. Spread 100 µL of spore solution evenly on a YE medium plate. Inoculate four antibiotic filter paper discs at four points 2.5 cm from the center of the plate. Repeat the experiment three times. Incubate at 28℃ for 7 days. Observe whether there is an inhibition zone around the filter paper disc. If there is, it indicates that the actinomycete is sensitive to the antibiotic. Otherwise, it indicates resistance.

[0072] Table 2. Characteristics of strain AMJ-169 on different culture media

[0073]

[0074] Note: +++ indicates good growth; ++ indicates average growth; + indicates poor growth.

[0075] Table 3. Growth of strain AMJ-169 under different pH and salt concentration conditions.

[0076]

[0077] Note:++ indicates good growth; ++ indicates average growth; + indicates poor growth; - indicates no growth.

[0078] Table 4 Biochemical characteristics of strain AMJ-169

[0079]

[0080] Note: + indicates a positive reaction; − indicates a negative reaction.

[0081] Table 5. Carbon and nitrogen source utilization of strain AMJ-169

[0082]

[0083] Note: +++, good growth; ++, moderate growth; +, weak growth; -, no growth.

[0084] Table 6 Antibiotic susceptibility testing

[0085]

[0086] Note: "S" represents sensitivity, and "R" represents resistance.

[0087] Example 3

[0088] Screening of antibacterial active ingredients

[0089] The tested pathogens were: *Colletotrichum fragariae* (ATCC 58718), *Colletotrichum musae* (ATCC 96726), *Colletotrichum gloeosporioides* (ATCC 58222), and *Colletotrichum papayae* (ATCC20438).

[0090] Referring to the experimental method in the "re-screening" section of Example 1, the antibacterial activity of VOCs produced by the strain AMJ-169 of the present invention against various pathogens was determined.

[0091] The activated strain AMJ-169 was inoculated into soybean flour medium and cultured with shaking at a suitable temperature of 28°C and a rotation speed of 180 rpm for 7 days. The fermentation broth was centrifuged at 8000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain sterile fermentation filtrate, which was used to determine the antibacterial activity of the strain's metabolites. 100-200 μL of the sterile fermentation filtrate was added to 500 mL of PDA medium, mixed thoroughly, and then plated. Finally, anthracnose sealing film was used to seal the plates. An equal volume of soybean flour medium was added to the control, and the plates were incubated at the pathogen-suitable temperature of 28°C. After 7 days of incubation, the antibacterial effect was observed. Figure 7 ), calculate the antibacterial rate (Table 7).

[0092] Table 7. Screening results of antibacterial active ingredients

[0093]

[0094] This shows that the VOCs activity of AMJ-169 is higher than that of its metabolites, and further analysis of the VOCs in AMJ-169 will be conducted later.

[0095] Example 4

[0096] I. Identification of VOCs Components in Antagonistic Actinomycetes

[0097] Prepare 100 μL of LYE liquid medium (YE medium without agar) and sterilize at high temperature for later use. Pick single colonies of strain AMJ-169 from a well-grown bacterial plate and inoculate them into LYE liquid medium. Place the plates on a shaker and incubate at 28°C and 180 rpm for 2 days. Afterward, mix thoroughly to use as seed culture. Add 100 μL of seed culture (10...) 6The volatile organic compounds (VOCs) were inoculated into a 50 mL sterile flask containing 15 mL of YE solid medium (CFU / mL). The flask was then sealed with sterile foil and a sealing film. Distilled water was used as a control instead of the strain. After incubation at 28°C for 7 days, volatile organic compounds (VOCs) were collected and analyzed by solid-phase microextraction (SPME) and gas chromatography-tandem mass spectrometry (GC-MS). The SPME fiber tip was inserted into the upper gas space of the headspace vial through the cap. The fiber tip was then extended to expose the headspace for adsorption. The adsorption time was typically 30 minutes. During this process, the headspace vial could be placed on a mild heated plate (40°C) or a magnetic stirrer to enhance VOC release. After adsorption was complete, the SPME fiber tip was retracted and quickly removed from the headspace vial. The SPME needle was immediately inserted into the GC-MS instrument inlet, the fiber tip was extended, and thermal desorption was performed for 5 minutes (the inlet temperature was typically set to 250-280°C), injecting the VOCs into the column. The program was set to the description of Zou et al. (2022). Compound identification was performed by comparing retention times and mass spectra with the NIST05 standard library. Peak areas were quantified as relative abundance. Results are shown below. Figure 8 See Table 8.

[0098] Table 8. Major compounds detected by GC-MS in strain AMJ-169

[0099]

[0100] Based on the peak area, the top 27 compounds with the highest relative content were selected (Table 8), and four more compounds were further screened for further antibacterial activity determination (Table 9).

[0101] Table 9 shows the four antibacterial compounds selected.

[0102]

[0103] II. Determination of the anti-anthrax activity of screened volatile organic compounds

[0104] Pour PDA medium into petri dishes. Inoculate the center with a 5mm diameter anthrax bacterial cake. Place a sterile filter paper disc on the inside of the petri dish lid. Add 10 μL of the pure liquid compound onto the filter paper disc. Immediately replace the lid and seal the petri dish completely with sealing film to ensure no VOC leakage. Add an equal volume of sterile water or solvent to the control. Incubate all petri dishes at 28°C for the pathogen. Observe regularly. When the control group colonies are about to cover the plate, measure the colony diameter of the pathogen using the cross-hatching method and calculate the inhibition rate of each compound.

[0105] Table 10. Antibacterial rate of volatile compounds in strain AMJ-169

[0106]

[0107] According to Table 10 and Figure 9 The results showed that levorotatory-α-pinene had the best antibacterial rate against strawberry anthracnose, and further experiments were conducted on its broad-spectrum antibacterial activity.

[0108] Example 5

[0109] Broad-spectrum antibacterial activity assay

[0110] The tested pathogens were: Fusarium oxysporum f. sp. cubenseRace 4 (ATCC 76255) (Foc TR4), the causal agent of banana wilt; Fusarium oxysporum f. sp. cucumerinum (ATCC 204378), the causal agent of cucumber wilt; Fusarium oxysporum f. sp. cubenseRace 1 (ACCC 76244) (Foc 1), the causal agent of tomato gray mold (Botrytis cinerea) (ACCC36055); Fusarium oxysporum f. sp. Lycopersic (ACCC 36264), the causal agent of tomato wilt; and Fusarium graminearum Schwabe (ATCC MYA-4620), the causal agent of wheat scab.

[0111] The inhibition rate of strain AMJ-169 against different pathogens was determined according to the "Primary Screening" method in Example 1, "II. Screening of Actinomycetes (Primary Screening and Secondary Screening)". The inhibition rate of L-α-pinene against different pathogens was determined according to the method in Example 4, "II. Determination of the Anti-Anthrax Activity of Identified Volatile Organic Compounds". Specific results are shown in Table 11 and... Figure 10 .

[0112] Table 11 Results of broad-spectrum antibacterial activity assay of strain AMJ-169 and L-α-pinene.

[0113]

[0114] As shown in Table 11, strain AMJ-169 showed no inhibitory activity against Fusarium wilt of wheat, but exhibited certain inhibitory activity against Fusarium wilt of banana race 4, Fusarium wilt of cucumber, Fusarium wilt of banana race 1, Botrytis cinerea of ​​tomato, and Fusarium wilt of tomato, with the best inhibitory activity against Botrytis cinerea of ​​tomato.

[0115] L-α-pinene has no inhibitory activity against Fusarium wilt race 1 of banana, but it also has some inhibitory activity against Fusarium wilt race 4 of banana, Fusarium wilt of cucumber, Botrytis cinerea of ​​tomato, Fusarium wilt of tomato, and Fusarium graminearum of wheat, but the inhibition rate is generally low.

[0116] Example 6

[0117] L-α-pinene strawberry fruit experiment

[0118] L-α-pinene EC 50 Determination of the value: Pour PDA medium into a petri dish. Inoculate the center with a 5mm diameter strawberry anthracnose fungal cake. Place a sterile filter paper on the inside of the petri dish lid. Calculate the value based on the total volume of the petri dish (120cm³). 3 Different amounts of pure liquid compounds were added to filter paper, and the concentration gradients are shown in Table 12.

[0119] Table 12 Concentration gradient of liquid compounds

[0120]

[0121] Immediately after adding the solution, cover the petri dish and seal it completely with sealing film to ensure no VOCs leakage. Add an equal volume of sterile water or solvent to the control group. Incubate all petri dishes at 28°C for the pathogen. Observe regularly. When the control group colonies are about to cover the entire plate, measure the colony diameter of the pathogen using the cross-sectional method and calculate the inhibition rate at different concentrations of levo-α-pinene. Figure 11 Inhibition rate curves of different concentrations of levo-α-pinene were established, and statistical analysis was performed using SPSS statistical software. The EC50 of the compound *Streptomyces cerevisiae* was calculated according to the method of Vanewijk and Hoekstra (1993). 50 Value, EC 50 The smaller the value, the stronger the compound's activity. The calculated EC value... 50 The value is 0.018 mL / L.

[0122] In equatorial regions of strawberry fruit, sterilized fruits were manually punctured with a sterile needle (2 mm deep, 3 mm wide). Each puncture was inoculated with a 5 mm diameter pathogen cake. After air-drying at room temperature for 3 hours, the fruits were placed in 20 cm sterile petri dishes. The experiment was repeated three times, with five fruits per treatment group. Five fruits were placed in a 2-liter plastic container. Then, 1 × EC was placed in each container. 50 (1EC) 50 ), 2 × EC 50 (2EC) 50 ), 4 × EC 50 (4EC) 50 ) and 6 × EC50 (6EC) 50 The compound was inoculated at a concentration of [missing information]. It was sealed with plastic wrap at 28°C for 9 days. Changes in the fruit were observed at 0, 5, 7, and 9 days post-inoculation. Figure 12 On day 9, the petri dishes were opened, and the plaque diameter was measured according to Oztekin and Karbancioglu (2021). Figure 13 ).

[0123] Depend on Figure 12 and Figure 13 It can be seen that levo-α-pinene has a significant inhibitory effect on the disease incidence of strawberry anthracnose, of which 6×EC 50 At the specified concentration, the smallest bacterial patch diameter of strawberry anthracnose was 0.833 cm, significantly lower than the control's 7.067 cm, with an inhibition rate of 88.21%, demonstrating the best inhibitory effect.

[0124] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A Streptomyces AMJ-169 strain capable of producing volatile antibacterial substances ( Streptomyces sp.AMJ-169), characterized in that, Category naming Streptomyces sp. is registered and preserved at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67083.

2. The application of Streptomyces AMJ-169 as described in claim 1 in the preparation of preparations that inhibit plant pathogens and / or diseases caused by said plant pathogens, wherein said plant pathogens are: strawberry anthracnose fungus, banana wilt pathotype 4, cucumber wilt fungus, banana wilt pathotype 1, tomato gray mold fungus and / or tomato wilt fungus.

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