Streptomyces D43-56 and its applications

Abstract

This invention provides a Streptomyces sp., registered and preserved at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:66719. The novel Streptomyces sp. D43-56 and its crude extract exhibit broad-spectrum antibacterial activity, showing good antagonistic effects against banana anthracnose fungi and collodion fungi. Furthermore, it can cause the mycelia and spores of banana anthracnose fungi to shrink and rupture, increasing the content of soluble solids, titratable acid, soluble sugars, and vitamin C in banana pulp, thus improving pulp quality. It can also increase the activity of oxidases and polyphenol oxidases in banana peel, while decreasing the activity of cellulase, pectin methyl esterase, and polygalacturonase, and reducing the reducing sugar content. Therefore, it is a potential biological agent for the prevention and control of anthracnose and the improvement of fruit pulp quality, with broad development potential and excellent application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to a Streptomyces D43-56 and its applications. Background Technology

[0002] Bananas are an important tropical fruit, serving as a staple food worldwide and a vital economic resource in many tropical and subtropical regions. They are also highly regarded for their rich nutritional value, including carbohydrates, vitamins, and minerals. However, the banana industry faces significant challenges. These include climate and pests and diseases. In terms of post-harvest preservation and transportation, bananas are considered a menopausal fruit because they ripen rapidly after harvest and are susceptible to post-harvest diseases such as anthracnose, which severely impact fruit quality. Banana anthracnose is a fungal disease caused by *Colletotrichum musae*. This disease primarily causes latent infection during the early stages of fruit development. In the early stages, it does not significantly affect the fruit. However, after harvest, as the fruit ripens, the pathogen multiplies rapidly, leaving black spots on the peel, which accelerates fruit decay. Therefore, reducing disease development is a crucial issue for post-harvest preservation of bananas.

[0003] Rhizosphere soil microorganisms play a crucial role in regulating plant growth and development. The interactions among soil, root microorganisms, and plants are highly complex. PGPF (Proteinized Plant Pathogens and Probiotics) are microorganisms widely distributed in the rhizosphere soil of various plants, improving soil conditions to promote plant growth. Biocontrol bacteria are also widely present in the rhizosphere soil, helping plants resist pathogen infection through direct or indirect actions. Streptomyces constitutes a large group of biocontrol bacteria. Streptomyces produces abundant secondary metabolites, many of which are rich in antimicrobial substances, such as antibiotics. Research indicates that Streptomyces also possesses a large number of undiscovered potential new compounds. Therefore, Streptomyces is an important resource for biological control, and developing new Streptomyces strains is of great significance for the control of plant diseases and pests and the improvement of agricultural product quality. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Streptomyces D43-56 and its applications.

[0005] The first aspect of the present invention is to provide a Streptomyces sp., which is registered and deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC NO:66719.

[0006] A second aspect of the invention is to provide a fermentation broth of Streptomyces as described in the first aspect of the invention, or a sterile supernatant of the fermentation broth.

[0007] A third aspect of the invention is to provide an ethanol extract of the fermentation broth of Streptomyces as described in the first aspect of the invention.

[0008] A fourth aspect of the present invention is to provide a crude extract of Streptomyces as described in the first aspect of the present invention, wherein the crude extract is obtained by filtering the ethanol extract of the third aspect of the present invention through a macroporous resin, eluting the adsorbed material on the column with methanol of different concentrations, and concentrating and drying the eluent.

[0009] A fifth aspect of the present invention is to provide a formulation comprising the Streptomyces of the first aspect of the present invention, or the fermentation broth or sterile supernatant of the second aspect of the present invention, or the ethanol extract of the third aspect of the present invention, or the crude extract of the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention in the preparation of formulations antagonizing banana anthracnose, and / or collodion anthracnose, and / or banana wilt race 4, and / or litchi anthracnose, and / or wheat scab, and / or pepper anthracnose, and / or banana long spot disease, and / or strawberry anthracnose.

[0011] The seventh aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention, in the preparation of formulations for the prevention and control of diseases caused by *Anthracnose fungus*, and / or *Colletotrichum gloeosporioides*, and / or *Fusarium wilt* race 4 of banana, and / or *Anthracnose fungus* of litchi, and / or *Fusarium graminearum* of wheat, and / or *Anthracnose fungus* of pepper, and / or *Anthracnose fungus* of banana long spot, and / or *Anthracnose fungus* of strawberry.

[0012] The eighth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention in the preparation of a formulation that causes the mycelia of *Anthracis chinensis* to shrink, deform, and break, and / or causes the spores of *Anthracis chinensis* to shrink and rupture.

[0013] A ninth aspect of the invention is to provide the use of Streptomyces as described in the first aspect of the invention, or the fermentation broth as described in the second aspect of the invention, or the ethanol extract as described in the third aspect of the invention, or the crude extract as described in the fourth aspect of the invention, or the formulation as described in the fifth aspect of the invention, in the preparation of formulations that cause leakage of soluble protein, and / or soluble sugar content, and / or nucleic acid from the cell membrane of Banana anthracnose fungus.

[0014] The tenth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention in the preparation of a formulation for preventing the colonization of banana anthracnose fungus on banana peel.

[0015] The eleventh aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention, in the preparation of formulations that increase the soluble solids content, and / or titratable acid content, and / or soluble sugar content, and / or vitamin C content in banana fruit.

[0016] The twelfth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention, in the preparation of a formulation that increases the activity of oxidase and / or polyphenol oxidase in banana peel without significantly affecting the activity of catalase.

[0017] The thirteenth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention, in the preparation of a formulation that reduces the activity of cellulase, and / or pectin methyl esterase, and / or polygalacturonase in banana peel.

[0018] The fourteenth aspect of the present invention is to provide the use of Streptomyces as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the ethanol extract as described in the third aspect of the present invention, or the crude extract as described in the fourth aspect of the present invention, or the formulation as described in the fifth aspect of the present invention in the preparation of a formulation that reduces the reducing sugar content in banana peel.

[0019] The novel *Streptomyces* species D43-56 and its crude extracts of this invention exhibit broad-spectrum antibacterial activity, showing good antagonistic effects against *Anthracnose fungus*, *Colletotrichum gloeosporioides*, *Fusarium wilt* race 4, *Anthracnose fungus*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Anthracnose fungus*, *Fungus graminearum*, and *Anthracnose fungus*. Furthermore, it can cause *Anthracnose fungus* mycelia to shrink, deform, and break, and spores to shrink and rupture, reducing the content of soluble proteins, soluble sugars, and nucleic acids in the *Anthracnose fungus* cell membrane. This substance can inhibit the growth of anthracnose fungus in bananas, and also increase the content of soluble solids, titratable acid, soluble sugar, and vitamin C in banana pulp, thus improving pulp quality. Furthermore, it can increase the activity of oxidases and polyphenol oxidases in banana peels, while decreasing the activity of cellulase, pectin methyl esterase, and polygalacturonase, and reducing reducing sugar content. Therefore, it is a potential biological agent for anthracnose control and improving pulp quality, with broad development potential and excellent application prospects. Attached Figure Description

[0020] Figure 1 The images show the anti-anthrax effect and inhibition rate of the strain and its fermentation broth, with the upper right image showing the strain and the lower right image showing the fermentation broth.

[0021] Figure 2 The morphology of strain D43-56 under SEM.

[0022] Figure 3 Phylogenetic tree of 16S rRNA for strain D43-56.

[0023] Figure 4 Calculate the ANI and DDH for 11 bacteria adjacent to each other in the phylogenetic tree.

[0024] Figure 5 The antibacterial activity of four methanol-eluted components of the fermentation broth of strain D43-56 was evaluated (A, antibacterial effect; B, mycelial diameter; C, antibacterial rate).

[0025] Figure 6 The antibacterial spectrum of bacteria D43-56 and its extracts.

[0026] Figure 7 Gradient antibacterial test of the extract (A, antibacterial effect; B, growth radius of the mycelial cake).

[0027] Figure 8 For 4×EC 50 Morphology of mycelia in concentration and control treatments under SEM and inverted microscope.

[0028] Figure 9 For 4×EC 50Effects of treatment on spore germination and morphology after control treatment (A, spore germination test; B, spore morphology under SEM; C, spore germination rate).

[0029] Figure 10 The effect of the extract on the cell membrane of C. musae.

[0030] Figure 11 The control effect of extract of fungus D43-56 on banana anthracnose (A, control effect; B, lesion area).

[0031] Figure 12 The effect of extract of fungus D43-56 on the colonization of C. musae on banana peel.

[0032] Figure 13 The effect of the extract of strain D43-56 on the quality of banana fruit was determined.

[0033] Figure 14 The activity of the extract of Bacillus D43-56 against banana peel-related defense enzymes was determined.

[0034] Figure 15 Determination of banana peel cell wall degrading enzymes by extract of bacteria D43-56.

[0035] Streptomyces strain D43-56 was named Streptomyces sp. and deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:66719, deposit date July 21, 2025, and deposit address 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0036] 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.

[0037] Example 1: Isolation of indigenous actinomycetes from banana rhizosphere soil and screening and identification of antagonistic bacteria

[0038] 1. Experimental Materials

[0039] 1.1 Soil Samples

[0040] Soil samples used for separation were collected from the rhizosphere soil of a banana plantation in Team Six of the Experimental Farm, Danzhou City, Hainan Province, China (109°30′1.998″E, 19°34′38.644″N). The samples were placed in sterile sealed bags and stored at 4°C before being brought back to the laboratory and preserved in an ultra-low temperature freezer at -80°C.

[0041] 1.2 Test Culture Medium

[0042] The main culture media required for this experiment include isolation medium, morphological observation medium, physiological and biochemical characteristic medium, potato dextrose agar (PDA) medium, and soybean flour fermentation medium (SLM). Some culture medium formulations are detailed in Table 1.

[0043] Table 1. Culture media and their formulations for observing culture characteristics

[0044] culture medium Element PDA 200g potatoes, 20g glucose, 20g agar, 1L distilled water SLM Soybean extract powder 20.0g, starch 5.0g, sucrose 10.0g, peptone 2.0g, yeast extract powder 2.0g, sodium chloride 2.0g, dipotassium hydrogen phosphate 0.5g, calcium carbonate 0.1g, distilled water 1L YE Yeast extract 4g, malt extract 10g, glucose 4g, agar 20g, distilled water 1L, pH=7.3

[0045] 1.3 Instruments and equipment used in the experiment

[0046] Table 2 Instruments and Equipment

[0047] Main instrument names model factory Vertical double-layer shaking bed ZWYR-D2403 Shanghai Smart City Autoclave HVE-2510 JapanHIRAYAMA Electrophoresis apparatus PowerPac Bio-Rad Biochemical incubator ZXSD-B1270 Shanghai Smart City Ultra-low temperature freezer MDF-382 Panasonic Double-person clean bench SW-CJ-2FD Suzhou Purification Thermostatic metal water bath HHS-11-2 Hangzhou Huier One ten-thousandth balance ME104E Shanghai Mettler pH meter Delta 320 Shanghai Mettler

[0048] 1.4 Main Reagents

[0049] Table 3. Major Biochemical Reagents and Their Sources

[0050] Reagent Name Manufacturer Agarose Sigma Aldrich

[0051] 2 Experimental Methods and Results

[0052] 2.1 Isolation and Screening of Soil Actinomycetes

[0053] (1) Separation

[0054] Carefully remove the top layer of soil from the banana plant and collect the soil from the root zone using a bag. Immediately place the bag in an ice box and store at -20°C in the laboratory. Weigh 5 g of fresh soil and dissolve it in 45 mL of sterile water. Incubate the solution at 28°C and 180 rpm for 1 h to obtain a soil suspension. Using a 10-fold serial dilution method, add 100 μL of the soil suspension to a centrifuge tube containing 900 μL of sterile water, mix thoroughly, and repeat this step to prepare 10... -1 10 -2 and 10 -3 Soil suspensions of different concentrations were collected and spread into isolation medium at 100 μL each. These suspensions were then incubated upside down at 28°C for 7 days, with three replicates. After colonies grew, single colonies with inconsistent morphological characteristics were streaked onto YE agar plates for purification and preservation. The isolated pure cultures were stored in 30% glycerol and then placed in a -80°C cryogenic freezer.

[0055] (2) Initial screening

[0056] The isolated bacteria were screened for antagonistic bacteria using the five-point confrontation method. The method was as follows: The same isolated actinomycetes were placed at four points 2.5 cm away from the center of the PDA medium. After incubation at 28°C for 2 days, once the actinomycetes had grown, a 3 mm diameter *Colletotrichum musae* (ATCC 96167) mycelial disc was inoculated at the center of the medium. The control group consisted only of mycelial discs of the pathogen. Figure 1 (See the top left image). The bacteria were placed in a biochemical incubator for 7 days, and the experiment was repeated three times. The inhibition rate was calculated using the following formula, in mm. 83 strains initially screened showed antibacterial activity, with strain D43-56 exhibiting the strongest resistance to anthrax, reaching an inhibition rate of 81.17%. Figure 1 (The image in the upper right corner).

[0057]

[0058] (3) Secondary screening

[0059] The actinomycetes obtained from the initial screening were inoculated into SLM medium and cultured at 28℃ and 180 r / min for 7 days. The cultured fermentation broth was then filtered through a 0.22 μm sterile microfiltration filter to obtain a sterile supernatant, i.e., sterile fermentation broth. Four wells (0.5 cm in diameter) were punched at 2.5 cm from the center of a PDA plate. The treatment group received 200 μL of the prepared sterile fermentation broth in each well of the PDA medium plate, while the control group received sterile water in the PDA plate. Figure 1 (See the lower left image). Use a punch to create 3mm diameter mycelial cakes, inoculate them in the center of a PDA plate, repeating each treatment three times. Incubate at 28℃ for 7 days. Measure the colony diameter using the cross-hatching method, and calculate according to the formula. The inhibition rate was calculated using this method. The inhibition rate of the aseptic fermentation broth of strain D43-56 was 65.95% ( Figure 1 (The image at the bottom right).

[0060] Example 2: Classification and identification of Streptomyces D43-56

[0061] 1. Cultivation characteristics

[0062] Streptomyces D43-56 was inoculated into various media, including YE, and incubated statically at 28°C for 7 days. The growth of Streptomyces D43-56 on different media was observed, including the color of aerial hyphae, intramural hyphae, and colony color and size. The results are shown in Table 4. Strain D43-56 grew on all seven media. It grew rapidly on ISP2, PDA, and Gause's No. 1 media, showed the weakest growth on ISP7, and grew well on the other three media. Under the growth conditions of all seven media, D43-56 did not produce soluble pigments. Colonies were mostly powdery and wrinkled, with aerial hyphae mostly white and intramural hyphae mostly yellowish-white.

[0063] Table 4. Culture characteristics of strain D43-56

[0064]

[0065] Note:++ indicates rapid growth; ++ indicates good growth; + indicates that it can grow on the culture medium.

[0066] 2. Scanning electron microscopy observation of Streptomyces D43-56 culture

[0067] Strain D43-56 was cultured on YE medium for 10-14 days. Colonies were carefully separated from the medium using a sterile syringe, selecting the flattest and thinnest possible colonies and cutting a 0.5cm square piece. After processing as described by Zhang, the culture was replaced with isoamyl acetate and then air-dried in a sterile laminar flow hood. Finally, after gold plating, the morphology of hyphae and spores was observed under a scanning electron microscope (Zeiss Sigma 500 / VP, Germany). The results are as follows. Figure 2 As shown, under a scanning electron microscope (SEM), the hyphae are well-developed and tightly intertwined, with spores scattered at the tips of the hyphae in a blocky shape, and chains can be observed.

[0068] 3. Physiological and biochemical characteristics of strain D43-56

[0069] The physiological and biochemical characteristics of the actinomycetes to be tested were identified in accordance with the book "Systematics of Actinomycetes - Principles, Methods and Practice". The main tests included single carbon and nitrogen source utilization tests, pH and NaCl tolerance tests, and metabolite tests (Xu Lihua et al., 2007).

[0070] 1) Carbon source utilization experiment

[0071] Using Pugol's medium as the basal medium, 1% of different carbon sources were added to prepare corresponding carbon source mediums, which were then sterilized and incubated. Actinomycetes were inoculated onto plates of different carbon source mediums, with a negative control included. The plates were incubated at 28℃ for 7 days, and their growth was observed and recorded. The results are shown in Table 5, indicating that this strain can utilize most carbon sources.

[0072] 2) Nitrogen source utilization experiment

[0073] Different nitrogen sources were mixed with 1% of the basal medium to prepare corresponding nitrogen source media. Actinomycetes were inoculated into these media, with a negative control included. The media were incubated at 28℃ for 7 days, and their growth was observed and recorded. The results are shown in Table 5. This strain can utilize most of the nitrogen source.

[0074] 3) pH tolerance test

[0075] Using liquid Bennett's medium as the basal medium, pH gradient media ranging from 4.0 to 10.0 were prepared, and actinomycetes were inoculated into each medium. The cultures were then incubated on a shaker (28℃, 180 r / min) for 7–14 days, and the growth was observed and recorded. The results are shown in Table 6. Strain D43-56 could survive at pH 7–8, with pH 7 being the optimal condition.

[0076] 4) NaCl tolerance test

[0077] Different concentrations of NaCl were added to liquid Bennett's medium to prepare media with NaCl concentrations of 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15%. Actinomycetes were inoculated into these media and cultured at 28°C for 7 days, and their growth was observed. The results are shown in Table 6. Strain D43-56 could survive under NaCl concentrations ranging from 1% to 5%, with an optimal NaCl concentration of 3%.

[0078] 5) Temperature tolerance test

[0079] YE medium was prepared, and actinomycetes were inoculated into it. The cultures were then placed in constant temperature incubators at different temperature settings (15℃, 20℃, 45℃, 55℃) to test their resistance to low and high temperatures. The results are shown in Table 6. Strain D43-56 could survive at temperatures ranging from 25 to 45 degrees Celsius.

[0080] 6) Metabolite assay

[0081] a. Hydrogen sulfide detection

[0082] Actinomycetes were inoculated onto hydrogen sulfide detection medium and incubated at 28°C for 2 weeks. Observation was performed; if the medium turned black, the result was positive; if it did not turn black, the result was negative. The results are shown in Table 6. Strain D43-56 could not produce hydrogen sulfide.

[0083] 7) Enzymatic metabolite assay

[0084] (1) Urease test

[0085] The ability of the strain to produce urease was tested. Actinomycetes were inoculated onto urease medium, with an uninoculated urease medium as the control. After incubation at 28℃ for 4 days, the color change of the medium was observed. A pinkish-red color indicated a positive result, suggesting the strain had the ability to produce urease; no color change indicated no urease production. The results are shown in Table 6. Strain D43-56 was found to produce urease.

[0086] (2) Starch hydrolysis

[0087] The presence of amylase activity in the strain was tested. Actinomycetes were inoculated onto starch hydrolysis medium and incubated at 28°C for 5–7 days. Iodine solution, covering the mycelia, was then added around the colonies for detection. The presence of a clear zone indicated a positive result, while the absence of a clear zone indicated a negative result. The results are shown in Table 6. Strain D43-56 can produce amylase.

[0088] (3) Gelatin liquefaction

[0089] The ability of the strain to produce protease was tested. Actinomycetes were 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. A control was prepared using the same medium but without inoculation. The degree of liquefaction was observed upon removal; flowability indicated a positive result, while coagulation indicated a negative result. The results are shown in Table 6, indicating that strain D43-56 could liquefy gelatin.

[0090] (4) Nitrate reduction

[0091] Actinomycetes were inoculated into nitrate-reducing medium and incubated at 28°C for one week. A small amount of the liquid was taken into a test tube, and one drop each of indicator solution A and B were added. The control was the medium without inoculation. A red color in the test tube indicated a positive result, while no change indicated a negative result. The results are shown in Table 6. Strain D43-56 can reduce nitrate.

[0092] (5) Cellulose decomposition test

[0093] Prepare sodium carboxymethyl cellulose medium and sterilize the plates. Inoculate actinomycetes onto the plates and incubate at 28℃ for 7 days. Remove the plates and pour 1 g / L Congo red solution into the plates to submerge the bacterial cells, staining for 30 min. Pour off the stain and then pour 1 mol / L NaCl solution into the plates to decolorize for 30 min. Observe whether a clear zone is formed on the plates. If a clear zone appears, it is a positive result, indicating the ability to produce cellulase; otherwise, it is not. The results are shown in Table 6. Strain D43-56 cannot decompose cellulose.

[0094] Table 5. Carbon and nitrogen source utilization of strain D43-56

[0095] carbon source Growth state nitrogen source Growth state D-glucose ++ Valine ++++ D-fructose ++++ Nysine - D-Mannitol + Hydroxymethionine ++++ L-arabinose +++ Dimethylamino acid + Ginsenosides ++ glycine - Trehalose ++ Thionine ++ dextrin +++ Tyrosine +++ Sorbitol ++++ Arginine - Galactose ++++ Phenylalanine ++ sucrose +++ L-histidine +++

[0096] Note: "++++" indicates promoted growth; "+++" indicates abundant growth; "++" indicates normal growth; "+" indicates minimal growth; "-" indicates no growth.

[0097] Table 6 Biochemical characteristics of strain D43-56

[0098] Urease production + Gelatin liquefaction + Ferrite carrier - Starch hydrolysis - Cellulose decomposition - Reduced nitrates + <![CDATA[Whether H2S is produced]]> - Temperature test 25-45℃ pH tolerance 7-8 (7 is the optimal number) Salt tolerance (%) 1-5 (Optimal 3)

[0099] Note: "+" indicates growth; "-" indicates no growth.

[0100] 4. Molecular biological identification of Streptomyces D43-56

[0101] (1) Constructing a phylogenetic tree based on 16S rRNA sequence

[0102] Actinobacterial DNA was extracted using the Biosharp Bacterial DNA Extraction Kit (BL1044A, Beijing Lanjieke, China). PCR amplification of the actinobacterial DNA was performed using universal bacterial primers: upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.

[0103] Table 7 PCR Reaction System

[0104]

[0105] Table 8 Reaction Procedure

[0106]

[0107] The PCR products obtained above were sent to Shanghai Sangon Biotech Co., Ltd. for 16S rRNA sequencing. After the sequencing company returned the data, the obtained sequences were uploaded to the EzBioCloud and GenBank databases for homology comparison and to search for and download highly similar 16S rRNA gene sequences. Multiple comparison analysis was performed on selected strains, and a phylogenetic tree was constructed using MEGA 7.0 software with neighbor-joining, and the bootstrap self-test value was set to 1000 (Qi et al 2021).

[0108] The 16S rRNA sequences predicted by the gene were compared with the NCBI 16S database using BLAST, with the parameter `identify>95`. Then, the top 30 16S rRNA sequences with the highest `identify` were selected (all were included if insufficient), and multiple alignments were performed using MAFFT software. After sequence cutting, a phylogenetic tree was constructed using FastTree / iqtree / raxml software. Sequencing: The 16S rRNA sequence of strain D43-56 was uploaded to the NCBI database and Ezbiocloud. After BLAST alignment, strain D43-56 was found to be the closest match to *Streptomyces showdoensis* strain 21411. A phylogenetic tree was constructed in MEGA 7.0 using the NJ method, as shown below. Figure 3 As shown, strain D43-56 is most closely related to Streptomyces showdoensis strain 21411, and has a development value of 71%, which preliminarily identifies this strain as belonging to the genus Streptomyces.

[0109] (2) Comparison of ANI and DDH values

[0110] The genomic data of the relevant strains were downloaded from NCBI or EzBicloud. The genomes of the relevant strains and strain D43-56 were uploaded to EzBicloud for average nucleotide identity (ANI) calculation and uploaded to (https: / / ggdc.mz.de) for DNA-DNA hybridization (DDH) calculation.

[0111] The results showed that strain D43-56 had an ANI value less than the 95% threshold for new species and a DDH value less than the 70% threshold for new species. Therefore, strain D43-56 was identified as a new species of Streptomyces. Figure 4The strain D43-56 was named Streptomycessp D43-56 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO:66719) on July 21, 2025. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0112] Example 3: Broad-spectrum antibacterial activity of strain D43-56

[0113] Streptomyces D43-56 was cultured on soybean meal medium (SLM), the pathogen (see Table 9) was cultured on PDB medium, and antagonism assays were performed on PDA plates. Streptomyces D43-56 was cultured on YE medium for GC-MS analysis.

[0114] Preparation of extract from strain D43-56: Streptomyces D43-56 was cultured in soybean liquid medium (SLM) at pH 6 at 28°C and 180 rpm for 8 days. The fermentation broth was obtained and mixed with an equal volume of anhydrous ethanol. Extraction was continued for 3 days on a shaker at 28°C and 180 rpm. After extraction, the bacterial cells were filtered through filter paper. The filtrate was distilled under reduced pressure at 40°C to 500 ml using a rotary evaporator (N-1300, EYELA, Ailang Instruments Co., Ltd., Shanghai, China) to obtain the crude fermentation extract. Macroporous resin was soaked in anhydrous ethanol for 24 hours and then wet-packed into a chromatographic column (6×80 cm, diameter×length), with a volume half the column volume. The column was rinsed with a large amount of sterile water from top to bottom until no ethanol odor could be detected by fan smell. The fermentation broth was carefully poured into the column and allowed to stand for 24 hours. The column was then eluted with a large amount of sterile water until the eluent was nearly colorless. Subsequently, four fractions were obtained by elution with a gradient of methanol (MeOH / H2O, 50%, 70%, 90%, 100% v / v). The distillates of the four fractions were subjected to vacuum distillation and dried into solid powders. The powders were weighed and dissolved in dimethyl sulfoxide solution, yielding a mother liquor with a final concentration of 20 g / L. The solution was filtered through a 0.22 μm sterile filter membrane before activity determination, and the experiment was repeated three times. At a concentration of 200 mg / L, the inhibition rates of the 50%, 60%, and 70% methanol extracts were 27.54%, 18.65%, and 14.56%, respectively. The 100% methanol-eluted fraction exhibited the strongest antibacterial activity, reaching 72.42%. Figure 5 This component was used to conduct subsequent antibacterial experiments.

[0115] Method for determining extract activity: Melt sterilized PDA medium by heating and slightly cooling until the liquid is freely flowing. Add an appropriate amount of the stock solution to the melted PDA medium according to the dilution formula until the final extract concentration is 1 g / L. Shake well and pour onto a PDA plate of uniform thickness. Place a 5 mm diameter *Anthracis bananais* mycelial cake in the center of the plate. Incubate the plate in a biochemical incubator at 28℃ for 5-7 days and calculate the inhibition rate.

[0116] The pathogens were collected for antifungal activity testing. The testing method followed the initial screening method described in Part 2, "Isolation and Screening of Soil Actinomycetes," to determine the broad-spectrum antifungal activity of the strains. The results showed that strain D43-56 exhibited good antagonistic effects against *Colletotrichum gloeosporioides*, *Colletotrichum lychee*, *Fusarium graminearum*, *Colletotrichum capsulatum*, and *Colletotrichum spp.* Figure 6 (See the left figure and Table 9).

[0117] The fraction with the strongest antibacterial activity (eluted with 100% methanol) was taken as the extract of strain D43-56. Antibacterial experiments were conducted according to the "Method for Extract Activity". The results showed that the extract of strain D43-56 had an inhibition rate of 41.56% to 95.73% against pathogens, with the strongest inhibitory activity against Fusarium graminearum, reaching 95.73%. Figure 6 (See the right figure and Table 9). Overall, the extract showed a higher inhibition rate than strain D43-56.

[0118] Table 9 Plant pathogens and their inhibition rates

[0119] sequence Pathogenic fungi Latin name ATCC number Antibacterial rate of strain (%) Antibacterial rate of extract (%) 1 Colloidal anthrax bacteria penz. ATCC 58222 64.76 84.57 2 Banana Fusarium wilt disease, race 4 f. sp. cubense Race 4 ATCC 76255 28.49 75.67 3 Lychee anthracnose fungus ATCC 16330 59.46 48.67 4 wheat scab ATCC MYA-4620 78.34 95.73 5 Anthracnose of pepper ATCC 48574 42.13 41.56 6 Banana long spot disease ATCC 38579 39.49 57.89 7 Strawberry anthracnose fungus ATCC 58718 49.22 50.13

[0120] Example 4: Effect of extract from strain D43-56 on *C. musae*, the anthracnose causal agent of banana anthracnose.

[0121] (1) Virulence regression equation test

[0122] The component with the strongest antibacterial activity was selected as the extract and prepared into PDA plates with concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg / L. Pathogens with a diameter of 5 mm were placed on the PDA plates, with the center of each plate placed separately. The plates were incubated at 28°C for 7 days, and the inhibition rate at each concentration was calculated (results are shown in the figure). Figure 7 (As shown). A virulence regression equation was established based on the concentration-inhibition rate relationship, and its EC50 was calculated. 50 The value is 170 mg / L.

[0123] (2) Effects of extracts on the growth and morphology of C. musae hyphae

[0124] The prepared concentration is 4×EC 50 (EC)50 Anthrax bacteria were inoculated onto PDA plates (4 times the effective value). Anthrax mycelial cakes with a diameter of 5 mm were inoculated at the center of each PDA plate, with methanol of the same concentration used as a control. After incubation at 28℃ for 5 days, the edges of the hyphae from both the treated and control groups were cut off using a sterile scalpel, and the effects of extract treatment on the structure and morphology of the hyphae were observed under SEM. The results are as follows: Figure 8 As shown, the control group had rounded hyphae with plump spores scattered around the hyphae, while the 4×EC... 50 The hyphae treated with high concentrations shrank, deformed, or even broke, and the spores scattered around them also shrank or even ruptured.

[0125] (3) Effects of extracts on spore germination and spore morphology of C. musae

[0126] Preparation of spore suspension: 5 ml of 0.05% Tween-80 solution was added to an anthracnose fungal plate that had grown for 7-10 days. The mycelia were crushed and scraped using a sterilized iron spoon to ensure thorough mixing. The filtrate was drawn up using a pipette tip with a cut end and filtered through a funnel lined with four layers of clean paper. 100 μL of the filtrate was collected and counted under an optical microscope. The solution was diluted as needed to obtain a final suspension of 10 μL. 6 A spore suspension at CFU / mL.

[0127] Mix 100 μL of spore suspension with 2 ml of sterile water, and add extract dissolved in methanol to final concentrations of 1×, 2×, 4×, and 8× EC. 50 The control group was treated with methanol. All liquid was transferred to 5 ml centrifuge tubes and treated at 28°C for 24 h. Each treatment was repeated three times. 30 μL of liquid was aspirated onto a coverslip, and spore germination was observed using an optical microscope. 100 spores were randomly counted as one field, and five fields were repeated. The spore germination rate was calculated according to the following formula. The spore germination rate of the control group was required to be greater than 90%.

[0128]

[0129] Preparation 10 6 CFU / mL spore suspension. Transfer 100 μL of spore suspension and 1 mL of sterile water to a 2 mL centrifuge tube, and add extract to a concentration of 4 × EC50. 50 A methanol solution was used as a control, and the mixture was incubated at 28°C for 6 hours in a biochemical incubator. 40 μL of the mixture was transferred to a glass slide, air-dried in a clean bench, and the effect of the extract on spore structure was observed under SEM. The results are as follows: Figure 9 As shown, using 4 × EC 50 Concentration treatment effectively inhibited anthracnose spore germination. In the control group, the spore germination rate was as high as 96%, while 4 × EC... 50After concentration treatment, the spore germination rate was only 1.67%. Meanwhile, under a scanning electron microscope, the spores in the control group were round and plump, while the spores in the treatment group were wrinkled and ruptured.

[0130] (4) Effects of the extract on the cell membrane of C. musae

[0131] The integrity of the anthrax cell membrane was determined based on several indicators, including the content of extracellular soluble protein, extracellular soluble sugar, A260, extracellular conductivity, hyphal dry weight, and malondialdehyde (MDA) content, after the extract was treated with the bacteria.

[0132] Pretreatment: Weigh the extract and dissolve it in sterile water, then sonicate at 60℃ until completely dissolved to a maximum soluble stock solution concentration of 5 g / L. Take 60 50 ml Erlenmeyer flasks and fill them with 20 ml of sterile water. Autoclave and cool the flasks. Add the extract to the Erlenmeyer flasks to final concentrations of 2×, 4×, and 8× EC50. Sterile water serves as the control group (CK). Each treatment is repeated three times. Subsequently, inoculate 100 μL of a 10% EC50 solution into PDB medium. 7 A spore suspension of CFU / mL was incubated at 28℃ and 180 rpm for 3 days. 1g of mycelium was carefully weighed in a clean bench, rinsed three times with sterile water, and then placed into Erlenmeyer flasks containing different extract concentrations. The flasks were incubated at 28℃ and 180 rpm on a shaker. Samples were collected at 0, 3, 6, 9, and 12 hours for each of the four treatments. The samples were transferred to 50ml centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected for the determination of soluble protein content, soluble sugar content, nucleic acid content (A260), and conductivity. The bacterial cells were collected, dried using a freeze dryer (SCIENTZ-18N), and weighed. The bacterial cells were also used for MDA determination.

[0133] Assay methods: Soluble protein was detected using the Bradford Protein Quantification Kit (W042-1-1, Nanjing Jiancheng); soluble sugar content was detected using a Soluble Sugar Content Assay Kit (BC0030, Solarbio); A260 absorbance was measured at 260 nm using a UV-Vis spectrophotometer (UV 1000, Shanghai Meipuda). Conductivity was measured using a portable handheld conductivity meter (DDB-305A, Shanghai Leici). MDA was measured using a micro-malondialdehyde assay kit (A003-2-2, Nanjing Jiancheng). All experiments were performed in triplicate, strictly following the manufacturer's recommended procedures.

[0134] The results are as follows Figure 10As shown, the amount of nucleic acid leaked from the cell membrane initially increased and then fluctuated slightly with time, showing a positive correlation between the amount and time during the 0-6 hour period. After 6 hours, the nucleic acid content fluctuated slightly. The amount of leaked nucleic acid in the CK group was lower than that in all treatment groups, and the amount of leaked nucleic acid further increased with increasing concentration. The content of soluble protein generally increased with increasing extract concentration, while 8 × EC... 50 The highest concentration of soluble protein was observed at the lowest concentration, reaching 198.57 mg / L at 12 hours after treatment. From 0 to 12 hours, the soluble sugar content generally showed a trend of first increasing and then fluctuating slightly. The extract concentration was positively correlated with the amount of soluble sugar released, with 8 × EC... 50 The soluble sugar content was highest after 12 hours of treatment, reaching 14.77 mg / L. MDA content increased with increasing extract concentration, indicating that 8 × EC... 50 After concentration treatment, the pathogen exhibited the most severe lipid peroxidation. The dry weight of the mycelium showed a negative correlation with the extract concentration (8 × EC50). 50 The treated mycelium had the lowest dry weight, at 0.04 g. The conductivity of the supernatant was measured using a portable handheld conductivity meter (DDB-305A, Shanghai Leici), and the treated group showed significantly higher conductivity than the control group. Conductivity increased with increasing concentration, reaching a maximum at 8 × EC⁻¹. 50 Maximum at concentration.

[0135] Example 5: Control effect of Streptomyces D43-56 on banana anthracnose

[0136] 1. Materials

[0137] 1.1 Test strains

[0138] The tested strain was Streptomyces D43-56.

[0139] 1.2 Pathogens

[0140] The pathogen that infects banana peels is C. musae.

[0141] 1.3 Culture medium

[0142] C. musae was cultured in PDB.

[0143] 1.4 Bananas tested

[0144] The bananas used in the experiment were all purchased from the Chengxi Farmers Market. Fruits of uniform ripeness and size were selected for the experiment. The fruits had no mechanical damage on their surface. After soaking in 75% ethanol (v / v) for two minutes, they were washed under running water for one minute. Finally, the fruits were air-dried at room temperature.

[0145] 1.5 Experimental Reagents and Equipment

[0146] (1) Main reagents

[0147]

[0148] (2) Main equipment

[0149]

[0150] 2. Experimental Methods and Results

[0151] 2.1 Inoculation test of banana fruit

[0152] Three holes, each 2 mm deep, were punched in the banana fruit using a sterile 2 mm punch. Each wound was injected with 30 μL of extracts at different concentrations (1×, 2×, 4×, and 8× EC). 50 An equal volume of methanol solution was used as a control group (CK). After air drying in a clean bench, 2mm anthrax mycelium cakes were affixed to the perforated areas. Six banana fruits were selected for each treatment and stored in artificial climate chambers at 28℃ and 85% relative humidity (Yongjie Technology Instrument Co., Ltd., Shanghai, China).

[0153] 2.2 Determination of the control effect of extracts on banana anthracnose

[0154] After 4-7 days of cultivation, the banana fruits were photographed, and the area of ​​lesions was calculated according to Zhou et al. (2022). The results are as follows: Figure 11 As shown in the figure. Four days later, the control group fruits showed obvious signs of rotting. At 4 and 5 days, the lesion area was 0.52 ± 0.23 cm², respectively. 2 and 0.55 ± 0.19cm 2 No obvious signs of rot were observed in any of the processed fruits. Figure 11 (A) As storage time increased, the area of ​​lesions gradually increased. At 7 dpi, the lesion area of ​​the control group fruit was 1.05 cm². 2 1 × EC 50 2 × EC 50 and 4 × EC 50 The area of ​​diseased spots on the treated banana fruits was 0.58 cm². 2 0.20 cm 2 and 0.14 cm 2 In fruits treated with 8 × EC50 extract, only 0.08 cm was observed. 2 The area of ​​the lesions.

[0155] 2.3 Determination of the effect of extracts on inhibiting the colonization of C. musae on banana peel

[0156] The colonization of *C. musae* in fruit wounds was observed using scanning electron microscopy (SEM). The results are as follows: Figure 12 As shown. In 4 × EC 50 In the control group, the extract treatment significantly inhibited the growth of the pathogen, and a large number of ungerminated spores were scattered around the holes in the treated fruit, indicating that the pathogen was in a defensive state under biological stress. In the control group, anthracnose hyphae colonized and penetrated the artificially punctured area around the peel, indicating that the banana had already colonized the peel and was in the disease stage of banana anthracnose.

[0157] 2.4 Determination of the effect of extracts on banana fruit quality

[0158] Four concentrations (1×, 2×, 4×, 8× EC) were prepared. 50 Extracts of the fungus were obtained. Seven days after inoculation with banana anthracnose according to method 2.1, the soluble solids (TSS), titratable acid (TA), soluble sugar (SCC), and vitamin C (VC) content of the pulp within a 1 cm radius centered on the puncture site were measured. The soluble solids (TSS) content (%) in the banana pulp was quantified using a portable refractometer (model N-1a, Atago Co., Ltd., Tokyo, Japan). SCC was measured using a plant soluble sugar content kit (BC0035, Beijing Solarbio). VC was measured using a vitamin C assay kit (phosphomolybdic acid colorimetric method, R22193-50T, Shanghai Yuanye), and TA was measured using a titratable acid assay kit (R30300, Shanghai Yuanye). Each experiment was repeated three times.

[0159] The results are as follows Figure 12 As shown. 1 ×, 2 ×, 4 ×, and 8 × EC were used. 50 After treatment with extracts of different concentrations for 7 days, the TSS content in banana fruits was determined. Treatment with different concentrations of extracts significantly increased the TSS content in banana fruits. Specifically, 8× EC... 50 The highest TSS (Total SSC) content was observed in the concentrated treatment, reaching 13.96%. After extract treatment, the SSC content in the banana fruit did not change significantly. After extract treatment, 2 × EC... 50 Compared to the control, the vitamin C content was significantly increased, and the vitamin C content of bananas was also higher after the other three concentration treatments. There was no significant difference in ta (taste precipitate) content after extract treatment, but the ta content in the treated groups was lower than that in the control group.

[0160] 2.5 Determination of the activity of extracts against banana peel-related defense enzymes

[0161] After culturing for 7 days according to method 2.1, the enzyme activities of the pericarp within a 1 cm radius centered on the punched hole were measured, including peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO). POD was measured using a peroxidase (POD) kit (Jiangsu, Edison). PPO was measured using a polyphenol oxidase (PPO) kit (Jiangsu, Edison). CAT was measured using a catalase (CAT) kit (Beijing, Solarbio). Each experiment was performed in triplicate.

[0162] The results are as follows Figure 13 As shown, enzyme activity increased with increasing extract concentration. Compared to the control, 4× and 8× EC 50 POD activity significantly increased in banana peel treated with the extract (P < 0.05). The enzyme activity showed an increasing trend with increasing extract concentration. Compared with the control, 4 × and 8 × EC 50 PPO activity significantly increased in banana peel treated with the extract (P < 0.05). CAT activity did not show a significant overall difference, but the CAT activity in the treated group was higher than that in the control group.

[0163] 2.6 Determination of the effect of extracts on banana peel cell wall degrading enzymes

[0164] To investigate whether the extract affects the activity of cell wall-related enzymes in banana peel, the activities of cellulase (CX), pectin methyl esterase (PME), and polygalacturonase (PG) in banana peel were measured on day 7. Simultaneously, the reducing sugar content of the peel was determined using a reducing sugar content assay kit (Shanghai Beibo Biotechnology Co., Ltd.) to assess the degree of degradation of banana peel cell wall polysaccharides.

[0165] (1) Determination of PG enzyme activity

[0166] Substrate preparation: 1 g of polygalacturonic acid was dissolved in 100 ml of Na-acetate buffer solution (50 mmol / L, pH=5.5).

[0167] Enzyme extraction: Take 1g of banana peel, grind it with liquid nitrogen until there are no granular powders, put it into a 10ml centrifuge tube placed on ice, add 5ml of Na-acetate buffer solution (0.1mol / L, pH=5), centrifuge at 4℃, 10000rpm for 10min using a refrigerated centrifuge, and take the supernatant for later use.

[0168] Standard curve determination: Dissolve 0.1g of galacturonic acid in 100ml of distilled water to prepare a concentration of 1mg / ml. Prepare 8 test tubes and dilute according to Table 6 to plot the standard curve. After adding the DNS reagent, shake well immediately. Incubate the test tubes in a 100℃ water bath for 5 minutes, then immediately stop the reaction in an ice bath. After cooling to room temperature, bring the total volume to 20ml with sterile water. Measure the absorbance at 540nm and plot the standard curve as concentration (x) - absorbance (y).

[0169] Enzyme activity assay: Take a test tube, add 1 ml of Na-acetate buffer solution (50 mmol / L, pH=5.5), 0.5 ml of enzyme solution, and 0.5 ml of substrate, and react at 40℃ for 1 h. Measure the absorbance at 540 nm according to the standard curve assay procedure described above. Set up a control group, where the enzyme solution is treated at 90℃ for 10 min, with all other conditions unchanged.

[0170] Table 12. Construction of galacturonic acid standard curve

[0171]

[0172] Table 13. Plotting the glucose standard curve

[0173]

[0174] Enzyme activity calculation:

[0175] M1: The mass (mg) of galacturonic acid converted from the absorbance of the test tube in the standard curve.

[0176] M_extinguished: The mass (mg) of galacturonic acid calculated by measuring the absorbance of the inactivated enzyme solution.

[0177] M2: The mass of the sample weighed, which is 1 (g) if the above steps are followed.

[0178] Vtotal: The total volume of the sample after homogenization. If the above steps are followed, it will be 5 (ml).

[0179] V_test: The volume of enzyme solution added during the test, which is 0.5 (ml) if the above steps are followed.

[0180] T: Enzyme reaction time, which is 60 (min) if the above steps are followed.

[0181] (2) Determination of CX enzyme activity

[0182] Substrate preparation: Weigh 1g of CMC and dissolve it in 100ml of pH 5.0 sodium citrate buffer (0.05mol / L). Heat to dissolve until the final concentration is 10g / L.

[0183] Enzyme extraction: Take 1g of banana peel, grind it with liquid nitrogen until there are no granular powders, put it into a 10ml centrifuge tube placed on ice, add 5ml of citric acid-sodium citrate buffer solution (0.1mol / L, pH=5.0), and incubate at 4℃, 10000rpm for 10min. Take the supernatant for later use.

[0184] Standard curve determination: Dissolve 0.1g glucose in 100ml distilled water to prepare a concentration of 1mg / ml. The remaining steps are performed according to the PG enzyme standard curve determination method.

[0185] Enzyme activity assay: Take a test tube, add 1 ml of pH 5.0 sodium citrate buffer (0.05 mol / L), 0.5 ml of enzyme solution, and 0.5 ml of substrate, and react at 40℃ for 1 h. Measure the absorbance at 540 nm according to the standard curve assay procedure described above. Set up a control group, where the enzyme solution is treated at 90℃ for 10 min, with all other conditions unchanged.

[0186] Enzyme activity calculation:

[0187] M1: The glucose mass (mg) converted from the absorbance of the test tube in the standard curve.

[0188] Mextinguished: The glucose mass (mg) calculated by measuring the absorbance of the inactivated enzyme solution.

[0189] M2: The mass of the sample weighed, which is 1 (g) if the above steps are followed.

[0190] Vtotal: The total volume of the sample after homogenization. If the above steps are followed, it will be 5 (ml).

[0191] V_test: The volume of enzyme solution added during the test, which is 0.5 (ml) if the above steps are followed.

[0192] T: Enzyme reaction time, which is 60 (min) if the above steps are followed.

[0193] (3) Determination of PME enzyme activity

[0194] Improvements were made to Kou Jingjing's method. 5 ml of crude enzyme solution was extracted using the PG enzyme extraction method. 1 g of pectin was dissolved in 100 ml of sterile water to prepare a 1% pectin solution. The reaction system consisted of 5 ml of enzyme solution, 20 ml of 1% pectin, and 0.05% bromothymol blue. Before the reaction, the pH of all reagents was adjusted to 7.6, and the mixture was quickly mixed and incubated at 37°C for 30 min. Titration was performed using NaOH (0.01 mol / L) while maintaining pH 7.6. The amount of NaOH was calculated, and the PME enzyme activity was calculated as the amount of NaOH (µmol) consumed per minute (min) per gram of pericarp (g).

[0195] The results are as follows Figure 15As shown. Treatment with the extract significantly reduced the PG enzyme activity in banana peel (P < 0.05), 8 × EC 50 The extract significantly reduced the activity of PG enzymes in the banana peel, slowing down the degradation of the peel cell wall. The extract treatment significantly reduced the PME enzyme activity in the banana peel (P < 0.05). Furthermore, the decrease in PME enzyme activity became more pronounced with increasing extract concentration, indicating that the extract treatment slowed down the degradation of the peel cell wall. The extract treatment also significantly reduced the CX enzyme activity in the banana peel (P < 0.05), and the enzyme activity further decreased with increasing concentration. 2× EC 50 4 × EC 50 and 8 × EC 50 Both significantly reduced the activity of CX in the pericarp and slowed down the degradation of pericarp cell walls. Regarding reducing sugar content, the content gradually decreased with increasing extract concentration, particularly 4 × EC. 50 and 8 × EC 50 The reducing sugar content did not differ significantly at different concentrations. This indicates that the degree of degradation of polysaccharides in the banana peel cell wall gradually increased with increasing extract concentration.

[0196] The above data show that the activity of PG enzyme, PME enzyme, CX enzyme and reducing sugar content decreased to varying degrees after the extract treatment, and the degradation of banana peel was slowed down. This further indicates that the banana fruit's resistance to banana anthracnose was improved after the extract treatment.

[0197] 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, characterized in that, Named as Streptomyces sp. and was deposited in Guangdong Microbial Culture Collection Center with the accession number of GDMCC NO: 66719.

2. The fermentation broth of the Streptomyces of claim 1.

3. A formulation characterized in that, A preparation comprising the Streptomyces of claim 1 or the fermentation broth of claim 2.

4. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for antagonizing Colletotrichum musae, and / or C. gloeosporioides, and / or race 4 of Fusarium oxysporum f. sp. cubense, and / or C. truncatum, and / or G. saubinetii, and / or C. sativum, and / or C. capsici, and / or Mycosphaerella musicola, and / or C. fragariae.

5. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for controlling diseases caused by Colletotrichum musae, and / or C. gloeosporioides, and / or race 4 of Fusarium oxysporum f. sp. cubense, and / or C. truncatum, and / or G. saubinetii, and / or C. sativum, and / or C. capsici, and / or Mycosphaerella musicola, and / or C. fragariae.

6. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for causing the hyphae of Colletotrichum musae to wrinkle, deform, and / or break, and / or for causing the spores of Colletotrichum musae to wrinkle and / or break.

7. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for causing the leakage of soluble proteins, and / or soluble sugars, and / or nucleic acids from the cell membrane of Colletotrichum musae.

8. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for preventing the colonization of Colletotrichum musae on the banana peel.

9. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for increasing the soluble solids content, and / or titratable acid content, and / or soluble sugar content, and / or vitamin C content in banana fruits.

10. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for increasing the activity of oxidase and / or polyphenol oxidase in the banana peel without significantly affecting the activity of catalase.

11. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for decreasing the activity of cellulase, and / or pectin methylesterase, and / or polygalacturonase in the banana peel.

12. Use of the Streptomyces of claim 1 or the fermentation broth of claim 2 or the preparation of claim 3 in the preparation of a preparation for decreasing the content of reducing sugars in the banana peel.

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