Streptomyces L1 and application thereof in prevention and treatment of Korla pear tree rot disease

Abstract

The invention relates to the field of microorganisms, in particular to streptomyces L1 and application thereof to prevention and treatment of Korla pear tree canker. According to the invention, Streptomyces sp. L1 is separated from Korla pear tree barks, and a plate confrontation experiment and a field test prove that the Streptomyces sp. L1 has a relatively good antagonistic effect on valsa ceratosperma of Korla pear trees, so that a new biocontrol bacterium resource is provided for prevention and treatment of the Korla pear tree rot disease.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to Streptomyces L1 and application thereof in preventing and treating Korla fragrant pear tree rot disease. Background Art

[0002] Korla fragrant pear tree rot is a fungal disease that mainly harms the phloem of the tree's trunk, main branches, side branches and twigs, causing them to necrotize. The symptoms of damage are mainly divided into two types: ulcer and branch dieback. The ulcer type mostly occurs on the trunk and main branches of the tree. The main manifestation is that the phloem tissue of the diseased part is reddish-brown in the early stage, slightly raised, and feels soft when pressed by hand. The diseased part often has brown juice with a wine-lees smell flowing out. In the later stage of the disease, the diseased part gradually loses water, shrinks and sinks, and the reddish-brown color gradually turns into black-brown. Small black spots appear on it, which are the spores of the rot disease. The branch dieback type mainly occurs on twigs, fruit mosses and dry branches. It is manifested by unclear boundaries of the phloem tissue at the diseased part, no obvious water-soaked lesions, and the phloem tissue of the branches dries up and shrinks, causing the death of the branches above the diseased part. The surface of the phloem tissue of the diseased part is densely covered with fungal fruiting bodies, which appear as small black particles.

[0003] Currently, the prevention and control of Korla fragrant pear tree rot primarily relies on prevention, supplemented by control, and treated as a rescue strategy. Specific prevention and control measures include pruning diseased branches, clearing the garden, scraping lesions, and grafting. However, these measures cannot completely control the rot; they can only mitigate the losses caused by the rot to a certain extent. Therefore, further rot control methods are needed. In recent years, biological control has attracted widespread attention from scholars due to its broad spectrum, high efficiency, environmental friendliness, harmlessness to crops, and high safety.

[0004] Although CN117844709A and CN116333889A respectively mention Streptomyces deccanensis F-04 and Streptomyces rochei, which can antagonize fragrant pear rot, the former is isolated from walnut orchard soil, and the latter is isolated from rhizosphere soil of healthy plants collected from pepper fields with continuous cropping for many years, and the source of the fragrant pear rot used in the detection is not specifically explained. Pear rot mainly harms the trunk, main branches, larger side branches and twigs, and the targetedness and antibacterial efficiency of the strains isolated from rhizosphere soil are relatively low. Therefore, even if the above-mentioned prior art provides plate antibacterial test data for fragrant pear rot, it is still difficult to ensure the prevention and control effect of pear rot in actual planting.

[0005] In addition, under the oasis agriculture model in southern Xinjiang (i.e., the production area of Korla fragrant pears), apple orchards, Korla fragrant pear orchards, and red date orchards are commonly planted adjacent to each other. In some orchards, multiple tree species even coexist, and poplars and euphratica are ubiquitous as street trees and orchard farmland shelterbelts. However, previous studies of the present invention have found that rot pathogens such as apple trees, pear trees, jujube trees, poplars, poplars, and walnut trees have an interactive infection phenomenon, which increases the severity of rot in Korla fragrant pear orchards and increases the difficulty of preventing and controlling rot in Korla fragrant pear trees. Summary of the Invention

[0006] In order to explore biocontrol strains that can prevent and control Korla fragrant pear tree rot, the present invention isolated strain L1 from the bark of Korla fragrant pear, which has a good antagonistic effect on Korla fragrant pear tree rot. Based on the comprehensive morphological characteristics of the strain, physiological and biochemical index determination, and 16S rDNA sequence identification, the antagonistic strain L1 was identified as Streptomyces.

[0007] Based on the above findings, the present invention first provides Streptomyces sp. L1, which was deposited on December 31, 2024 at the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was referred to by the number ALELMJ2406 during the deposit. Its classification name is Streptomyces sp., and the deposit number is CGMCC No. 33264.

[0008] Furthermore, the present invention also provides a bacterial agent containing the Streptomyces (Streptomyces cess p.) L1.

[0009] Furthermore, the present invention also provides a fermentation broth, which is obtained by culturing the Streptomyces sp. L1.

[0010] Furthermore, the present invention also provides a biocontrol preparation containing the Streptomyces (Streptomyces cess p.) L1, or the bacterial agent, or the fermentation broth.

[0011] Furthermore, the present invention also provides use of the Streptomyces sp. L1, or the bacterial agent, or the fermentation liquid, or the biocontrol agent in preventing and controlling pear tree rot.

[0012] Furthermore, the present invention also provides the use of the Streptomyces sp. L1, or the bacterial agent, or the fermentation liquid, or the biocontrol agent in inhibiting the pathogens of pear tree rot.

[0013] The present invention isolates Streptomyces sp. L1 from the bark of Korla fragrant pear trees, and confirms through plate confrontation experiments and field tests that it has a good antagonistic effect on Korla fragrant pear tree rot pathogens, providing a new biocontrol bacteria resource for the prevention and treatment of Korla fragrant pear tree rot disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a phylogenetic tree established by the ML method based on 16S rDNA sequences in the examples of the present invention.

[0016] Figure 2 The results of the plate confrontation experiment in the embodiment of the present invention are shown from left to right: the morphological characteristics of Streptomyces on LB medium, the morphological diagram of the pathogen of fragrant pear rot, and the confrontation diagram of Streptomyces and the pathogen of fragrant pear rot.

[0017] Figure 3 This is the growth curve of Streptomyces L1 in the embodiment of the present invention.

[0018] Figure 4 1 is the metabolic enzyme test result of Streptomyces L1 in the embodiment of the present invention; in this figure, (a), (b), (c), and (d) are the metabolic test results of the strain for protease, amylase, cellulase, and siderophore, respectively.

[0019] Figure 5 The sizes of Korla fragrant pear tree rot lesions in different concentrations of biocontrol bacteria fermentation liquid in the examples of the present invention are shown. DETAILED DESCRIPTION

[0020] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0021] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0023] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0024] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0025] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0026] In the present invention, the term "pear tree" is scientifically known as Pyrus, which refers to a deciduous fruit tree belonging to the genus Pyrus in the Rosaceae family. Common varieties include, but are not limited to, Pyrus pyrifolia, Pyrus communis, Pyrus elaeagnifolia, Pyrus calleryana, Pyrus malus, Pyrus sinkiangensis Yü, Pyrus bretschneideri, and Pyrus ussuriensis.

[0027] In the present invention, the term "prevention and control" refers to the process of reducing or eliminating pear tree rot disease through preventive and control measures, which covers both prevention and control. The purpose of the former is to reduce or avoid the occurrence of pear tree rot disease, and the purpose of the latter is to alleviate the impact or inhibit its spread after the rot disease occurs in pear trees.

[0028] The present invention first provides Streptomyces sp. L1, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.33264.

[0029] Furthermore, the present invention also provides a bacterial agent containing the Streptomyces (Streptomyces cess p.) L1.

[0030] In some embodiments, the bacterial agent may be a liquid bacterial agent or a solid bacterial agent.

[0031] In some embodiments, the bacterial agent further contains other functional bacteria.

[0032] In some embodiments, the microbial agent also contains excipients permitted in the field of microbial preparations. In specific implementation, the excipients may refer to any added ingredients in the product, in addition to the active ingredients, used to increase product stability, improve effects, or facilitate use. As an example, the excipients generally include the following types: (1) Carrier materials: a) Organic carriers: such as corn flour, wheat flour, rice bran, defatted powder, etc. These materials can provide nutritional support and provide a good environment for the growth and reproduction of microorganisms; b) Inorganic carriers: such as bentonite, silicate, etc., which are commonly used for the fixation and protection of microorganisms. (2) Protective agents: a) Glycerol, propylene glycol: used to maintain microbial activity and prevent inactivation or death during storage; b) Polyvinyl alcohol, polyvinyl pyrrolidone: protect microorganisms by forming a film and increase their stability. (3) Stabilizers: a) Sugars (such as sucrose, glucose): can improve the stability of active ingredients and promote the growth of microorganisms as a nutrient source; b) Amino acids and proteins: such as casein, gelatin, etc., can provide microorganisms with necessary nitrogen sources and promote their metabolism. (4) Thickeners and paste agents: Gelatin, carrageenan, xanthan gum: These thickeners can enhance the adhesion and stability of microbial preparations and ensure homogeneity. (5) Regulators: a) pH regulators: such as citric acid, sodium hydroxide, etc., can be used to adjust the pH of microbial preparations and optimize the growth environment of microorganisms; b) Salts: such as sodium chloride, potassium phosphate, etc., help maintain osmotic pressure and promote the survival of microorganisms. (6) Preservatives: such as certain herbal plant extracts (such as rosemary extract), which effectively prevent the deterioration of microbial culture media. (7) Surfactants: such as lecithin and polyether emulsifiers, which can improve the dispersibility and bioavailability of microorganisms. (8) Flavors and pigments: In view of market demand, a small amount of natural flavors and pigments are sometimes added to microbial preparations to improve the sensory properties of the product.

[0033] In the specific implementation scheme, the bacterial agent can be prepared by conventional technical means.

[0034] Furthermore, the present invention also provides a fermentation broth, which is obtained by culturing the Streptomyces sp. L1.

[0035] In a specific embodiment, the fermentation broth can be prepared by conventional technical means.

[0036] In some specific embodiments, when obtaining the fermentation broth, the Streptomyces (Streptomyces p.) L1 can be inoculated into a liquid culture medium (generally containing a carbon source, a nitrogen source, minerals and growth factors, etc.), maintaining a suitable temperature, pH, oxygen concentration and time to promote the growth and metabolism of the strain, that is, to promote the fermentation process.

[0037] In some embodiments, the fermentation broth is obtained by culturing the Streptomyces sp. L1 in PDB medium.

[0038] In some embodiments, the fermentation conditions for obtaining the fermentation broth are 150-200 r·min -1 (e.g. 150 r / min -1 、160r·min -1 、165r·min -1 、170r·min -1 、175r·min -1 、180r·min -1 、190r·min -1 , or 200r·min -1 etc.), 25-30°C (such as 25°C, 26°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, or 30°C, etc.).

[0039] In some specific embodiments, the fermentation broth obtained by culture can be diluted with sterile water to obtain an appropriate administration concentration.

[0040] In some specific embodiments, those skilled in the art may also dry or extract the obtained fermentation broth to concentrate and purify the fermentation product, thereby obtaining a product with the same antibacterial effect. Such a solution also falls within the scope of protection of the present invention.

[0041] Furthermore, the present invention also provides a biocontrol preparation containing the Streptomyces (Streptomyces cess p.) L1, or the bacterial agent, or the fermentation broth.

[0042] In some specific embodiments, the biocontrol agent further comprises one or more selected from other functional bacteria, plant extracts, disease-fighting factors, and natural minerals. For example, the plant extracts may be herb and spice extracts, such as garlic extract, curcumin, and rosemary extract, which possess natural antibacterial and antifungal properties; or flavonoids, such as green tea extract and red wine extract, which possess antioxidant and antimicrobial properties. Disease-fighting factors may be insect hormones, such as antibiotics and hormones (e.g., jasmonic acid), which improve plant physiology and enhance resistance; or inducible antimicrobial factors, such as hydrogen peroxide and amino acids, which stimulate the plant's own defense mechanisms. Natural minerals may be silicates, which strengthen plant cell walls and enhance disease resistance; or sulfur and copper compounds, which possess antimicrobial properties and, at appropriate concentrations, can control a variety of diseases.

[0043] Furthermore, the present invention also provides use of the Streptomyces sp. L1, or the bacterial agent, or the fermentation liquid, or the biocontrol agent in preventing and controlling pear tree rot.

[0044] Furthermore, the present invention also provides the use of the Streptomyces sp. L1, or the bacterial agent, or the fermentation liquid, or the biocontrol agent in inhibiting the pathogens of pear tree rot.

[0045] Based on the control effect of Streptomyces sp. L1 of the present invention on Korla fragrant pear tree rot disease, people in this field can confirm that this strain has a better control effect on other pear tree rot diseases, especially pear tree rot diseases related to Korla fragrant pear tree rot pathogen (Cytospora ambiens).

[0046] In some embodiments, the pear tree is a Korla fragrant pear tree (Pyrus sinkiangensis Yü).

[0047] In some embodiments, other plants susceptible to rot are also planted near the planting environment of the pear tree, and the other plants include at least one of apple trees, jujube trees, poplar trees, poplar trees, and walnut trees.

[0048] In some embodiments, the pathogen of pear tree rot includes Cytospora ambiens.

[0049] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0050] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0051] Example

[0052] 1. Research Content

[0053] 1.1 Research Methods

[0054] 1.1.1 Sampling and separation

[0055] 1.1.1.1 Sampling Information

[0056] In September 2023, 5-7 year old branches with obvious rot disease were collected from the Korla fragrant pear orchard in Jiutuan, Aral City, Xinjiang (81°9′47″ east longitude, 40°32′45″ north latitude), and the phloem about 20 cm at the junction of the diseased and healthy branches was cut off with a knife for later use.

[0057] 1.1.1.2 Separation method

[0058] Take the bark of Korla fragrant pear and wash it with pure water. Place the bark on a sterile table for surface disinfection. Cut the bark into small pieces, soak it in 75% alcohol for 3 minutes, rinse it with sterile water 3 times, 2% sodium hypochlorite solution for 3 minutes, rinse it with sterile water 6 times, take 100 μL of the last sterile water and apply it to beef extract peptone culture medium. If there are no colonies, the surface is sterilized. 2 Place the bark pieces in a sterilized mortar, grind them into a homogenate, let them stand for 1 minute, draw the supernatant, and dilute them to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Apply 100 μL of the solution to a beef extract peptone medium. Incubate the culture at 28°C for 2-5 days. Once colonies form in the medium, pick them with an inoculating loop and purify them in the appropriate culture medium using the streak method. Repeat the purification three times.

[0059] 1.1.2 Determination of strain morphological characteristics and physiological and biochemical indicators

[0060] A single colony of strain L1 was inoculated on LB solid medium and cultured at 28°C for 24 hours. The morphological characteristics and physiological and biochemical indices of the strain were observed and determined according to the "Common Bacterial Systematic Identification Manual" and "Bergey's Manual of Bacterial Identification".

[0061] 1.1.3 Identification of 16S rDNA of L1 strain

[0062] DNA from strain L1 was extracted using a genomic extraction kit. PCR amplification of 16S rDNA genes 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3') was performed using total DNA as a template. The amplification reaction system consisted of 25 μL of 2X TaqMan Fast qPCR premix, 21 μL of ddH2O, 2 μL of DNA template, and 1 μL of each upstream and downstream primer. Reaction conditions were: 95°C for 2.5 min; 35 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 10 min.

[0063] The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequences with high similarity were downloaded, and a phylogenetic tree was constructed using the maximum likelihood method (ML) in MEGA11 software with Actinokineospora fastidiosa (NR_118890) as the outgroup.

[0064] 1.2 Antibacterial effect of antagonistic bacteria L1 on Korla fragrant pear tree rot

[0065] The pathogen causing Korla fragrant pear tree rot was isolated by our research group and identified as Cytospora ambiens through morphological observation and molecular biological identification. The inhibitory effect of L1 against the Korla fragrant pear tree rot pathogen was tested using the plate standoff method. A 5 mm diameter cake of the Korla fragrant pear tree rot pathogen was inoculated in the center of a PDA medium plate. Then, 5 μL of a suspension of the test strain with an OD600 of 1.0 was dripped 2.5 cm from the center of the plate. Using sterile medium as a control, five replicates were incubated in a 28°C incubator for 5–7 days. After the blank strain grew to the edge of the plate, the plate was photographed and the inhibition rate was calculated using Image-Pro Plus 6.0.

[0066] Inhibition rate = (control colony area - treated colony area) / control colony area × 100%.

[0067] The hyphae at the edge of the pathogen colonies in the control group and the treatment group were picked and their morphology was observed under a microscope.

[0068] 1.3 Biocontrol bacteria growth curve

[0069] The biocontrol bacteria were picked after activation culture for 24 hours and inoculated into 100 mL of PDB culture medium. -1 Under sterile conditions, 1 mL of the biocontrol bacteria seed solution was aspirated and inoculated into a conical flask containing 99 mL of PDB culture medium. Three replicates were set up and placed at 28°C and 170 rpm. -1 In the shaking culture on a shaker, samples were taken after culturing for 12h, 24h, 48h, 72h, 96h, 120h and 144h, and the OD600 value of the biocontrol bacteria culture solution was determined using an ultraviolet spectrophotometer.

[0070] 1.4 Determination of metabolic enzymes of biocontrol bacteria

[0071] 1.4.1 Protease detection

[0072] A: Weigh 6.4g of skim milk powder (DSM) and dissolve it in 240mL of distilled water. Sterilize in an autoclave at 121°C for 1 minute. B: Weigh 6.4g of agar, add water to 240mL, and sterilize in an autoclave at 121°C for 20 minutes. Allow to cool and set aside. Once A is sterilized, remove and cool. Simultaneously, melt B in a microwave oven, cool to 50-60°C, and mix the two ingredients. Pour the mixture onto a plate. After inoculating the biocontrol bacteria, incubate in a 30°C incubator for 3 days. Observe for the appearance of a clearing zone and photograph.

[0073] 1.4.2 Cellulase detection

[0074] Weigh 10g of peptone, 10g of sodium carboxymethyl cellulose (CMC-Na), 10g of yeast extract, 5g of sodium chloride (NaCl), 18g of agar, and 1g of potassium dihydrogen phosphate (KH2PO4), dilute to 1000mL with water, adjust the pH to 7.0, sterilize in an autoclave, and pour into plates after cooling. Inoculate the biocontrol bacteria and culture them in a 30℃ incubator for 2 days. Take them out and use a pre-prepared concentration of 1mg·mL -1 Congo red solution was dyed, and the solution was allowed to stand for 1 hour. The dye solution was discarded and then 1 mol·L -1 Soak in NaCl solution for 1 hour, take photos and observe whether there is a transparent circle.

[0075] 1.4.3 Amylase detection

[0076] Prepare amylase identification medium: Weigh 2g of soluble starch, 0.05g of FeSO4·7H2O, 3.1g of KNO3, 0.05g of NaCl, 0.05g of K3PO4·3H2O, 0.05g of MgSO4·7H2O, 2g of agar, and 100mL of distilled water. Sterilize with high-pressure steam (121°C, 20min). Soak a filter paper in the fermentation broth to moisten it, then place it in the amylase medium. Incubate at 37°C for 2–3 days, then cover the entire medium plate with dilute iodine solution. After 15 minutes, observe for the presence of a clearing zone.

[0077] 1.4.4 Siderophore detection

[0078] A: Weigh 60.5 mg of chrome azurol sulphonate (CAS) and dissolve it in 50 mL of deionized water; B: Prepare 10 mL of ferric iron solution (prepare 1 mmol FeCl3·6H2O, which is equivalent to 10 mmol·L -1 Hydrochloric acid); C: Weigh 72.9 mg of hexadecy-ltrimethyl-ammonium bromide (HDTMA) and dissolve it in 40 mL of deionized water. Combine A and B and stir thoroughly with a glass rod. Then add C, adjust the pH to neutral, add 20.0 g of agar, and distilled water to 1000 mL. Sterilize in an autoclave (121°C, 20 minutes). After cooling to 50-60°C, mix thoroughly and pour onto a plate. Inoculate the center of the plate with the biocontrol bacteria and incubate in a 30°C incubator for 5 days. Take photos and observe for the presence of a clearing zone.

[0079] 1.5 Biocontrol efficacy in field trials

[0080] 1.5.1 Preparation of biocontrol bacteria fermentation broth

[0081] As shown in Table 1, three different concentrations of biocontrol bacteria (strain L1) fermentation liquid treatment groups and one water control group were set up. The L1 fermentation liquid with different treatments was evenly sprayed on the branches of fragrant pear. Preparation of biocontrol bacteria fermentation liquid: The biocontrol bacteria seed liquid was transferred to fresh PDB medium at a 1% inoculation rate and the mixture was incubated at 170 r·min. -1 The cells were cultured at 28°C for 2 days and diluted with sterile water to treatment 1: OD600 = 0.1; treatment 2: OD600 = 0.5; treatment 3: OD600 = 1; treatment 4: sterile water.

[0082] Table 1 Fermentation liquid fertilization experimental treatment

[0083]

[0084] One-year-old branches of Korla fragrant pear (Pyrus communis) of uniform thickness were collected from the same tree on the Tarim University campus. The branches were cut into approximately 20 cm lengths, leaving 4-5 leaves at the top. The branches were washed with clean water to remove surface dust, soaked in 4% sodium hypochlorite for 30 seconds, and rinsed three times with sterile water. The pre-prepared biocontrol bacteria fermentation liquid was poured into a conical flask, sealed with sterile plastic wrap, and the treated branches were inserted into each conical flask. Five branches were inserted into each conical flask, and three replicates were used for each concentration. The conical flasks were placed in an artificial incubator at 28°C, with a natural light cycle and a humidity of 70% ± 5%. The leaves were sprayed with the biocontrol bacteria fermentation liquid every 24 hours.

[0085] 1.5.2 Rot inoculation

[0086] The test pathogen was Cytospora ambiens, the pathogen causing Korla fragrant pear tree rot, isolated earlier by our research group. After 5 days of incubation, the branch surface was sterilized by wiping with 75% alcohol. A 5mm punch was used to create a wound in the branch, followed by inoculation with a Cytospora ambiens bacterial cake. The cake was obtained using a punch of the same diameter from a previously cultured fragrant pear tree rot strain. This cake was then inoculated onto the injured branch (with the mycelium facing the xylem). The inoculation site was then securely sealed with parafilm, which was then removed after 2 days. The rate of lesion expansion was observed.

[0087] 2. Results and Analysis

[0088] 2.1 Isolation and identification of strain L1

[0089] Strain L1 appears pale yellow on the front in beef extract peptone medium. The colonies dry out, become opaque, and are difficult to pick up. After one day of culture, a silver-gray powder appears. Strain L1 was sequenced using 16S sequencing, and the amplified sequence was compared with the NCBI database for homology analysis. A 16S gene phylogenetic tree was constructed using the maximum likelihood (ML) method using MEGA11 software, with Actinokineospora fastidiosa (NR_118890) as the outgroup. This strain clustered with Streptomyces bacillaris on a branch (e.g., Figure 1 The strain L1 was identified as Streptomyces sp. by comprehensive morphological observation and molecular biological methods.

[0090] The strain was deposited on January 20, 2025 in the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was referred to by the number ALELMJ2406 during the deposit. Its classification name is Streptomyces sp., and the deposit number is CGMCC No. 33264.

[0091] 2.2 Antibacterial activity of strain L1 against Korla fragrant pear tree rot

[0092] Plate confrontation experiments showed that Streptomyces had a significant antibacterial effect on the pathogen of Korla fragrant pear tree rot ( Figure 2 ), the inhibition rate reached 67.32% on the 8th day.

[0093] 2.3 Growth curve of Streptomyces

[0094] By measuring the OD600 value of the fermentation culture, it was shown that with the increase of inoculation culture time, the OD600 value of the biocontrol bacteria suspension increased rapidly within 12 to 96 hours, and entered a decline period from 96 to 144 hours.

[0095] 2.4 Effects of strain L1 on the prevention and growth promotion of Korla fragrant pear rot

[0096] like Figure 4 As shown in (a) to (d), the detection of biocontrol bacteria-related metabolic enzymes proved that strain L1 can secrete protease, amylase, cellulase and siderophore.

[0097] 2.5 Effect of strain L1 on the prevention of Korla fragrant pear tree rot

[0098] Different treatments were used to inoculate the Korla fragrant pear tree rot disease. The lesion sizes are shown in Table 2 and Figure 5 As shown in the figure, 5 days after pathogen inoculation, the average lesion size showed significant differences among the different treatments. This indicates that the biocontrol agent L1 has a significant preventive effect on Korla fragrant pear tree rot.

[0099] Table 2 The size of rot lesions treated with different concentrations of L1 fermentation liquid

[0100]

[0101] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible without departing from the spirit of the present invention, and all such variations and modifications fall within the scope of the present invention.

Claims

1. Streptomyces sp. L1, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit number being CGMCC No. 33264.

2. A bacterial agent comprising the Streptomyces sp. L1 according to claim 1.

3. A fermentation broth obtained by culturing the Streptomyces sp. L1 according to claim 1.

4. The fermentation broth according to claim 3, wherein The fermentation broth is obtained by culturing the Streptomyces sp. L1 in PDB medium.

5. A biocontrol agent comprising the Streptomyces sp. L1 according to claim 1, the bacterial agent according to claim 2, or the fermentation broth according to claim 3 or 4.

6. Use of the Streptomyces sp. L1 according to claim 1, or the bacterial agent according to claim 2, or the fermentation liquid according to claim 3 or 4, or the biocontrol agent according to claim 5 in preventing and controlling pear rot.

7. Use of the Streptomyces sp. L1 according to claim 1, or the bacterial agent according to claim 2, or the fermentation broth according to claim 3 or 4, or the biocontrol agent according to claim 5 in inhibiting pathogens of pear rot.

8. The use according to claim 6 or 7, wherein: The pear tree is the Korla fragrant pear tree (Pyrussinkiangensis Yü).

9. The use according to any one of claims 6 to 8, wherein Other plants susceptible to rot are also planted near the planting environment of the pear tree, and the other plants include at least one of apple trees, jujube trees, poplar trees, poplar trees, and walnut trees.

10. The use according to any one of claims 6 to 9, wherein The pathogenic bacteria of the pear rot disease include Cytospora ambiens.

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