Streptomyces olivaceus nigrum and application thereof in prevention and control of solanaceous vegetable root rot

Abstract

This invention provides a strain of Streptomyces olfelsii. Streptomyces atroolivaceus This strain was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24482. This strain is cold- and drought-tolerant and can stably colonize solanaceous vegetables and their rhizosphere soil. It contains a rich variety of active substances in its fermentation broth, including esters and coumarins. It achieves its antibacterial effect by disrupting the cell membrane stability of pathogens and inhibiting the activity of protective enzymes in pathogen cells, thereby exacerbating cellular lipid peroxidation. Simultaneously, it activates the stress resistance system of solanaceous vegetables, enhancing their resistance to pathogen infection and abiotic stress. The fermentation broth of this strain exhibits inhibitory activity against various pathogens, including Fusarium, Phytophthora, and Pythium, and demonstrates a strong control effect on root rot diseases in solanaceous vegetables caused by these pathogens.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology and relates to a dark olive streptomyces and its application in the prevention and control of root rot in solanaceous vegetables. Background Technology

[0002] Solanaceae vegetables, mainly including tomatoes, eggplants, peppers, and potatoes, are staple ingredients on people's daily tables, ranking first in market sales and production among vegetables. High demand has led to a dramatic increase in planting area. Due to long planting periods, limited variety cultivation, severe continuous cropping, and difficulties in crop rotation, diseases occur frequently and are diverse, but are widespread and cause damage year-round, resulting in losses exceeding 15%. Root rot, caused by various plant pathogens such as pathogenic monotypic bacteria, Fusarium, Phytophthora, and Pythium, is relatively difficult to control and easily develops resistance to currently used pesticides. Most of these pathogens thrive in mature soil conditions and directly invade through stomata, epidermis, and rhizomes, causing primary infection. Sporangia produced on diseased plants migrate and cause multiple reinfections, easily leading to large-scale outbreaks and severe losses. Both seedlings and mature plants can be affected, making it one of the cancerous diseases of Solanaceae vegetables. Due to the short production cycle of Solanaceae vegetables and the variety and severity of diseases, chemical pesticide control remains the primary method of disease control. However, the unrestrained use of chemical pesticides has led to a vicious cycle of increased disease resistance, rising control costs, severe environmental pollution, and harm to natural enemies. To effectively control root rot in solanaceous vegetables, achieve a reduction in both chemical fertilizers and pesticides, significantly improve vegetable quality, and ensure the safety of vegetable products and the environment, biological control technology has become the preferred alternative. Biological control utilizes organisms or biological products obtained through biological metabolism and biotechnology to manage harmful organisms. This includes the protection, utilization, and release of natural enemies (frogs, insects, birds, etc.), the application of biopesticides, utilizing the natural interactions of plants, and the research and cultivation of superior insect-resistant varieties. Among these, biopesticides, especially microbial pesticides, are one of the most important methods of biological control. They have relatively long-lasting effects, are less likely to induce resistance in pests, are harmless to natural enemies, and do not pollute the environment, giving them significant advantages in the production of green and pollution-free vegetables. Streptomyces are a major group of soil actinomycetes and an important source of natural active substances. They are among the earliest studied and successfully applied microorganisms for plant disease control, rich in various antibiotics, enzymes, and active substances from their fermentation broths. They possess multiple antibacterial mechanisms, including inhibiting the synthesis of macromolecular compounds, disrupting cell membrane structure, inhibiting the metabolic processes and mitosis of pathogenic microorganisms, affecting their morphology, or competing for nutrients and living space. Therefore, they have significant application value and development potential in the biological control of plant diseases. This invention isolates and selects superior Streptomyces strains with high activity and high resistance against root rot in solanaceous vegetables from poisonous weeds in special habitats with insecticidal and antibacterial activities, providing excellent strain resources and fundamental theoretical support for the biological control of root rot in solanaceous vegetables. Summary of the Invention

[0003] The first objective of this invention is to provide a *Streptomyces violaceum* that is drought-resistant, cold-resistant, and can stably colonize tomato roots and the surrounding soil. It achieves its antibacterial effect by inhibiting the biosynthesis of ergosterol in the cell membrane of pathogenic fungi, thereby disrupting the cell membrane structure and its stability; inhibiting the activity of protective enzymes in pathogenic fungi, and exacerbating the degree of lipid peroxidation in cells.

[0004] The second objective of this invention is to provide the application of the aforementioned *Streptomyces olfleurone* and its fermentation broth in the prevention and control of root rot in solanaceous vegetables. *Streptomyces olfleurone* and its fermentation broth can enhance the photosynthesis and carbon assimilation of solanaceous vegetables, activate their stress resistance system, significantly increase the activity of their disease-fighting enzymes, and reduce the intracellular malondialdehyde content, thereby improving the resistance of solanaceous vegetables to pathogen infection and their ability to resist abiotic stress.

[0005] To address the aforementioned problems, this invention first provides a *Streptomyces olfelsii* strain, which was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center. Streptomyces atroolivaceus The accession number is CGMCC No.24482.

[0006] The dark olive streptomyces Streptomyces atroolivaceus The highly active antagonistic strain STRA-2518, isolated and purified from the rhizome of the poisonous weed *Ligustrum lucidum* at an altitude of 3900 meters in Maqu County, Gansu Province, was finally identified as *Streptomyces atroolivaceus* after morphological observation, physiological and biochemical identification, and 16S rDNA molecular identification.

[0007] The dark olive streptomyces Streptomyces atroolivaceus It is cold- and drought-resistant and can reproduce and grow at temperatures of 5℃ to 20℃; it can tolerate severe drought in simulated environments, that is, it can grow and reproduce at a PGE6000 concentration of 150 to 270 g / L.

[0008] The dark olive streptomyces Streptomyces atroolivaceus It can stably colonize in the root zone and surrounding soil of tomatoes, and its colonization ability is strong. The colonization bacteria count is maintained at 104 cfu / ml to 105 cfu / ml within 20 days. It can also colonize in tomato leaves and stems, but the colonization is weaker, with the colonization bacteria count around 103 cfu / ml.

[0009] The dark olive streptomyces Streptomyces atroolivaceusThe antibacterial mechanism is as follows: It inhibits the growth of fungal hyphae and spore germination; it inhibits the biosynthesis of ergosterol in the cell membrane of pathogenic fungi, destroys the cell membrane structure and its stability, and causes the cell contents to leak out, resulting in the growth and development of pathogenic fungi being hindered or death; it inhibits the activity of cell-protective enzymes such as PAL, SOD, and CAT in pathogenic fungi, and aggravates the degree of cell lipid peroxidation, causing pathogenic fungal cells to die rapidly, thereby achieving the purpose of antibacterial action. The dark olive streptomyces Streptomyces atroolivaceus The live bacteria and the active substances in the fermentation broth work together to inhibit pathogenic Fusarium, Rhizoctonia solani, Phytophthora, Pythium spp., Pseudomonas spp., Cladosporium and other plant pathogens.

[0010] Dark olive streptomyces Streptomyces atroolivaceus The fermentation medium for preparing the fermentation broth consisted of 20g millet, 10g glucose, 5.0g peptone, 1.2g dipotassium hydrogen phosphate, 0.8g magnesium sulfate, 0.01g ferrous sulfate, 1000mL distilled water, and a pH of 7.4. This fermentation medium improved the sporulation rate of *Streptomyces olfleurone* and the antibacterial activity of its fermentation broth.

[0011] The second objective of this invention is the application of the above-mentioned strains and their fermentation broth in the prevention and control of root rot in solanaceous vegetables.

[0012] The dark olive streptomyces Streptomyces atroolivaceus The active substances in the fermentation broth can effectively enhance the photosynthesis and carbon assimilation of tomatoes, increasing their chlorophyll content by 16.27%; it also activates the tomato stress resistance system, significantly increasing the activity of disease-fighting and defense enzymes such as POD, SOD, CAT, PAL, and β-1,3-glucanase, while reducing the malondialdehyde content in plant cells. This improves the plant's resistance to pathogen infection and stress, and significantly reduces the risk of disease and damage. The Dark Olive Streptomyces provided by this invention Streptomyces atroolivaceus The fermentation broth and its product are highly effective against root rot of solanaceous vegetables caused by pathogenic Fusarium, Rhizoctonia solani, Phytophthora, Pythium spp., Pseudomonas aeruginosa, and Cladosporium, with an average control effect of 86% to 95%, which is quite good. There is no significant difference between the product and the control pesticide at the 0.05 level (P≥0.05). Attached Figure Description

[0013] Figure 1 The colony morphology of Streptomyces olfurtherus STRA-2518 of this invention; Figure 2 The mycelial morphology of Fusarium oxysporum treated with fermentation broth of Streptomyces olfurtherum STRA-2518 of the present invention; Figure 3 The mycelial morphology of Fusarium oxysporum that was not treated with the fermentation broth of Streptomyces olfurtherum STRA-2518 of this invention; Figure 4 This invention relates to the effect of fermentation supernatant of Streptomyces olfurtherum STRA-2518 on the protective enzymes in pathogenic fungi. Figure 5 This invention relates to the effect of fermentation supernatant of Streptomyces olfurtherum STRA-2518 on protective enzymes in pathogenic bacteria. Figure 6 The main antimicrobial spectrum of the dark olive streptomyces STRA-2518 of this invention is as follows: (The pathogens are, in order, Phytophthora capsici, Fusarium oxysporum, Fusarium solani, Pythium cucumeris, Rhizoctonia solani, Pseudomonas stolonifera, Pseudomonas stolonifera, Cladosporium, and pathogenic monotyphi). Figure 7 This invention demonstrates the drought resistance of the dark olive streptomyces STRA-2518 strain. Figure 8 This invention demonstrates the temperature tolerance of the black olive streptomyces STRA-2518. Figure 9 This invention demonstrates the colonization ability of Streptomyces olfurtherus STRA-2518 in tomatoes and their rhizosphere soil. Figure 10 This is one of the effects of Streptomyces olfurtherus STRA-2518 on the tomato defense enzyme system of the present invention; Figure 11 This is the second aspect of the effects of Streptomyces olfurtherus STRA-2518 on the tomato defense enzyme system of the present invention. Figure 12 This invention relates to the effect of Streptomyces olflacrios STRA-2518 on tomato disease-resistant enzymes. Figure 13 This invention relates to the effect of Streptomyces tarda STRA-2518 on malondialdehyde (MDA) content in tomatoes. Detailed Implementation

[0014] I. Isolation, purification, and classification of antagonistic strains Isolation and purification of strain 1 1.1 Main Culture Medium Gao's No. 1 medium, PDA medium, and NA medium are currently available with standard formulations.

[0015] Strains Isolation and Purification Collection of *Ligustrum lucidum* plants: In mid-July, healthy *Ligustrum lucidum* plants were collected from alpine meadows at an altitude of 3900m in Maqu County, Gansu Province. During sampling, the entire plant was pulled up by the roots, placed in a sterile bag, refrigerated and stored, and brought back to the laboratory for endophytic bacteria isolation.

[0016] Isolation and purification of endophytic bacteria from the rhizomes of *Ligularia gracilis*: Complete and healthy *Ligularia gracilis* plants were thoroughly cleaned with running water and rinsed under running water for 1 hour. The entire rhizome was then cut off in a clean bench and disinfected by rinsing with 75% alcohol for 2 minutes, rinsing three times with sterile water, soaking in 3% NaClO for 1 minute, rinsing three times with sterile water, rinsing with 75% alcohol for 15 seconds, and rinsing five times with sterile water. The surface moisture was then absorbed with sterile filter paper, cut into approximately 0.5 cm pieces with sterile scissors, and ground into a paste. 0.2 ml of the paste was diluted 10 times with sterile water and spread onto Gao's No. 1 medium. After standing for 1 hour, the culture dish was inverted and incubated at 28°C. Starting from the third day after culture, single colonies of actinomycetes were selected based on colony morphology and aerial hyphae characteristics, and transferred to Gao's No. 1 plates. After culturing for 7–14 days, the colonies were examined. Single strains that had undergone multiple purifications were numbered and cultured on Gao's No. 1 slant agar for 7–14 days, then stored at 4°C for later use. Furthermore, an appropriate amount of sterile water from the final rinse of the *Ligustrum lucidum* rhizomes was spread onto Gao's No. 1 agar. After culturing at 28°C for 15 days, no colonies grew, indicating that the surface of the *Ligustrum lucidum* rhizomes used for isolating endophytic bacteria was thoroughly disinfected.

[0017] Screening of highly antagonistic strains The agar diffusion method was used. Pathogenic Fusarium oxysporum was inoculated onto PDAs for activation, and then washed with an appropriate amount of sterile water to prepare 1×10⁻⁶ samples. 8 CFU / ml pathogen suspension. Add 200µl of pathogen suspension to a sterile PDA plate, spread evenly, and allow to dry at room temperature for 5 min. Then, use a sterile punch to vertically punch a 0.6cm diameter well in the prepared plate. Remove the agar block with a sterile toothpick, and seal each well by heating it under an alcohol lamp flame for 10 seconds. Inject 100µl of the fermentation broth or supernatant of the strain isolated and purified in step 1.1 into each well, using sterile water as a control. Perform four replicates for each strain's fermentation broth or supernatant. After incubation at 28℃ for 7 days, observe for the presence of inhibition zones and measure the diameter of the inhibition zones to determine the presence and strength of antibacterial activity. The pathogen was changed to *Ralstonia solanacearum*, and the inoculation medium was changed to LB medium; all other aspects remained unchanged.

[0018] Table 1: Screening results of highly antagonistic strains Note: The table lists the top 3 strains with the strongest antagonistic activity isolated and purified from 1.1.2; different lowercase letters in the same column indicate significant differences at the 0.05 level (p≤0.05).

[0019] Based on the results in Table 1, strain 2518, which exhibited the strongest antagonistic activity, was selected for further classification and identification.

[0020] Classification and identification of strain 2518 with high antagonistic activity 1.4.1 Morphological identification The slide insertion method was used. Strain 2518 was inoculated on Gao's No. 1 medium using the streak method and incubated at 28℃ for 7 days. Colony morphology was observed. After incubating at 28℃ for 3 days, sterile coverslips were inserted at a 45° angle into Gao's No. 1 medium, with 3 coverslips per plate. After continuing to incubate for 14 days, the slides were removed and observed under an optical microscope to observe hyphal morphology and the presence or absence of spores.

[0021] 1.4.2 Physiological and Biochemical Assays Referring to "Rapid Identification and Systematic Classification of Actinomycetes", physiological and biochemical characteristics such as carbon source utilization, cellulose utilization, nitrogen source utilization, gelatin liquefaction, starch hydrolysis, nitrate reduction, and milk coagulation or peptone formation were observed.

[0022] 1.4.3 16S rDNA Sequence Analysis Bacterial DNA was extracted using the protease-SDS method. Amplification primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-TACGGYTACCTTGTTACGACTT-3', sequencing and homology analysis were completed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0023] 1.4.4 Identification Results Colonies of strain 2518 are round, white, opaque, slightly raised, and with regular edges (see Figure 1). Microscopic examination reveals abundant hyphae of strain 2518, without septa, highly branched, with microspiral filaments and nearly oval conidia (see Figure 1). Figure 1 The strain tested positive for milk coagulation and peptone reaction, glucose fermentation, fructose fermentation, maltose fermentation, sucrose fermentation, starch hydrolysis, nitrate reduction, tyrosine fermentation, and histidine fermentation; and negative for gelatin liquefaction, mannitol fermentation, cellulose decomposition, and dihydrogen sulfide reaction. The 16S rDNA sequence of this strain was compared with NCBI data and belonged to the same cluster as *Streptomyces atroolivaceus*, showing 100% homology. Based on morphological characteristics, physiological and biochemical characteristics, and 16S rDNA molecular identification results, strain 2518 was identified as *Streptomyces atroolivaceus*, final identification number STRA-2518. II. Characteristics and Effects Tests: 1. Preparation method of fermentation broth of Streptomyces urophylla STRA-2518: 10 μL of Streptomyces urophylla STRA-2518 was fermented... 8 The CFU / ml bacterial suspension was inoculated into the fermentation medium at an 8% inoculum and cultured at 27±1℃ with constant temperature shaking at 200 rpm for 10 days for later use.

[0024] The above fermentation medium formula is: 20g millet, 10g glucose, 5.0g peptone, 1.2g dipotassium hydrogen phosphate, 0.8g magnesium sulfate, 0.01g ferrous sulfate, 1000mL distilled water, pH 7.4.

[0025] The above-mentioned fermentation medium improved the sporulation of Streptomyces atroolivaceus and the antibacterial activity of its fermentation broth, with specific effects as follows.

[0026] Mechanism of action of Streptomyces urophylla STRA-2518.

[0027] Effects of fermentation supernatant of strain STRA-2518 on mycelial growth of pathogenic fungi Sterile PDA culture medium and fermentation broth of strain STRA-2518 were mixed at a ratio of 8:1 and poured into plates. The mycelial cake (5 mm in diameter) of *S. scutellaria baicalensis* was then placed in the center of the medium. The plates were left to stand for 5 minutes, then inverted and incubated at 26±1℃ for 10 days. Plates without fermentation broth of strain STRA-2518 were used as controls. The growth status and diameter of the colonies were observed, and the mycelial growth inhibition rate was calculated.

[0028] Mycelial growth inhibition rate % = (average colony diameter of control - average colony diameter of treatment) / average colony diameter of control Table 2: Effects of fermentation broth from strain STRA-2518 on the mycelial growth of the target pathogen. mycelial growth inhibition rate % 83.19a 70.32b Note: Different lowercase letters in the same row of the table indicate a significant difference at the 0.05 level (p≤0.05).

[0029] Effects of STRA-2518 fermentation broth on spore germination of pathogenic fungi Take 50 µL of *Bacillus scutellatus* spore suspension (10⁸ cfu·mL⁻¹) -1 Add the sample to the center of a concave glass slide, then add 50µL of the fermentation broth of strain STRA-2518. Use sterile water as a control. Repeat each treatment 3 times. After incubating in the dark at 28℃ for 72 h, record the number of spores that germinate (spores with a germ tube length greater than 1 / 2 of the spore are considered to have germinated) and calculate the spore germination rate.

[0030] Spore germination rate (%) = (Number of germinating spores / Total number of spores) × 100 Spore germination inhibition rate (%) = (Control spore germination rate - Treated spore germination rate) / Control spore germination rate × 100 Table 3: Effects of fermentation broth from strain STRA-2518 on spore germination of target pathogens mycelial growth inhibition rate % 89.09a 71.63b Note: Different lowercase letters in the same row of the table indicate a significant difference at the 0.05 level (p≤0.05).

[0031] Data analysis: The results in Table 2 show that the fermentation broth of strain STRA-2518 inhibited the mycelial growth and spore germination of Fusarium oxysporum, with inhibition rates of 83.19% and 89.09%, respectively, which were significantly different from those of the same strain (P≥0.05).

[0032] Effects of STRA-2518 fermentation broth on hyphal morphology of target pathogens The effect of biocontrol bacteria fermentation broth on the hyphal morphology of *Fusarium oxysporum* was observed using the double-layer Oxford cup method. PDA was heated until melted, and 15 mL was poured into a petri dish. After solidification, another 5 mL of melted PDA was poured in, and an Oxford cup was placed in the center of the dish. After the culture medium solidified, *Fusarium oxysporum* mycelial cakes (5 mm in diameter) were inoculated symmetrically at 1 cm from the edge of the dish. 100 µL of the target biocontrol bacteria fermentation broth was added to the Oxford cup, while the control group received an equal volume of sterile distilled water. The petri dishes were placed upright in a constant temperature incubator and incubated at 26℃ for 3-5 days. After incubation, the petri dishes were removed, and the *Fusarium oxysporum* culture at the boundary between the *Fusarium oxysporum* and the inhibition zone was cut and placed on a glass slide. The hyphal morphology was observed using an optical microscope and compared with the control group.

[0033] analyze: Figure 2 , 3 The results showed that, compared with the untreated Fusarium oxysporum mycelial morphology, the fermentation broth of Streptomyces olfurniture STRA-2518 caused the growth point at the tip of the Fusarium oxysporum mycelium to swell, the mycelium to severely lose water and shrink, bend and deform, and break and shrink in large numbers, which severely restricted its reproduction and growth.

[0034] 2.4 Determination of Ergosterol Content in Fermentation Supernatant of Strain STRA-2518 against Pathogen (1) Preparation of ergosterol standard curve Determination of the maximum absorption peak of ergosterol: A standard solution of ergosterol with a concentration of 50 µg / mL was prepared, and its maximum absorption peak was determined by scanning with a UV spectrophotometer. Scan range: 220-320 nm; Recording range: -0.3010-3.0000 Å; Scan speed: medium speed; Sampling interval: 1 nm. High-performance liquid chromatography (HPLC) conditions: Waters C18 column (5 µm, 4.6 mm × 250 mm), mobile phase: 100% methanol, flow rate: 0.4 mL / min, column temperature: 28℃, injection volume: 20 μL, detection wavelength: 282 nm. Construction of the standard curve: Accurately weigh an appropriate amount of ergosterol standard, place it in a 25 mL brown volumetric flask, add an appropriate amount of ethanol to dissolve and dilute to the mark, and prepare a standard stock solution with a concentration of 500 µg / mL. Accurately pipette this stock solution and dilute it with methanol to prepare ergosterol standard solutions with concentrations of 250, 125, 50, 5, and 2.5 µg / mL, respectively. Pipe 50 µL of each solution into the liquid chromatograph and determine it according to the preset conditions. Plot the peak area as the ordinate and the concentration as the abscissa to obtain the standard equation: Y = 15737X + 68148. r =0.9994.

[0035] (2) Determination of ergosterol content in pathogens High performance liquid chromatography (HPLC). Several 5.0 mm Fusarium oxysporum canisters were collected and inoculated into PDA culture medium, with 10 canisters per 100 mL. The mixture was incubated at 26±1℃ and 180 r.min. -1 After 48 hours of shaking culture, a bacterial suspension was prepared. The bacterial suspension was inoculated into sterile PDA medium at a 10% inoculum size and cultured under the same conditions for 48 hours. Then, 10% (v / v) of the fermentation supernatant of strain STRA-2518 was added, with a blank control included. Culture was continued for 120 hours. The mycelia were filtered through four layers of gauze, washed with pH 7.5 PBS, and the water was absorbed with filter paper. 0.6 g of wet bacterial cells were weighed and placed in a mortar, 1.35 mL of PBS buffer was added, and the mixture was homogenized. The homogenate was transferred to a glass tube containing 50 mL of 10% KOH ethanol solution and shaken rapidly. The mixture was then extracted in an 85°C water bath for 1 hour, followed by extraction with 25 mL of petroleum ether. The sample was washed with distilled water, and the ether layer was evaporated to dryness in a 60°C water bath. The ether layer was washed down with anhydrous methanol and the volume was adjusted to 0.5 mL for analysis. Three replicates were performed per treatment. The concentration of ergosterol in the sample was determined under the same conditions as the standard curve, and the ergosterol content in the mycelia was calculated based on the standard curve. Results are expressed as g / kg mycelium.

[0036] Table 4: Effects of fermentation broth from strain STRA-2518 on the target pathogen ergosterol Ergosterol content (g / kg) 0.702a 1.683b Note: Different lowercase letters in the same row of the table indicate a significant difference at the 0.05 level (p≤0.05).

[0037] Data Analysis: Table 4 shows that the ergosterol content in the mycelia of *Fusarium oxysporum* treated with the fermentation supernatant of strain STRA-2518 decreased to 0.702 g / kg, which was significantly different from the ergosterol content in the untreated *Fusarium oxysporum* culture (p≤0.05). This indicates that the fermentation supernatant of strain STRA-2518 inhibited the synthesis of ergosterol in *Fusarium oxysporum* mycelia. Ergosterol is a sterol specifically expressed on the fungal cell membrane, which can affect the integrity, fluidity, and asymmetry of the cell membrane. Its synthesis is inhibited, disrupting the structure and stability of the pathogenic fungal cell membrane, causing leakage of cell contents, and leading to inhibition of fungal growth and development or cell death.

[0038] 2.5 Effect of fermentation supernatant of strain BAC-198 on protective enzymes in pathogens. Enzyme solution preparation The pathogen was a fungus: *Bacillus scutellatus* was activated and cultured on PDA medium at 26°C for 10 days. After washing away the spores with sterile water, 6% of the inoculum was added to the PDA medium and incubated at 26°C and 180 rpm. -1 After culturing for 72 hours, an equal volume of the fermentation supernatant of strain STRA-2518 was inoculated into *Bacillus scutellarioides* culture medium, with sterile water added as a control. The culture was continued for 120 hours. Mycelia were collected by filtration through four layers of gauze and purified using pH 7.8 PBS (25 mmol / L). -1 Rinse the mycelium, absorb the water with filter paper, take 0.5g of mycelium in a pre-cooled mortar, add quartz sand and 3.5mL of pre-cooled pH 7.8 PBS, grind in an ice bath to form a homogenate, 4℃, 8000r.min. -1 Centrifuge for 10 min, the supernatant is the crude enzyme solution, stored at -20℃ for later use. The pathogen is bacteria: *Rauvolfia solanacearum* was activated and cultured on Gao's No. 1 medium at 28℃ for 2 days. The bacterial cells were washed away with sterile water and inoculated into NA culture medium at 6% concentration, incubated at 28℃ and 180 rpm. -1 After culturing for 12 hours, an equal volume of the fermentation supernatant of strain STRA-2518 was inoculated into the culture medium of *Rahuella solani*, with sterile water as a control. The culture was continued for another 36 hours. The culture medium was then incubated at 4°C and 10,000 rpm. -1 Centrifuge, collect the bacterial sludge, and treat the rest in the same way as the pathogenic fungus.

[0039] Enzyme activity assay (1) Effect of fermentation supernatant of strain STRA-2518 on the activity of phenylalanine ammonia-lyase (PAL) of pathogenic bacteria Add 1 ml of enzyme solution, 1 ml of 0.02 mol / L phenylalanine, and 2 ml of distilled water to a total volume of 4 ml. For the control, add 1 ml more distilled water instead of substrate. Incubate the reaction mixture in a constant temperature water bath at 30°C for 0.5 h. Measure the absorbance at 290 nm using a UV spectrophotometer. One unit is defined as the amount of enzyme required to produce a 0.01 μg change in absorbance at 290 nm per hour (equivalent to the formation of 1 μg cinnamic acid per milliliter of reaction mixture). Protein content is determined using the Folin-phenol method.

[0040] PAL activity (U / (mg.min)) = (A) CK -A E ) / (C*×0.01*60) A CK : Absorbance of the control group without substrate; A E : Absorbance of the sample tube; C: Protein content; 0.01: 0.01 U unit is defined as a change in OD value of 0.01 per hour; 60: Reaction time is 60 minutes.

[0041] (2) Effect of fermentation supernatant of strain STRA-2518 on superoxide dismutase (SOD) activity of pathogenic bacteria Table 5: Amounts required for colorimetric reactions of various solutions <![CDATA[0.05 mol·L -1 Phosphate buffer 1.5 1.5 1.5 <![CDATA[130 mmol·L -1 Met Solution]]> 0.3 0.3 0.3 <![CDATA[750 µmol·L -1 NBT solution 0.3 0.3 0.3 <![CDATA[100 µmol·L -1 EDTA-Na2 solution 0.3 0.3 0.3 <![CDATA[20 µmol·L -1 Riboflavin solution 0.3 0.3 0.3 enzyme solution 0.1 0 0 distilled water 0.5 0.6 0.6 Take clean test tubes, add solutions according to Table 5, place one control tube in the dark, and react the other tubes under 4000 Lx sunlight for 20 min. For SOD activity determination, use the unlit control tube as a blank and measure the absorbance of the other tubes.

[0042] Total SOD activity (U / g) = (A ck A E ) × V / 1 / 2 × A ck × W xVt In the formula: total SOD activity is expressed as enzyme units per milligram of sample (U.mg) -1 ) ; A ck : Absorbance of the control tube under illumination; A E V is the absorbance of the sample tube; V is the total volume of the sample solution (mL); Vt is the amount of sample used during the determination (mL); w is the fresh weight of the sample (mg).

[0043] (3) Determination of CAT enzyme activity of strain STRA-2518 fermentation supernatant against pathogens CAT enzyme activity was determined by UV spectrophotometry. 2.9 mL of phosphate buffer (0.1 mol / L, pH 7.0) containing 0.1% H₂O₂ was incubated at 30 °C for 30 min. Immediately after incubation, the mixture was placed in a spectrophotometer, and 0.1 mL of diluted enzyme solution was added. The change in absorbance of the mixture over 1 min was measured at 240 nm. The OD₂ value over 1 min was used as the metric. 240 A reduction of 0.1 in enzyme amount equals one enzyme activity unit (U).

[0044] CAT enzyme activity (U / mg protein) = ΔOD / [0.1 × protein content in crude enzyme solution (mg)] △OD = ODso - ODs1,; 0.1: OD 240 Each decrease of 0.1 represents one enzyme activity unit.

[0045] Data analysis: Intracellular superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), catalase (CAT), etc., are important components of the antioxidant system and play a protective role when cells are damaged. Figure 4 , 5 The results showed that treatment with the fermentation supernatant of strain STRA-2518 significantly inhibited the activity of SOD, PAL and CAT in the pathogen cells, disrupting the reactive oxygen metabolism balance of mycelial cells, aggravating the degree of lipid peroxidation of mycelial cell membranes, severely damaging the structural integrity of the cell membrane system, causing leakage of contents, resulting in mycelial cell shrinkage and rupture, leading to severe mycelial breakage and twisting, affecting its growth and reproduction or causing death.

[0046] The main active substances in the fermentation broth of Streptomyces olfurtherum STRA-2518 Active substances were extracted from the fermentation broth of *Streptomyces urophylla* STRA-2518 using a methanol (containing a mixture of isotope-labeled internal standards)-ultrasonic extraction method. The extracted substances were then sent to Shanghai Aqu Biotechnology Co., Ltd. for non-target metabolomics analysis using LC-MS and GC-TOF-MS. The results are shown in Table 6.

[0047] Table 6: Main active substances in the fermentation broth of strain STRA-2518

[0048] Table 6 shows that the active substances in the fermentation broth of *Streptomyces olfleurone* STRA-2518 mainly include aromatic compounds, alkaloids, phenylpropanoids, organic acids, organic heterocyclic compounds, phenols, terpenes, sterols, coumarins, anthracenes, sugars, nucleic acids, and amino acids. Among them, 3,4-Dihydroxyphenylglycol; Acetaminophen; (-)-Fumigaclavine B; (3xi,6E)-1,7-Diphenyl-6-hepten-3-ol; 4R,5S,7R,11S)-11,12-Dihydroxy-1(10)-spirovetiven-2-one 11-glucoside; Biochanin A; Cyprodinil; Dihydrocapsaicin; Adipicacid; 3-Hydroxyquinine; (ent-2b,4S,9a)-2,4,9-Trihydroxy-10(14)-oplopen-3-one 2-(2-methylbutanoate) 9-(3-methyl-2E-pentenoate); Erythroskyrin;Alantolactone;1'-O-Acetylpaxilline; Fenbendazole; Androsterone sulfate; 2-(5-Methyl-2-furanyl)pyrrolidine; Estrone; 2-Amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline; 3-Hydroxy-beta-ionone; Gingerol;3-Hydroxy-6,8-dimethoxy-7(11)-eremophilen-12,8-olide; Hygromycin B; Isoeugenol benzyl ether; Lactaroviolin;Mesalazine;6-Hydroxymelatonin; Perilloside C; Arecaidine; 6-Methylquinoline;Piperidine; Psoralen; Pyridoxine; 6,10,14-Trimethyl-5,9,13-pentadecatrien-2-one; Quercetin;Tetrahydrocurcumin; 7-Aminoflunitrazepam; S-Furanopetasitin;Mesalazine; (-)-Farnesiferol C; alpha-Solanine; 7-Ethoxy-4-methyl-2H-1-benzopyran-2-one; L-Arginine;Natamycin; L-Glutamic acid; L-Lysine;L-Norleucine; Isonicotinic acid; Isopimpinellin; L-Phenylalanine; L-Proline; L-Serine; L-Threonine; Pseudoionone; Pyridoxal and Pyridoxamine are compounds unique to this strain. Other antibacterial and stress-resistant compounds include adipic acid, styracil lactone, androstenone monosulfate, salsamolol, psoralen, furanyltetrahydropyrrole, aminotrimethylimidazoquinoline, methylquinoline, ethoxymethylcoumarin, arecoline, pyrimethanil, fenbendazole, isodesin, isofenzin, isonicotinic acid, mesalazine, protodiosgenin, and myristic acid. These compounds also contain amino acids and plant hormones, which help activate the plant's resistance system and enhance its stress resistance.

[0049] 4. Main antibacterial spectrum of live Streptomyces urophylla STRA-2518.

[0050] The inhibition spectrum of Streptomyces urophylla STRA-2518 was determined by either the inhibition zone method (for bacteria) or the plate confrontation method (for fungi) in the diffusion method.

[0051] Plate confrontation method. Pathogenic fungi such as *Fusarium oxysporum*, *Fusarium solani*, *Rhizoctonia solani*, *Phytophthora capsici*, *Pythium spp.*, *Pseudomonas aeruginosa*, and *Cladosporium* were inoculated onto PDA plates for activation, and several mycelial cakes were collected for later use. A cross was drawn on the bottom of a sterile PDA plate, with the pathogenic fungal cakes placed in the center of the cross. Four 0.5cm diameter wells were made 1.0cm from the center of the cross on the PDA plate, and 50µl of *Streptomyces urophylla* STRA-2518 fermentation broth was inoculated into each well. Sterile water served as a control plate. The experiment was repeated four times. After incubation at 28℃ for 12 days, the presence and size of inhibition zones were observed to determine whether *Streptomyces urophylla* STRA-2518 had an inhibitory effect and the strength of its inhibitory ability.

[0052] Inhibition zone method: Pathogenic bacteria such as pathogenic monotypic bacteria are activated and cultured in NA medium at 28℃ for 2-3 days. 5 mL of sterile water containing 0.3% Tween 80 is added to the slant agar. The bacterial growth is scraped off and placed in a 50 mL Erlenmeyer flask containing sterile glass bulbs. After shaking thoroughly for 2 hours on a shaker, it is diluted to a bacterial count of 1 × 10⁻⁶. 8 CFU / mL, for later use. Spread 200 μL of pathogen suspension evenly on a NA medium plate. Place a sterile steel ring (0.6 cm in diameter) in the center of the plate, add 100 μL of Streptomyces urophylla STRA-2518 fermentation broth, and use sterile water as a blank control. Incubate at 28℃ for 2 days. Observe the presence and size of the inhibition zone to determine whether Streptomyces urophylla STRA-2518 has antibacterial activity against it and the strength of the activity.

[0053] Figure 6 The results showed that the main antibacterial spectrum of Streptomyces davidiana STRA-2518 was pathogenic monotypic bacteria, Fusarium oxysporum, Fusarium solanum, Rhizoctonia solani, Phytophthora capsici, Pythium spp., Pseudomonas spp., Cladosporium and other plant pathogens.

[0054] 5. Determination of drought resistance of Streptomyces urophylla STRA-2518 Different concentrations of aseptically treated PEG6000 were added to 100 mL of sterilized fermentation medium to achieve final concentrations of 0, 30, 60, 90, 120, 150, 180, 210, 240, and 270 g / L. The medium was then inoculated with 6% *Streptomyces olfleurone* STRA-2518 seed culture and cultured at 28℃ and 200 r / min for 10 days with shaking. The OD value at 700 nm was then read using pure fermentation medium to zero the culture. A PEG6000 concentration of 0-60 g / L represents mild drought, 90-150 g / L represents moderate drought, and greater than 150 g / L represents severe drought.

[0055] Figure 7 The results showed that *Streptomyces olfur* STRA-2518 exhibited strong drought tolerance, capable of growth and reproduction at PGE6000 concentrations below 270 g / L, with better growth and reproduction observed at lower PGE6000 concentrations. Under simulated severe drought conditions with PGE6000 concentrations ranging from 150 g / L to 270 g / L, the OD of the treated solution... 700nm A value between 0.895 and 0.075 indicates that Streptomyces urophylla STRA-2518 can also grow and reproduce.

[0056] Determination of the cold resistance of Streptomyces urophylla STRA-2518.

[0057] Colonies of *Streptomyces olfelsii* STRA-2518 were picked using an inoculation loop and inoculated into Gao's No. 1 liquid medium. The medium was cultured at 28°C and 180 rpm for 24 h to prepare a seed culture. The *Streptomyces olfelsii* STRA-2518 seed culture was then inoculated at a 6% inoculation rate into Gao's No. 1 liquid medium (pH 7.2, containing 5 g / L NaCl). The medium was then cultured at 0, 5, 10, 20, 30, 37, 42, 50°C, and 60°C at 180 rpm for 48 h on a shaker. Uninoculated control cultures were used. The OD value of each treatment culture was measured at 600 nm using sterile Gao's No. 1 liquid medium to determine the temperature tolerance of *Streptomyces olfelsii* STRA-2518.

[0058] Figure 8 The results showed that *Streptomyces olfelsii* STRA-2518 can grow normally at temperatures ranging from 20℃ to 37℃, and also grows well at temperatures ranging from 0℃ to 20℃. Growth and reproduction slow down at lower temperatures, although a small amount of growth and reproduction still occurs at 0℃. It can also grow at temperatures ranging from 37℃ to 50℃, but at a slower rate, it dies at 50℃. This demonstrates that *Streptomyces olfelsii* STRA-2518 can tolerate temperatures below 50℃, and is particularly resistant to low temperatures.

[0059] Colonization capacity of Streptomyces urophylla STRA-2518 in tomatoes and its rhizosphere soil was determined. Colonization capacity of strain STRA-2518 in tomato roots, stems, leaves, and rhizosphere soil was determined: Rifampicin and kanamycin-labeled strain of *Streptomyces olfleurone* STRA-2518 was inoculated into Gao's No. 1 culture medium containing 300 μg / mL rifampicin and 200 μg / mL kanamycin, and cultured at 28±1℃ with shaking at 180 r / min for 72 h. The concentration was then diluted to 10. 8 CFU / mL, 10.0 mL / plant was used for root irrigation of tomato plants in the experimental standard, and 5.0 mL / plant was sprayed on the plant surface. Sterile culture medium was used as a control. A total of 500 plants were treated. 1.0 g of root, stem, and leaf tissue and rhizosphere soil (soil closely attached to the root system was taken) were collected at 1, 5, 10, 15, 20, 25 and 30 days after inoculation. The root, stem, and leaf samples of the treated plants were each divided into two equal portions (0.5 g). One portion was wiped with 70% alcohol, then soaked in 0.1% mercuric chloride for 1.5–2.0 min, washed five times with sterile water, dried, chopped, and ground with 1 mL of sterile water. The other portion was directly shaken with 5 mL of sterile water five times for 15 min each time. The shaken liquids were combined and set aside. The rootstock (1.0 g) was dispersed in 10 mL of sterile water, shaken at 200 r / min for 10 min, and allowed to stand. The supernatant was then diluted to 10. －1 10 －２ 10 －３ 10 －４Then, 200 μl of each of the above sample solutions was evenly spread onto Gao's No. 1 culture medium plates containing 300 μg / mL rifampicin and 200 μg / mL kanamycin. Each treatment was repeated three times, and the samples were incubated at 28±1 ℃ for 48 h before counting. Based on the average number of colonies in each treatment, the bacterial count (cfu / g) in each gram of fresh leaves, roots, stems, and rhizosphere soil was calculated.

[0060] Data analysis: Excellent biocontrol strains must possess broad-spectrum activity and high toxicity. Furthermore, they must be able to colonize the host and its rhizosphere, occupying favorable sites, and survive for a considerable period in competition with the natural environment and its rhizosphere microbiota. Only then can they potentially be developed into biopesticides. Therefore, in current research on biocontrol microorganisms, their colonization ability in crops and their rhizosphere soil is used as an important evaluation indicator for screening superior biocontrol strains. Figure 9 The results showed that *Streptomyces illus var. tarda* STRA-2518 could stably colonize tomato roots, stems, leaves, and rhizosphere soil, with the strongest colonization ability observed in the rhizosphere soil. From 1 day to 25 days after inoculation, the colonization count remained at 10. 5 The bacterial count reached 10 CFU / ml, and it also had a strong ability to colonize the plant roots, with the bacterial count reaching 10 CFU / ml within 20 days after inoculation. 4 The colonization rate is CFU / ml, and it can also colonize in leaves and stems, but weakly, with the colonization count generally around 10. 3 Approximately CFU / ml.

[0061] Effects of Streptomyces urophyllum STRA-2518 on tomato chlorophyll and its ability to induce stress resistance 8.1 Effects of Streptomyces urophylla STRA-2518 fermentation broth on tomato chlorophyll Chlorophyll content was determined using an extraction method. Plump tomato seeds were selected, disinfected with 75% ethanol for 20 minutes, then with 0.5% sodium hypochlorite for 1 minute, and finally rinsed thoroughly with sterile water and dried. After germination, the seeds were soaked in 40℃ warm water for 60 minutes and then sown in nutrient pots filled with composite substrate. After emergence, seedlings were treated with 20 mL of *Streptomyces urophylla* STRA-2518 fermentation broth per plant via root drenching and spraying every 7 days, for a total of 4 treatments. Sterile water served as a blank control. Each treatment group consisted of 20 seedlings, and each treatment was replicated 4 times. The seedlings were cultured at room temperature under a 12 h / 12 ​​h photoperiod. Five days after the last application, tomato leaves were collected, rinsed, and dried with filter paper. The midrib was removed, and 0.1 g of the extract was added to 20 mL of the extract. The extract was prepared using acetone:ethanol:distilled water in a ratio of 4.5:4.5:1 and mixed thoroughly. Soak the leaves in the dark at room temperature for 8-12 hours until they turn completely white. Mix thoroughly with a vortex mixer, and bring the volume of the upper green solution to 20 mL. Pour the extract into a cuvette with a light path of 1 cm, zero the container with the prepared extract, and read the absorbance at 652 nm. Calculate the tomato chlorophyll content using the following formula: Chlorophyll content (mg / g) = OD 652 ×V / 34.5×m OD 652 The absorbance value was read at 652 nm; V represents the total volume of the extract (mL); m represents the fresh weight of the leaf (g).

[0062] Table 7: Effects of fermentation broth of Streptomyces urophylla STRA-2518 on chlorophyll content in tomatoes

[0063] Note: △t represents the growth rate.

[0064] Effects of live Streptomyces urophylla STRA-2518 and active substances in fermentation broth on the activity of tomato-related defense enzymes and disease-related enzymes. 8.2.1 Treatment of Test Plants: Plump tomato seeds were selected and disinfected with 75% ethanol for 20 minutes, followed by 0.5% sodium hypochlorite for 1 minute. Finally, the seeds were rinsed thoroughly with sterile water and dried. After germination, the seeds were soaked in 40℃ warm water for 60 minutes and then sown into composite substrate nutrient pots containing BSS-142 fermentation broth (500 ml / g). After emergence, the seedlings were treated with 25 mL of the broth per plant by root irrigation and spraying every 5 days for a total of 5 treatments. No watering was applied in between. Sterile water served as a blank control. Each treatment group consisted of 50 seedlings, and each treatment was replicated 4 times. The experimental seedlings were cultured at room temperature under a 12h / 12h photoperiod. The experiment began 3 days after the last treatment.

[0065] 8.2.2 Extraction of enzyme solution After the tomato seedlings for the test were completed, they were uprooted and divided into different treatment groups. Leaves from the same part of the plant were cut off. Two-sixths were used immediately, and four-sixths were frozen with liquid nitrogen and stored in a -80℃ refrigerator for later use.

[0066] Take 0.5 g of leaves from the same part of the tomato seedling, put them in a pre-cooled mortar, add 5 mL of pre-cooled 0.05 M pH 6.8 phosphate buffer (containing 2% polyvinylpyrrolidone) and a small amount of quartz sand, grind them into a slurry in an ice bath, centrifuge at 8000 rpm for 20 min at 4℃, and the supernatant is the crude enzyme solution. Make up to 10 mL and use it to determine the activities of peroxidase and polyphenol oxidase. Store it in a -20℃ refrigerator for later use.

[0067] Take 0.5g of leaves from the same part from a -80℃ freezer and place them in a pre-cooled mortar. Add 5 mL of pre-cooled 0.05 M borate buffer (pH 8.8, containing 5mM mercaptoethanol, 1mM EDTA and 1% PVP) and a small amount of quartz sand. Grind the mixture into a slurry in an ice bath. Centrifuge at 8000rpm for 20 min at 4℃. The supernatant is the crude enzyme solution. Make up to 10 mL and use it to determine the activity of phenylalanine ammonia-lyase (PAL). Store the solution in a -20℃ freezer for later use.

[0068] Take 1g of leaves from the same part from a -80℃ freezer and place them in a pre-cooled mortar. Add 5mL of pre-cooled 0.05M pH 7.8 phosphate buffer (containing 2% polyvinylpyrrolidone) and a small amount of quartz sand. Grind in an ice bath to form a slurry. Centrifuge at 8000 rpm for 20min at 4℃. The supernatant is the crude enzyme solution. Make up to 25mL and use it for SOD and CAT enzyme activity assays. Store in a -20℃ freezer for later use.

[0069] Take 0.5g of leaves from the same part from a -80℃ freezer and grind them into powder in a pre-cooled mortar. Then add 7mL of 50mmol / L sodium acetate buffer (pH 5.0) to homogenize. Centrifuge the mixture at 15000 rpm for 15min at 4℃. Use the supernatant for β-1,3-glucanase activity assay and store it at -20℃ for later use.

[0070] 8.2.3 Activity determination of plant-related defense enzymes and disease-related enzymes (1) Peroxidase (POD) activity assay Take four test tubes and add 0.5 mL of 0.05 M phosphate buffer (pH 6.8), 0.5 mL of 1% guaiacol, 100 µL of crude enzyme solution (with inactivated crude enzyme solution added to the control tube), and 5 mL of distilled water to each tube. After adding 0.5 mL of 2% H₂O₂, immediately start a stopwatch and measure the OD value at 470 nm, reading every 30 seconds for 8-9 minutes (until the OD value no longer increases or changes little). Calculate the enzyme activity.

[0071] POD activity = ΔA 470 ×(V / Vt) / 0.01 × t × W In the formula, △A 470 V: Change in absorbance during reaction time; V: Total volume of extract (ml); Vt: Volume of enzyme solution used during determination (ml); t: Reaction time (min); W: Sample weight (g).

[0072] (2) Assay of phenylalanine ammonia-lyase (PAL) activity Take four test tubes and add 3.8 mL of 0.1 M borate buffer (containing 5 mM mercaptoethanol) and 1 mL of 0.02 M L-phenylalanine to each tube, followed by 100 µL of crude enzyme solution (the control tube contains inactivated crude enzyme solution). Incubate at 40 °C for 60 min, then add 1 mL of 6 M hydrochloric acid to terminate the reaction. Measure the OD value at 290 nm. Calculate enzyme activity.

[0073] PAL activity (U / (g.min)) = (A CK -A E ) / (C*×0.01*60) In the formula A CK : Absorbance of the control group without substrate; A E : Absorbance of the sample tube; C: Protein content; 0.01: 0.01 U unit is defined as a change in OD value of 0.01 per hour; 60: Reaction time is 60 min.

[0074] (3) Assay of superoxide dismutase (SOD) activity Take 7 test tubes and add 1.5 mL of phosphate buffer (pH 7.8, 0.05 mol / L), 0.3 mL of methionine solution (130 mM), 0.3 mL of 750 µM nitrocyanate tetrazolium chloride (NBT), 0.3 mL of 100 µM EDTA-Na, and 0.5 mL of distilled water to each tube, for a total of 2.9 mL (the above reagents can be mixed in proportion and prepared fresh). Then add 100 µL of crude enzyme solution to three of the tubes and inactivated crude enzyme solution to the other four tubes. Add 0.3 mL of 200 µM riboflavin. Place the three tubes of crude enzyme solution and the three tubes of inactivated enzyme solution in a light incubator with 4000 Lux daylight lamp at 25°C for 15 min, and place the tube of inactivated enzyme solution in the dark for 15 min. The tube of inactivated enzyme solution in the dark is used as a control for zeroing. Measure the OD of each light treatment. 560 nm. Enzyme activity is calculated with 50% inhibition of NBT photoreduction as one unit.

[0075] Total SOD activity (U / g) = (A ck A E ) × V / 1 / 2 × A ck × W x Vt In the formula: Total SOD activity is expressed in enzyme units per gram of sample (Ug) -1 ) ; A ck : Absorbance of the control tube under illumination; A E V is the absorbance of the sample tube; V is the total volume of the sample solution (mL); Vt is the amount of sample used during the determination (mL); w is the fresh weight of the sample (g).

[0076] (4) Determination of catalase (CAT) activity Take four test tubes and add 5 mL of phosphate buffer (pH 7.8, 0.05 mol / L), 1 mL of distilled water, and 50 µL of crude enzyme solution to each tube (add inactivated crude enzyme solution to the control tube). Immediately after adding 2% H₂O₂ (add an equal volume of distilled water to the control tube), quickly pour the solution into a quartz cuvette and measure the absorbance at 240 nm. Read the absorbance every 30 seconds for a total of 9–10 minutes. Calculate the enzyme activity.

[0077] CAT enzyme activity (U / g protein) = ΔOD / [0.1 × protein content (g) in crude enzyme solution] In the formula, △OD=ODso-ODs1,; 0.1: OD 240 Each decrease of 0.1 represents one enzyme activity unit (U).

[0078] (5) Effect on β-1,3-glucanase activity Protein content was determined using bovine serum albumin as the standard protein and the kelp polysaccharide method.

[0079] Preparation of glucose standard curve: Take 1 mg / mL glucose solution (0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 mL), add water to a volume of 1.5 mL, add 2 mL of DNS solution to terminate the reaction, place in a boiling water bath for 5 min, cool rapidly, then add 4.5 mL of distilled water, and measure the OD. 540 Plot a standard curve with sugar content on the x-axis and OD value on the y-axis: Y = 2.7259X - 0.0.1208, r = 0.9991. Take 0.1 mL of enzyme solution, add 0.4 mL of kelp polysaccharide substrate solution, and then add 1 mL of acetate-sodium acetate buffer (0.1 M, pH 5.0). For the control, use 0.1 mL of buffer solution instead of enzyme solution. React in a 37°C water bath for 30 min, then add 2 mL of DNS solution to terminate the reaction. Place in a boiling water bath for 5 min, cool rapidly, and measure the OD. 540 The amount of reducing sugar was calculated from the standard curve. One enzyme activity unit (U) was defined as the amount of enzyme that produces 1 µg of reducing sugar per gram of protein per minute, and the value was expressed as U / mg protein.

[0080] A standard curve was established based on the absorbance (x) and concentration (y, mg / ml) of the standard tube. ΔA was then substituted into the formula to calculate the sample absorbance. The content of reducing sugars produced in the process, y value (mg / ml). β-1,3-glucanase activity (U / mg) = y ÷ Cpr In the formula, Cpr represents the protein concentration in the sample. (6) Effect on malondialdehyde content The thiobarbituric acid method was used for determination. 0.5 g of leaves from the same part of a tomato seedling was added to 20 mL of 0.05 mol / L solution. -1 The mixture was homogenized with 1.0 g of quartz sand in pH 7.8 PBS in an ice bath at 4°C and 8000 rpm. -1 Centrifuge for 10 min, collect the supernatant and store at -20℃ or below for later use. Take 2 mL of the supernatant, add 2 mL of 0.67% TBA, shake well, place in a boiling water bath for 30 min, then quickly cool in an ice-water mixture to terminate the reaction. Continue cooling at 4℃ and 8000 rpm. -1 Centrifuge for 10 min, collect the supernatant, and use 0.5% TBA solution as a blank to measure the absorbance at 600, 532 and 450 nm to calculate the malondialdehyde content.

[0081] MDA (µmol·kg) -1 ) = 6.45 (A53 2 -A 600 -0.56 × A 450 Data Analysis: Table 7 shows that the fermentation broth of *Streptomyces purpureus* STRA-2518 significantly increased the chlorophyll content of tomato plant leaves, reaching 4.903 mg / g, a growth rate of 16.27%. This enhanced photosynthesis in tomato plants, resulting in larger, stronger plants and increased disease resistance. Plant-induced resistance is the phenomenon of resistance to harmful pathogens that plants develop after being induced by physical, chemical, or biological factors. Figures 10, 11, 12, and 13 show that the fermentation broth of *Streptomyces purpureus* STRA-2518 significantly increased the activities of defensive enzymes such as POD, PAL, SOD, and CAT, as well as the activities of plant disease-resistant enzymes such as β-1,3-glucanase, in tomato plants. Compared with untreated tomato plants, the activities of related defensive and disease-resistant enzymes were more than doubled. This strengthens the cell wall structure of tomato plants and promotes the production of disease-resistant substances such as phytoalexins, lignin, and phenolic compounds, thereby enhancing the plant's resistance to stress and disease. The malondialdehyde (MDA) content in tomato cells decreased by more than half, indicating that it inhibited lipid peroxidation in tomato cells, improved the plant's resistance to abiotic stress, and significantly reduced the degree of disease infection and damage.

[0082] 9. The control effect of Streptomyces violaceum STRA-2518 on root rot in solanaceous vegetables. Plant preparation: Select plump pepper and tomato seeds, disinfect them with 70% alcohol for 1 minute, then disinfect them with 0.5% sodium hypochlorite for 1 minute, rinse them with sterile water 5-6 times, and then soak them in sterile water at 40 ℃ for 2 hours. Germinate them at a constant temperature of (27±1) ℃ in the dark. After most of the seeds have germinated, select the seeds with consistent germination and sow them.

[0083] The fermentation broth of Streptomyces urophylla STRA-2518 was prepared as described in Section II.1 above.

[0084] Preparation of pathogenic fungal spore (cell) suspensions: Pathogenic monotypic bacteria and other pathogenic fungi were activated on NA medium for 72 h; Fusarium oxysporum, Fusarium solanum, Rhizoctonia solani, Phytophthora capsici, Pythium spp., Pseudomonas aeruginosa, Cladosporium, and pathogenic monotypic bacteria were activated on PDA medium at 26±1℃ for 10-15 days; after a large number of cells or spores were generated, they were washed with an appropriate amount of sterile water to prepare suspensions containing more than 10 spores (cells). 8 cfu·ml -1 The spore (fungus) suspension is prepared for later use.

[0085] Test reagents: Fermentation broth of Streptomyces urophylla STRA-2518 was used as the test reagent; 800-fold dilution of 68% metalaxyl-manganese zinc suspension was used as the positive control (fungus); 800-fold dilution of 20% thiamethoxam zinc suspension was used as the positive control (bacteria); and water treatment was used as the blank control.

[0086] Control Experiment: The seedling substrate was sterilized. 30% by volume (by weight) of the test agent, control agent, and water (blank control) were inoculated into the sterile substrate and kept at a temperature (28±1℃) and humidity for 3 days. Then, 10% by volume (by weight) of each pathogenic fungal spore (fungus) suspension was inoculated into the mixed substrate and kept at a temperature (26±1℃) and humidity for 12 days (fungi) or 3 days (bacteria). The substrate was then filled into 50-cell seedling trays and labeled. Seeds of the tested peppers and tomatoes with uniform germination were sown in the seedling trays. Two trays were treated with each pathogen suspension and labeled. Each tray contained 50 seedlings. Tomatoes were sown in trays inoculated with Fusarium, Pythium citrinum, and pathogenic monotypic mycelial (body) suspensions; peppers were sown in trays inoculated with Rhizoctonia solani, Phytophthora, Pseudomonas, and Cladosporium mycelial suspensions. Routine management was implemented, and the trays were prepared for use. The experimental agent, control agent, and sterile water were then applied to the roots by drenching and scoring at a rate of 30 ml / plant. All treatments were managed with insulation (28±1℃) and humidity (75%~80%). A total of three treatments were conducted, with the first two treatments spaced 3 days apart and the last treatment 7 days apart. Daily observations and records of seedling emergence, plant growth, and disease incidence (including those that had not yet emerged; the cause of disease in those that had not emerged was investigated promptly) were kept. Thirty-five days after emergence (with a disease incidence rate greater than 10% in the blank control), the number of diseased plants and their disease severity were tallied, and the control effect was calculated. The experimental results are shown in Table 8.

[0087] Calculation formula: Disease incidence rate (%) = Number of diseased plants / Total number of plants surveyed × 100 Control efficacy (%) = (Incidence rate of diseased plants in the control area - Incidence rate of diseased plants in the treatment area) / Incidence rate of diseased plants in the control area × 100 Table 8: Control effect of Streptomyces urophylla STRA-2518 fermentation broth on root rot in solanaceous vegetables

[0088] Note: Different lowercase letters in the same row of the table indicate a significant difference at the 0.05 level (p≤0.05); positive controls were 68% metalaxyl-manganese zinc suspension diluted 800 times (fungus) and 20% thiamethoxam zinc suspension diluted 800 times (bacteria); no bacterial wilt was observed.

[0089] Data Analysis: Table 8 shows that the fermentation broth of *Streptomyces illus var. illus* STRA-2518 can effectively control various diseases in solanaceous vegetables. It is particularly effective against root rot in solanaceous vegetables caused by pathogenic fungi such as *Fusarium*, *Rhizoctonia solani*, *Phytophthora*, *Pythium spp.*, *Pseudomonas*, *Cladosporium*, and others, with an average control efficacy of 86%–95%. There were no significant differences compared to the control pesticide at the 0.05 level (P≥0.05).

Claims

1. A strain of *Streptomyces olfelsii*, which was deposited on March 7, 2022, at the China General Microbiological Culture Collection Center. Streptomyces atroolivaceus The accession number is CGMCC No.24482.

2. The application of *Streptomyces urophylla* as described in claim 1 in the prevention and control of root rot in solanaceous vegetables.

3. The application of the fermentation broth of Streptomyces urophylla as described in claim 1 in the prevention and control of root rot in solanaceous vegetables.

4. The use according to claim 3, wherein the compound is ###0002### The fermentation medium formula for preparing the fermentation broth is as follows: 20g millet, 10g glucose, 5.0g peptone, 1.2g dipotassium hydrogen phosphate, 0.8g magnesium sulfate, 0.01g ferrous sulfate, and distilled water to a final volume of 1000mL, pH 7.

4.

5. Use according to claim 2 or 3, wherein the compound is ###0002### The application of *Streptomyces urophylla* in the prevention and control of root rot in solanaceous vegetables caused by one or more of pathogenic Fusarium species, *Rhizoctonia solani*, *Phytophthora*, *Pythium spp.*, *Pseudomonas spp.*, and *Cladosporium*.

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